Page 1 [OPERATION] FV10-SW Ver 5.0c Petition This user’s manual is for the software to be run on Olympus FLUOVIEW FV1000 Confocal Laser Scanning Biological Microscope. To ensure safety, obtain optimum performance and familiarize yourself fully with this product, we recommend that you study this manual thoroughly before operation.
Page 3 CAUTION CAUTION 1. Reproduction, copying or duplication of a part or all of this software and manual is prohibited. 2. The information described in this manual may be subject to change without notice. Registered Trademarks Microsoft, Microsoft Windows, Excel for Windows are registered trademarks of Microsoft Corporation. Other brand names and product names are trademarks or registered trademarks of their respective owners.
Page 4: Table Of Contents
FLUOVIEW MANUAL CONFIGURATION FLUOVIEW MANUAL CONFIGURATION The FLUOVIEW system uses two manuals including this “User’s Manual” and the on-screen manual built into the software (“Online Help”). The User’s Manual is composed of the five following volumes and subject matter: OPERATION INSTRUCTIONS Describes the operation procedures of the FLUOVIEW system, for example, methods for image acquisition and various image processing.
Page 5 NOTATIONS IN THIS MANUAL NOTATIONS IN THIS MANUAL This manual complies with the following notations. Notation of Caution, Notes and Tips Notation Description Caution to prevent injuries to the user or damage to the product (including surrounding objects). Note for the user. NOTE Hint or one-point advice for user reference.
Page 6 NOTATIONS IN THIS MANUAL Notation of key operations Notation Description Enter The name of a key is enclosed inside The positive sign (+) expresses the combination of more than one key operation. For example, refers to pressing the key while holding the key down.
Page 7 Software Functional Configuration Software Functional Configuration This software uses panel-type windows. Usually, it is required to “select a menu then select the command to be executed” in order to execute a function provided by software. With the panel system, a software function can be executed easily by “selecting the panel page tab of the function to be executed”, just like when using a system notebook or file folder.
Page 8 Software Functional Configuration Panel Structure of the Software This software cannot show the all function panels at a glance.Please use the following list of the panels for reference in scrolling. Acquire Sets up the image acquisition and executes actual acquisition. Settings Sets the zooming ratio and observation mode for image acquisition.
Page 9 Software Functional Configuration Icons Executed by Dragging & Dropping This software selects image files and observation methods (dye name) by means of dragging & dropping. This allows simple selection based on an intuitive operation of “selecting an icon (image file or observation method), dragging it to the desired position and dropping it there”.
Page 10 Software Functional Configuration Identification of Images Depending on the Observation Methods On many occasions, FLUOVIEW displays image Image Icon Significance icons to allow identification of the observation XZ observation method used when each image is acquired. (See XZ observation, 2-channel mode table on the left.) When the [File I/O], [Tile], [Process], [Analyze] or Xt observation...
Page 11 On This Volume This volume describes the operating procedures of the FLUOVIEW FV1000 system. “Getting Started FLUOVIEW” contains information on the basic operation flow until acquisition of XY images. “APPLIED OPERATIONS” provides detailed operating procedures of the system. Please read this volume so that you can understand the system...
Page 13 CONTENTS 1 Getting Started FLUOVIEW 1-1 Basic Operations ................1-1 1-1-1 Microscope ....................1-1 1-1-2 General Mouse Operation Procedures............1-7 1-1-3 Names of Major Panel and Window Controls and Their Functions ....1-8 1-2 Outline of LSM Observation Procedures ........1-9 1-2-1 Turning Power On ..................1-11 1-2-2 Focusing on the Specimen................
Page 14 CONTENTS 1-3 Online Help..................1-36 1-3-1 Function Help ....................1-36 1-3-2 Microscope Help ..................1-37 1-3-2-1 Configuring the Microscope ................1-40 1-3-2-2 Parfocality Correction and Jog Sensitivity Adjustment ........1-46 1-3-2-3 Configuring the Filters (When using a filter system) ........1-52 1-3-2-4 Configuring the filters (When using a spectral detecting system)....1-56 1-3-2-5 Setting the C.A.
Page 15 CONTENTS Images......................2-61 2-2-3-1 Monochrome Image..................2-61 2-2-3-2 Dual-Fluorochrome Image ................2-64 2-2-3-3 Transmitted Image...................2-67 2-2-4 Image Acquisition by Rotating It (Rotation Scan) ........2-71 2-2-5 Image Acquisition of Only the Rectangular Position (Clip Scan)....2-72 2-2-6 Image Acquisition by Magnifying the Rectangular Position (Zoom-In Scan)2-75 2-2-7 High-Speed Image Acquisition ..............
Page 16 CONTENTS 2-3-7-1 Saving the Region File .................. 2-145 2-3-7-2 Reading the Region File ................2-147 2-4 Protocol processor..............2-150 2-4-1 Starting the Protocol Processor ..............2-151 2-4-2 Editing the Protocol ...................2-152 2-4-2-1 Description of Setting Items ................2-153 2-4-2-2 Command List ....................2-160 2-4-2-3 Protocol Repetition Processing..............
Page 17 CONTENTS 2-5-4-2 Displaying Merged Image of Multiple Channels (Single View)......2-206 2-5-5 Changing the Number of Divided Images ..........2-208 2-5-5-1 Increasing the Number of Divided Images ............2-208 2-5-5-2 Decreasing the Number of Divided Images...........2-210 2-5-6 Switching the Display Method of Multiple Images ........2-211 2-5-7 Displaying Multiple Image Slices Together ..........
Page 18 CONTENTS 2-6-4-2 Colocalization for series image data set ............2-254 2-6-4-3 Image measurement ..................2-255 2-6-5 Appending image (Append) ...............2-258 2-6-5-1 Appending two images.................. 2-258 2-6-5-2 Appending image from several image data set ..........2-262 2-6-6 Extract image (Crop)..................2-267 2-7 Image Analysis................2-274 2-7-1 Checking the Intensity of a Specific Part ...........2-275 2-7-1-1 Intensity Values on a Line (Line Profile) ............
Page 19 CONTENTS 2-10-1 Displaying Images Together..............2-316 2-10-2 Displaying Images Successively ............. 2-316 2-11 Transferring Data to Another Application .......2-319 2-11-1 Transferring Analysis Data to Another Application ........2-319 2-11-2 Transferring the Plot Image of Analysis Data to Another Application..2-322 2-11-3 Transferring Image Data to Another Application (Paint, etc.) ....
Page 20 CONTENTS Appendix B Glossary Appendix C USER REGISTRATION OF FV1000 Appendix C-1 User Registration............C-1 Appendix C-2 Logging into the FV1000..........C-4 Appendix C-3 Deleting a User ............. C-5 Appendix D Change of Default Folder for [File I/O] Panel Appendix E List of Functions in the [Active Overlays] Dialog Box Appendix E-1 Coordinate Position Data..........E-1 Appendix E-1-1 X-Coordinate ................
Page 21 CONTENTS Allocation Appendix F-1 Hand Switch Functions ..........F-1 Appendix F-1-1 BX/BXWI ..................F-1 Appendix F-1-2 IX....................F-2 Appendix F-2 Microscope Frame Functions ........F-3 Appendix F-2-1 BX ....................F-3 Appendix F-2-2 IX....................F-4 Appendix F-2-3 Focus Adjustment Knob ..............F-5 1 BX ..........................F-5 2 IX ..........................F-5...
Page 23: Getting Started Fluoview
Getting Started FLUOVIEW Basic Operations 1 Getting Started FLUOVIEW 1-1 Basic Operations 1-1-1 Microscope The following figure shows the major controls of a microscope. The actual configuration of the modules including the specimen stage, revolving nosepiece and lighting equipment may differ from those shown below.
Page 24 Getting Started FLUOVIEW /Basic Operations Light path selector knob Select the light path between the visual observation and photography observation. • See the following table and set the knob to the position corresponding to the required light path. Light path selector knob Symbol Intensity Ratio Pushed in...
Page 25 Getting Started FLUOVIEW /Basic Operations With transmitted light differential interference observation using an NOTE immersion objective, set the microscope’s field diaphragm so that it circumscribes the field of view. Otherwise the contrast may degrade. (This applies to both visual observation and laser differential interference observation.) Filters These filters are used to adjust the transmitted light.
Page 26 Getting Started FLUOVIEW /Basic Operations Combination with IX81 FVF (5) Filters (4) Condenser (4) Transmitted light DIC slider U-DICTS (optional) (6) Light path selector button (1)Fluorescence mirror unit ((2) Analyzer IX-MDICT Optionally installed) (6)Hand switch U-HSTR2 (U-FH is optionally available.) OPERATION INSTRUCTIONS Page...
Page 27 Getting Started FLUOVIEW /Basic Operations Fluorescence mirror unit Select the fluorescence observation tube by rotating the turret. • Engage the desired cube in the light path for visual fluorescence observation. • For laser microscopy, rotate the turret to page tab .
Page 28 Getting Started FLUOVIEW /Basic Operations Condenser, polarizing plate Condenser for transmitted lighting. In addition, the rotary turret for the transmitted light DIC and the polarizing plate for differential interference observation (analyzer) are also provided. • To perform differential interference observation, engage the transmitted light DIC (optional) matching the objective in use in the light path (For both visual observation and laser differential interference observation).
Page 29: General Mouse Operation Procedures
Getting Started FLUOVIEW /Basic Operations 1-1-2 General Mouse Operation Procedures Use the mouse to select a command, character string or button. Use the left button of the mouse unless otherwise specified. To select or execute something: Clicking To click the mouse, place the mouse pointer on the desired function and press the mouse button once.
Page 30: Names Of Major Panel And Window Controls And Their Functions
Getting Started FLUOVIEW /Basic Operations 1-1-3 Names of Major Panel and Window Controls and Their Functions The window as shown below is displayed when FLUOVIEW starts up. FLUOVIEW uses panel-type windows. This section describes the names of the major controls displayed in panels and windows. Minimize button <...
Page 31: Outline Of Lsm Observation Procedures
Getting Started FLUOVIEW /Outline of LSM Observation Procedures 1-2 Outline of LSM Observation Procedures • Fluorescence observation procedure Start the system. Change the ND filter with a filter • Turn the system power ON. with higher transmittance (Section 1-2-1) • Start the FLUOVIEW software.
Page 32 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures • Transmitted light observation procedure Start the system • Turn the system power ON. (Section 1-2-1) Change the ND filter with a higher • Start the FLUOVIEW software. transmittance filter. (Section 1-2-1) (Section 1-2-6) Set the observation condition.
Page 33: Turning Power On
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-1 Turning Power On Set the power switch of each unit to ON, then start the software. For details, see sections 1-1 “Turning the Power On” and section 1-2 “Starting the Software” in Volume II [PREPARATION For OPERATION] of [HARDWARE GUIDE] of the FV1000 User’s Manual.
Page 34: Focusing On The Specimen
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-2 Focusing on the Specimen 1-2-2-1 Combination with BX Select the light path for 100% eyepiece by pushing in the light path selector knob (1) fully to the stop position. From the page tabs on the bottom right of the [Acquire] panel , select the [Settings] sub-panel.
Page 35 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures When focus is aligned with use of focus handle of microscope or U-FH, uncheck checkbox of [Locked] on [Z Stage] sub panel inside [Acquire] panel. (See 2-2-1-4-7 of this manual.) When [Locked] checkbox is checked, the handle cannot be operated.
Page 36: Combination With Ix81 Fvf
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-2-2 Combination with IX81 FVF 1. Push the light path selector button (1) on the front of the microscope to (when using Manual microscope). From the page tabs on the bottom right of the [Acquire] panel, select the [Scan] sub-panel, and Select the <BI>...
Page 37 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures When focus is aligned with use of focus handle of microscope or U-FH, uncheck checkbox of [Locked] on [Z Stage] sub panel inside [Acquire] panel. (See 2-2-1-4-7 of this manual.) When [Locked] checkbox is checked, the handle cannot be operated. NOTE The specimen may float during oil-immersed observation.
Page 38: Setting The Lsm Light Path
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-3 Setting the LSM Light Path 1-2-3-1 Combination with Upright Microscope (BX) From the page tabs on the bottom right of the [Acquire] panel, select the [Settings] sub-panel. Fig. 1-5 [Settings] Sub-panel Select the <LSM>...
Page 39 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures When only fluorescence observation is required, disengage the transmitted DIC slider (3) by setting the switch to the pulled-out position. When transmitted light differential interference observation or simultaneous fluorescence + transmitted light differential interference observation is required, engage the transmitted DIC slider and the optimum transmitted light DIC for the objective in the light path by operating the universal condenser (5).
Page 40: Combination With Inverted Microscope (Ix81 Fvf)
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-3-2 Combination with Inverted Microscope (IX81 FVF) From the page tabs on the bottom right of the [Acquire] panel, select the [Settings] sub-panel, and .select the <LSM> button in the [Light Path] group box. The <LSM>...
Page 41 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures During the transmitted light observation, be sure to disengage the filter (5) from the light path. (5) Filters (4) Condenser (3) Transmitted DIC U-DICTS (1) Fluorescence mirror unit ((2) Analyzer IX-MDICT Optionally installed.) (6) Hand switch U-HSTR (U-FH is optionally...
Page 42: Selecting The Dyeing Method
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-4 Selecting the Dyeing Method From the page tabs on the bottom right of the [Acquire] panel, select the [Dyes] sub- panel. Fig. 1-6 [Dyes] Sub-panel Select the specimen dyeing method by dragging [FITC] and [TRITC] in the [Available Dyes] list box in the [Selected Dyes] group box to the field immediately above the list box.
Page 43 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures When the dyeing method is selected from the [Available Dyes] list box and the <Apply> button is clicked, a channel for acquiring fluorescence is set automatically according to the switched filter. And the dyeing method is shown in the [Ch] group box.
Page 44 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures One Point! The [Assign dyes manually] check box can also be used to set the dyeing method to the desired channel. Check the [Assign dyes manually] check box in the [Dyes] sub-panel. Select the dyeing method in the [Available Dyes] list box and drag it directly to the field of the [Ch] check box.
Page 45: Selecting The Filters
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-5 Selecting the Filters The excitation dichroic mirror, beam splitter and barrier filters are set automatically to the light path according to the dyeing method selected for the specimen. To change filters, see section 1-3-2-4, “Configuring the Filters” in this volume and follow instructions in the [Optical System Configuration] window.
Page 46 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures Excitation Beam Beam Beam Barrier Barrier Barrier Filter 1 Barrier Filter 2 Laser Combination Dichroic Mirror Splitter 1 Splitter 2 Splitter 3 Filter 3 Filter 4 LD440, Multiline Ar, (1) BS20/80 (1) Mirror (1) Mirror (1) BA465-495 (1) BA505IF...
Page 47 A single type of excitation filter can be equipped. Replace it if necessary. Up to two types of barrier filter can be equipped per channel. If the equipment of another filter set for laser configuration is required, please contact your local Olympus representative. OPERATION INSTRUCTIONS...
Page 48: Setting The Nd Filters
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-6 Setting the ND Filters When you use the laser combiner, you can set the laser intensity by setting ND filter on the laser combiner. Display the [Acquire] panel. Set each laser intensity by sliding the scale bar in the [Laser Intensity] group box of the [Lasers] sub-panel, in accordance with specimen’s brightness, fluorescence crosstalk and photo-bleaching.
Page 49: Setting The Observation Condition
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures One Point! After the dyeing method has been set with the [Dyes] sub-panel, the ND filters can be set using the [Ch] group box in the upper part of the [Acquire] panel. In the upper part of the [Acquire] sub-panel, open the [Ch] group box for the ND to be changed.
Page 50: Setting The Zoom Ratio To 1X
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-7-2 Setting the Zoom Ratio to 1X Use the [Zoom] scale in the [Acquire] panel to set the zoom ratio to “X1”. Using the UV-Ar laser, set the zoom ratio to “X2”. 1-2-7-3 Setting the Channels In the [Ch] group box, make sure that the check boxes showing the applicable dyeing methods are check-marked to indicate that the channels are ready for image...
Page 51: Setting The Highest Scan Speed
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-7-4 Setting the Highest Scan Speed Set the scan speed to the fastest speed by using the scale in the [Scan Speed] group box in the [Acquire] panel [Scan Speed] group box Set the scan speed by clicking a point on the scale line.
Page 52: Setting The Xy Observation Mode
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-7-5 Setting the XY Observation Mode In the [Mode] group box in the [Acquire] panel, select the [Surface] option button. In the [Mode] group box in the [Acquire] panel, select [800 by 600] from the [Scan Size] drop-down list.
Page 53: Setting The Cross-Section To Be Observed
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-7-7 Setting the Cross-section to be Observed While acquiring image, move the Z stage to select the cross-section to be observed. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Z Stage] sub-panel.
Page 54: Setting The Area To Be Observed
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-7-8 Setting the Area to be Observed When the observation targets are concentrated in a narrow area or when observation of a specific area detail is required, the image of a limited area can be selected. Clicking a point in the scale area The 4 buttons represent directions, and to change the value on a large...
Page 55: Stopping Repeated Scanning
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-7-10 Stopping Repeated Scanning After the brightness and gain have been adjusted, select the <STOP SCAN> button in the [Acquire] panel to stop scanning temporarily. 1-2-8 Acquiring Image Select the <Once> button. The acquired image will be displayed in the [Live] panel. <Once>...
Page 56: Saving Image
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-9 Saving Image Display the [File I/O] panel. When saving images acquired with more than one channel, it is possible to select whether images from more than one image are saved simultaneously or only one of the <Display channel switch>...
Page 57: Exiting From The Software, Turning Power Off
Getting Started FLUOVIEW/ Outline of LSM Observation Procedures Click <Experiment> button in the [Save] group box. The [Save Experiment As] dialog box will open. Fig. 1-11 [Save Experiment As] Dialog Box Enter the file name in the [File name:] text box. Select “FLUOVIEW MultiTif”...
Page 58: Online Help
Getting Started FLUOVIEW/ Online Help 1-3 Online Help The FLUOVIEW application comes with two kinds of online help facility: • function help for referencing the function and operation procedure description while controlling the application, and • microscope help providing information on the system setup. 1-3-1 Function Help This section describes a simple method for displaying and consulting the online help on the functions and operation procedures.
Page 59: Microscope Help
Getting Started FLUOVIEW /Online Help When the mouse pointer is placed on a word in enhanced display, the mouse pointer turns into a “finger pointer”. One Point! Click the <Contents> button to the initial display. Click the <Back> button to return to the previous information page.
Page 60 Getting Started FLUOVIEW/ Online Help 3. Click the <Apply> button to apply the selected dyeing method to the [Ch] group box on the upper part of the [Acquire] panel. When the dyeing method is selected from the [Available Dyes] list box and the <Apply>...
Page 61 Getting Started FLUOVIEW /Online Help One Point! The [Assign dyes manually] check box can also be used to set the dyeing method to the desired channel. Check the [Assign dyes manually] check box in the [Dyes] sub-panel. Select the dyeing method in the [Available Dyes] list box and drag it directly to the field of the [Ch] check box.
Page 62: Configuring The Microscope
Getting Started FLUOVIEW/ Online Help 1-3-2-1 Configuring the Microscope When the combination with BX61 or IX81 is in use, the microscope and scan unit can be configured on the FLUOVIEW software. Use the following procedure to configure the microscope. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Settings] sub-panel.
Page 63 Getting Started FLUOVIEW /Online Help Select the <Scope> button on the bottom of the [Settings] sub-panel. The [Microscope Control] window as shown below appears. [Shutter] group box Clicking inside the box switches the [EPI lamp] Indicates the EPI lamp. EPI shutter to be closed/opened, [Filter Turret] group The shutter is opened.
Page 64 Getting Started FLUOVIEW/ Online Help [Lamp] group box Clicking inside the box switches the TD lamp ON/OFF. The TD lamp is set to OFF. The TD lamp is set to ON. [Options] group box Used for optional IX settings. <Link Setting> button Selects the function to change settings corresponding to the change of IX settings.
Page 65 Getting Started FLUOVIEW /Online Help Clicking the <Link Setting> button displays the [Link Setting] dialog box as shown below. Checking here links the objective in the [Nosepiece] group box with the condenser turret. Checking here escapes the stage or revolving nosepiece when the objective is selected and changed in the [Nosepiece] group box.
Page 66 Getting Started FLUOVIEW/ Online Help Open the setting Open the file to which the microscope settings were exported with the <Export Setting> button. The Settings configured by another user or configured for another combination can be applied. Selecting the <Import Setting> button of the [Link Setting] dialog box displays the [Open] dialog box as shown below.
Page 67 Getting Started FLUOVIEW /Online Help Save the setting Settings in the [Link Setting] dialog box can be applied to other users or other combinations. Selecting the <Export Setting> button of the [Link Setting] dialog box displays the [Save As] dialog box as shown below. To change the save destination drive or directory, use the [Save in: ] drop-down list.
Page 68: Parfocality Correction And Jog Sensitivity Adjustment
Getting Started FLUOVIEW/ Online Help 1-3-2-2 Parfocality Correction and Jog Sensitivity Adjustment When the system use the BX or IX microscope, the parfocality correction and jog sensitivity can be set per objective. Open the [Microscope Control Panel] window. For the method of displaying this window, see steps 1 and 2 in section 1-3-2-1, “Configuring the Microscope ”...
Page 69 Getting Started FLUOVIEW /Online Help The following option button menu is displayed in the [Pfcl Setup Wizard] group box. If you want to begin the parfocality correction value setting with the objective with the maximum magnification, click the [Switch to...] option button then click the <Next> button.
Page 70 Getting Started FLUOVIEW/ Online Help The objective set as the specified in the [Objectives] group box is selected, and the [Pfcl Setup Wizard] group box displays the message shown below. Bring the specimen in focus by observing it visually or on the scanned image, and then click the <Next> button.
Page 71 Getting Started FLUOVIEW /Online Help The [Pfcl Setup Wizard:] group box displays the option button menu shown below. If you want to set the parfocality correction values of objectives with other power values, simply click the <Next> button. Click to proceed to the setting of the objective with next higher power to the current objective.
Page 72 Getting Started FLUOVIEW/ Online Help The [Pfcl Setup Wizard] group box displays the option button menu shown below. Adjust the focal position and click the <Next> button. Click to set the current focal position as the parfocality correction value for the current objective and proceed to the setting of the next objective.
Page 73 Getting Started FLUOVIEW /Online Help Set the parfocality correction values of objectives by repeating steps 6 and 7 for each. The [Pfcl Setup Wizard] group box displays the option button menu shown below. Click the <Pfcl> button to activate the parfocality correction. [Jog Fine] group box Select the jog sensitivity of each objective from...
Page 74: Configuring The Filters (When Using A Filter System)
Getting Started FLUOVIEW/ Online Help 1-3-2-3 Configuring the Filters (When using a filter system) The barrier filters, excitation filter and beam splitter are set automatically to the light path according to the dyeing method selected for the specimen. Use the following procedure to change these filters. Select the [Settings] sub-panel.
Page 75 Getting Started FLUOVIEW /Online Help 2. Click the <Scan Unit> button at the bottom of the panel. The window as shown below will appear. [Laser Unit] group box Shows the type of laser to be used. (With the laser combiner operation, the laser type is set and displayed automatically.) When the <On>...
Page 76 Getting Started FLUOVIEW/ Online Help One Point! The formulas of resolution of X and Y are as follows; FWHM : Resolution of X and Y L : The length of a diagonal line of Square Pinhole NA = nSINθ :NA of Objective lens MG : The objective Magnification N : The refractive index of a medium Air : 1.0...
Page 77 Getting Started FLUOVIEW /Online Help One Point! The formula of resolution of Z is as follows; FWHM : Resolution of Z L : The length of a diagonal line of Square Pinhole NA = nSINθ :NA of Objective lens MG : The objective Magnification N : The refractive index of a medium Air : 1.0 Water : 1.3...
Page 78: Configuring The Filters (When Using A Spectral Detecting System)
Getting Started FLUOVIEW/ Online Help 1-3-2-4 Configuring the filters (When using a spectral detecting system) When the system incorporates the spectral detector unit that is composed of a 2-channel spectral detector and 1-channel filter, it is possible to set the detection conditions more flexibly, acquire the fluorescence spectral data and use the fluorescence isolation function.
Page 79 Getting Started FLUOVIEW /Online Help The parameters of the spectral detecting system can also be set in the [Optical System Configuration] window as well as in the [Spectral-Control] sub panel. Select the [Settings] sub panel. <Scan Unit> button Display the filter and laser types to be set.
Page 80 Getting Started FLUOVIEW/ Online Help For other functions available in the [Optical System Configuration] window, see section 1-3- 2-3, “Configuring the Filters (When using a filter system),” After completing the setup, click the <Close> button to close the window. OPERATION INSTRUCTIONS Page 1 - 5 8...
Page 81: Setting The C.a. Diameters
Getting Started FLUOVIEW /Online Help 1-3-2-5 Setting the C.A. Diameters The pinhole diameters are set automatically according to the selected dyeing method. Use the following procedure to change the pinhole diameters. Display the [Acquire] panel. [Ch] group box Sets whether the image of each channel acquired or not, the PMT...
Page 82 Getting Started FLUOVIEW/ Online Help Right-click the mouse inside the [Ch] group box to display the [Ch] group boxes of all channels. [Ch1], [Ch2], [Ch3], [Ch4] and [Ch5] group boxes Select the channels from which you want to acquire images. [PMT], [Gain] and [Offset] LED sliders Set these values independently.
Page 83 Getting Started FLUOVIEW /Online Help 3. Click the < > button of the channel you want to change the pinhole diameter. The group boxes for setting the pinhole diameters and laser ND filter values of the channels are displayed below the [Ch] group box. (They are not displayed for channels set for transmitted light observation.) Ch1], [Ch2], [Ch3], [Ch4] and [Ch5] group boxes...
Page 84 Getting Started FLUOVIEW/ Online Help While observing the images in the [Live] panel, change their pinhole diameter settings. Clicking this button allows fine adjustment of the value. Clicking this field allows the value to be changed on a large scale. The ND value which are usually used are displayed in green.
Page 85: Applied Operations
APPLIED OPERATIONS /General Operation Procedure 2 APPLIED OPERATIONS 2-1 General Operation Procedure This section describes the general image acquisition procedure with the aim to get accustomed with the operation. Begin using the FLUOVIEW system by acquiring an image or opening an image from a file.
Page 86 APPLIED OPERATIONS /General Operation Procedure Turn power ON and start the FLUOVIEW software. (Sections 1-2-1 & 1-2-2) Acquire an image. (According to the selected Open an image in a file. observation mode) (Section 2-3-2) For detailed operation procedures for image acquisition, see section 2-1-1, “Image Acquisition Procedure (Section A)”.
Page 87: Image Acquisition Procedure (Section (A))
APPLIED OPERATIONS /General Operation Procedure 2-1-1 Image Acquisition Procedure (Section (A)) This section describes the procedure for acquiring images. See sections 2-2 and after for the actual operation methods. The detailed operation methods of each item in the procedure are described in the section specified in parentheses (( )). After “Turn power ON and start the FLUOVIEW software”,...
Page 88: Image Acquisition Procedure In An Observation Mode (Section (B))
APPLIED OPERATIONS /General Operation Procedure 2-1-2 Image Acquisition Procedure in an Observation Mode (Section (B)) As an example of “Acquire an image in an observation mode”, this section describes the procedure in the XY observation mode. For the procedures in other observation modes, see section 2-2-2, “Image Acquisition in Other Observation Modes”...
Page 89: Examples Of Operation Procedures
APPLIED OPERATIONS /General Operation Procedure 2-1-3 Examples of Operation Procedures Begin using the FLUOVIEW system by acquiring an image or opening an image in a file. For the detailed operation method of each item in the procedure, see the section specified in parentheses (( )).
Page 90 APPLIED OPERATIONS /General Operation Procedure Example 3) To acquire an image and compared it with a Example 4) To open an image in a file, improve its previously acquired image: contrast and create a presentation image by entering comment, etc. Turn power ON and start the Turn power ON and start the FLUOVIEW software.
Page 91: Image Acquisition
APPLIED OPERATIONS /Image Acquisition 2-2 Image Acquisition Confirm the image to be acquired using the microscope, and acquire its image using FLUOVIEW. The image can be saved in a file as required. NOTE If FLUOVIEW window is moved while an image is being acquired, it may cause image acquisition failure.
Page 92: Image Acquisition In Xy Observation Mode
APPLIED OPERATIONS /Image Acquisition 2-2-1 Image Acquisition in XY Observation Mode This section describes the basic operation procedure from the system configuration to the image acquisition in the XY observation mode and image saving in a file as shown in the following chart.
Page 93: Configuring The Microscope
APPLIED OPERATIONS /Image Acquisition 2-2-1-1 Configuring the Microscope Set the light path so that the image can be observed through the microscope. Display the [Acquire] panel. <Once> button Acquires an image in the currently <Focus> button selected observation mode and display The repetition images can be acquired at a high speed.
Page 94 APPLIED OPERATIONS /Image Acquisition Setting the dyeing method From the page tabs on the bottom right of the [Acquire] panel, select the [Dyes] sub-panel. Place the pointer on the icon [Assign dyes manually] displayed [Selected check box Dyes], and the dyeing method Checking this enables the is shown in the pop-up display.
Page 95 APPLIED OPERATIONS /Image Acquisition Click the <Apply> button to apply the selected dyeing method to the [Ch] group box on the upper part of the [Acquire] panel. When the dyeing method is selected from the [Available Dyes] list box and the <Apply>...
Page 96 APPLIED OPERATIONS /Image Acquisition One Point! The [Assign dyes manually] check box can also be used to set the dyeing method to the desired channel. Check the [Assign dyes manually] check box in the [Dyes] sub-panel. Select the dyeing method in the [Available Dyes] list box and drag it directly to the field of the [Ch] check box.
Page 97 APPLIED OPERATIONS /Image Acquisition From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. <Scope> button Displays useful information for the system setup. Fig. 2-4 [Optics] Sub-panel OPERATION INSTRUCTIONS Page 2 - 1 3...
Page 98 APPLIED OPERATIONS /Image Acquisition Select the <Scope > button at the bottom of the panel. The window as shown below will appear (in case of a combination with the IX81). Fig. 2-5 [Microscope Control Panel] Window Configure the microscope in [Microscope Control Panel] window. After completing the setup, click the <Close>...
Page 99: Setting The Filters
APPLIED OPERATIONS /Image Acquisition 2-2-1-2 Setting the Filters The excitation filter, spectral filter and barrier filters are set automatically to the light path according to the dyeing method selected for the specimen. To change filters, use the following procedure. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Settings] sub-panel.
Page 100 APPLIED OPERATIONS /Image Acquisition Click the <Scan Unit> button at the bottom of the panel. The window as shown below will appear. [Laser Unit] group box Shows the type of laser to be used. (With the laser combiner operation, the laser type is set and displayed automatically.) When the <On>...
Page 101: Setting The Nd Filters
APPLIED OPERATIONS /Image Acquisition 2-2-1-3 Setting the ND Filters When you use the laser combiner, you can set the laser intensity by setting ND filter on the laser combiner. Display the [Acquire] panel. Set each laser intensity by sliding the scale bar in the [Laser Intensity] group box of the [Lasers] sub-panel, in accordance with specimen’s brightness, fluorescence crosstalk and photo-bleaching.
Page 102 APPLIED OPERATIONS /Image Acquisition One Point! After the dyeing method has been set with the [Dyes] sub-panel, the ND filters can be set using the [Ch] group box in the upper part of the [Acquire] panel. In the upper part of the [Acquire] sub-panel, open the [Ch] group box for the ND to be changed.
Page 103: Setting The Observation Condition
APPLIED OPERATIONS /Image Acquisition 2-2-1-4 Setting the Observation Condition 1 Setting the Objective Magnification From the drop-down list on the center of the [Acquire] panel, select the objective being used with the microscope. NOTE The measurement results will be inaccurate if the objective magnification set here does not match the actual magnification of the objective in use.
Page 104 APPLIED OPERATIONS /Image Acquisition 3 Setting the Channels In the Channel 1 group box, check the check box showing the applicable dyeing method to make the image acquisition ready. In the Channel 2 group box, check the check box showing the applicable dyeing method to ready the image acquisition.
Page 105 APPLIED OPERATIONS /Image Acquisition 4 Setting the Highest Scan Speed Set the scan speed to the fastest speed by using the scale in the [Scan Speed] group box in the [Acquire] panel [Scan Speed] group box Set the scan speed by clicking a point on the scale line.
Page 106 APPLIED OPERATIONS /Image Acquisition 5 Setting the XY Observation Mode In the [Mode] group box in the [Acquire] panel, select the [Surface] option button. In the [Acquire] panel, select the XY observation mode option button. 6 Repeated Scanning Operation Select the <XY Repeat> button. The acquired image will be displayed in the [Live] panel.
Page 107 APPLIED OPERATIONS /Image Acquisition The moving amount assigned to the <Z stage fine adjustment> and <Z stage coarse adjustment> buttons can be changed. See section 1-3 in MAINTENANCE, “Setting the System Configuration” for detailed operations. Check the [Locked] check box in the [Z Stage] sub-panel. While observing the image in the [Live] panel, locate the plane to be observed by displacing the stage using the <Z stage coarse adjustment>...
Page 108 APPLIED OPERATIONS /Image Acquisition 8 Setting the Area to be Observed When the observation targets are concentrated in a narrow area or when observation of a specific area detail is required, the image of a limited area can be selected. Blue frame appears.
Page 109 APPLIED OPERATIONS /Image Acquisition 9 Adjusting the image brightness Clicking this button allows fine adjustment of the value. Clicking this field allows the value to be changed on a large scale. The ND value which are usually used displayed in green. Clicking the <+>...
Page 110 APPLIED OPERATIONS /Image Acquisition Each click of the <+> or <-> button of the [PMT] LED slider increases or decreases the PMT voltage by 5 V. Each click of the slider section of the [PMT] LED slider varies the PMT voltage by 25 V.
Page 111 APPLIED OPERATIONS /Image Acquisition [Hi-Lo] LUT can be utilized to adjust image intensity easily. Click <LUT> button from tool bar. Dialog box – [Color Tool] will appear. Click [Hi-Lo] LUT from group box of [Standard Color LUTs]. <LUT> button The intensity value 0 is colored with Blue, and the maximum intensity is colored with Red.
Page 112: Acquiring Image
APPLIED OPERATIONS /Image Acquisition 10 Setting a Lower Scan Speed The scan speed can be decreased using the scale in the [Scan Speed] group box on the [Acquire] panel. In general, setting a lower scan speed allows the acquired image quality to be improved.
Page 113: Acquiring Image In Accumulation Mode
APPLIED OPERATIONS /Image Acquisition 2-2-1-6 Acquiring Image in Accumulation Mode When the image is dark or noisy, use an accumulation mode in image acquisition to improve the image quality. Kalman Accumulation and Peak Accumulation The Kalman accumulation acquires images for the specified number of times while averaging the images.
Page 114 APPLIED OPERATIONS /Image Acquisition 1 Acquiring Image in Accumulation Mode (Frame mode) From the page tabs on the bottom right of the [Acquire] panel, select the [Settings] sub-panel. [Filter Mode] group box Select the accumulation mode. Two accumulation modes, [Kalman] and [Accumulate To Peak] are available.
Page 115 APPLIED OPERATIONS /Image Acquisition When [Accumulate] is selected, enter the addition count in the text box. Click the <Once> button in the [Acquire] panel. The acquired image will be displayed in the [Live] panel. 2 Acquiring Image in Accumulation Mode (Line mode) NOTE Image acquisition in the line mode can be performed when you use the FV1000 system with AOTF (FV5-COMBA).
Page 116 APPLIED OPERATIONS /Image Acquisition Enter the accumulation count in the text box and select the <Line> button displayed. Enter the accumulation count. <Line> button The accumulation count can be set up a maximum of 64 times. When 0 is set as the number of times of accumulation, the number is set to 1 automatically ..
Page 117: Saving The Acquired Image In File
APPLIED OPERATIONS /Image Acquisition 2-2-1-7 Saving the Acquired Image in File Display the [File I/O] panel. Click the page tab of the [Live] panel showing the image to be saved, so that the image is displayed at the front. Click the <Experiment> button in the [Save] group box in the [File I/O] panel. <Experiment>...
Page 118: Image Acquisition In Other Observation Modes
APPLIED OPERATIONS /Image Acquisition 2-2-2 Image Acquisition in Other Observation Modes 2-2-2-1 XZ Observation Mode The description in this section will be focused on the image acquisition operations in the XZ observation mode that are not used in the XY observation modes (which are the operations enclosed in in the chart on the next page).
Page 119 APPLIED OPERATIONS /Image Acquisition 1 Setting the Z-direction scanning range While acquiring image, move the Z stage according to the range of the multiple sections to be observed (i.e. the Z-direction scanning range). From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Z <Go>...
Page 120 APPLIED OPERATIONS /Image Acquisition While observing the image in the [Live] panel, locate the upper edge of the range to be observed by moving down the stage using the <Z stage coarse adjustment> and <Z stage fine adjustment> buttons in the [Z Stage] sub-panel. When using the FLUOVIEW system with an inverted microscope, locate the bottom edge of the range to be observed by moving down the revolving nosepiece using the <Z stage coarse adjustment>...
Page 121 APPLIED OPERATIONS /Image Acquisition 3 Setting the observation line A line is displayed on the image in the [Live] panel. Place the mouse pointer arrow on the line and drag it to the position you want to observe. 4 Setting the numbers of steps and acquired image slices From the page tabs on the bottom right of the [Acquire] panel, select the [Z Stage] sub-panel.
Page 122: Xt Observation Mode
APPLIED OPERATIONS /Image Acquisition 2-2-2-2 XT Observation Mode The description in this section will be focused on the image acquisition operations in the XT observation mode that are not used in the XY observation modes (which are the operations enclosed in in the chart on the next page).
Page 123 APPLIED OPERATIONS /Image Acquisition 1 Setting the observation mode In the [Mode] group box in the [Acquire] panel, select the [Line] option button. In the [Acquire] panel, select the XT observation mode option button. 2 Setting the observation line A line is displayed on the image in the [Live] panel. Place the mouse pointer arrow on the line and drag it to the position you want to observe.
Page 124: Xzt Observation Mode
APPLIED OPERATIONS /Image Acquisition 2-2-2-3 XZT Observation Mode The description in this section will be focused on the image acquisition operations in the XZT observation mode that are not used in the XY observation modes (which are the operations enclosed in in the chart on the next page).
Page 125 APPLIED OPERATIONS /Image Acquisition 1 Setting the Z-direction scanning range While acquiring image, move the Z stage according to the range of the multiple sections to be observed (Z-direction scanning range). From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Z Stage] sub-panel.
Page 126 APPLIED OPERATIONS /Image Acquisition Check the [Locked] check box in the [Z Stage] sub-panel. While observing the image in the [Live] panel, locate the upper edge of the range to be observed by moving down the stage using the <Z stage coarse adjustment> and <Z stage fine adjustment>...
Page 127 APPLIED OPERATIONS /Image Acquisition 3 Setting the observation line A line is displayed on the image in the [Live] panel. Place the mouse pointer arrow on the line and drag it to the position you want to observe. 4 Setting the numbers of steps and acquired image slices From the page tabs on the bottom right of the [Acquire] panel, select the [Z Stage] sub-panel.
Page 128 APPLIED OPERATIONS /Image Acquisition 5 Setting the interval time From the page tabs on the bottom right of the [Acquire] panel, select the [Time Series] sub-panel. Set the interval time using the < > or < > button in the [Interval] text box. [Interval] text box Set the interval time using the <...
Page 129 APPLIED OPERATIONS /Image Acquisition 7 Acquiring image Click the <XZT> button in the [Acquire] panel. The acquired image will be displayed in the [Live] panel. While acquiring an image in the XZT observation mode, clicking the <STOP SCAN> button changes the buttons at the upper part of the [Acquire] panel as shown below. The <Resume>...
Page 130: Xyz Observation Mode
APPLIED OPERATIONS /Image Acquisition 2-2-2-4 XYZ Observation Mode The description in this section will be focused on the image acquisition operations in the XYZ observation mode that are not used in the XY observation modes (which are the operations enclosed in in the chart on the next page).
Page 131 APPLIED OPERATIONS /Image Acquisition 1 Setting the Z-direction scanning range While acquiring image, move the Z stage according to the range of the multiple sections to be observed (Z-direction scanning range). From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Z <Go>...
Page 132 APPLIED OPERATIONS /Image Acquisition Check the [Locked] check box in the [Z Stage] sub-panel. While observing the image in the [Live] panel, locate the upper edge of the range to be observed by moving down the stage using the <Z stage coarse adjustment> and <Z stage fine adjustment>...
Page 133 APPLIED OPERATIONS /Image Acquisition 2 Setting the numbers of steps and acquired image slices 1. Set the number of steps using the < > or < > button in the [Step Size] text box. The number of steps shown in the [Step Size] text box has been calculated by [Step Size] text box the system so that the depth scale of the acquired image is identical to the horizontal scale.
Page 134 APPLIED OPERATIONS /Image Acquisition 5 Appending image XYZ image can be added after the image acquisition. Immediately after acquisition of an image in the XYZ observation mode, the buttons at the upper part of the [Acquire] panel changes as shown below. <Append Next>...
Page 135: Xyt Observation Mode
APPLIED OPERATIONS /Image Acquisition 2-2-2-5 XYT Observation Mode The description in this section will be focused on the image acquisition operations in the XYT observation mode that are not used in the XY observation modes (which are the operations enclosed in in the chart on the next page).
Page 136 APPLIED OPERATIONS /Image Acquisition 1 Setting the observation mode In the [Mode] group box in the [Acquire] panel, select the [Surface] option button. In the [Acquire] panel, select the XYT observation mode option button. 2 Setting the interval time From the page tabs on the bottom right of the [Acquire] panel, select the [Time Series] sub-panel.
Page 137 APPLIED OPERATIONS /Image Acquisition 3 Setting the number of scans Set the number of scans using the < > or < > button in the [N] text box in the [Time Series] sub-panel. 4 Acquiring image Click the <XYT> button in the [Acquire] panel. The acquired image will be displayed in the [Live] panel.
Page 138 APPLIED OPERATIONS /Image Acquisition 5 Appending image XYT image can be added after the image acquisition. Immediately after acquisition of an image in the XYT observation mode, the buttons at the upper part of the [Acquire] panel changes as shown below. <Series Done>...
Page 139: Xyzt Observation Mode
APPLIED OPERATIONS /Image Acquisition 2-2-2-6 XYZT Observation Mode The description in this section will be focused on the image acquisition operations in the XYZT observation mode that are not used in the XY observation modes (which are the operations enclosed in in the chart on the next page).
Page 140 APPLIED OPERATIONS /Image Acquisition 1 Setting the Z-direction scanning range While acquiring image, move the Z stage according to the range of the multiple sections to be observed (Z-direction scanning range). From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Z <Go>...
Page 141 APPLIED OPERATIONS /Image Acquisition Check the [Locked] check box in the [Z Stage] sub-panel. While observing the image in the [Live] panel, locate the upper edge of the range to be observed by moving down the stage using the <Z stage coarse adjustment> and <Z stage fine adjustment>...
Page 142 APPLIED OPERATIONS /Image Acquisition 2 Setting the numbers of steps and acquired image slices Set the number of steps using the < > or < > button in the [Step Size] text box. The number of steps shown in the [Step Size] text box has been calculated by the system so that the depth scale of the acquired image is identical to the [Step Size] text box horizontal scale.
Page 143 APPLIED OPERATIONS /Image Acquisition Set the interval time using the < > or < > button in the [Interval] text box. An image with no interval can be acquired by entering [0] in the [Interval] text box. In this case, the [Interval] text box shows “Free Run” message. Using <...
Page 144 APPLIED OPERATIONS /Image Acquisition 7 Appending image XYZT image can be added after the image acquisition. Immediately after acquisition of an image in the XYZT observation mode, the buttons at the upper part of the [Acquire] panel changes as shown below. <Append Next>...
Page 145: Differences In Image Acquisition Method Between Fluorescent And Transmitted
APPLIED OPERATIONS /Image Acquisition 2-2-3 Differences in Image Acquisition Method Between Fluorescent and Transmitted Images 2-2-3-1 Monochrome Image The wavelength obtained by monochrome dyeing can be acquired and observed as an image of a channel (either Ch1, Ch2, Ch3 or Ch4). The subsequent description deals with the differences in operation between the monochrome dyeing and dual-fluorochrome dyeing.
Page 146 APPLIED OPERATIONS /Image Acquisition One Point! The [Assign dyes manually] check box can also be used to set the dyeing method to the desired channel. Check the [Assign dyes manually] check box in the [Dyes] sub-panel. Select the dyeing method in the [Available Dyes] list box and drag it directly to the field of the [Ch] check box.
Page 147 APPLIED OPERATIONS /Image Acquisition With types of dyeing of the specimen to observe, the barrier filter will be set automatically to the optical path. To change the barrier filter types, see section 1-3-2-3, “Configuring the Filters (When using a filter system)” and change by [Optical System Configuration] window. Make the channel ready for image acquisition.
Page 148: Dual-Fluorochrome Image
APPLIED OPERATIONS /Image Acquisition 2-2-3-2 Dual-Fluorochrome Image The wavelength obtained by dual-fluorochrome dyeing can be acquired and observed as images of the channels (Ch1 or Ch2). The subsequent description deals with the differences in operation between the monochrome dyeing and dual-fluorochrome dyeing. Set the two or more dyeing methods.
Page 149 APPLIED OPERATIONS /Image Acquisition One Point! The [Assign dyes manually] check box can also be used to set the dyeing method to the desired channel. Check the [Assign dyes manually] check box in the [Dyes] sub-panel. Select the dyeing method in the [Available Dyes] list box and drag it directly to the field of the [Ch] check box.
Page 150 APPLIED OPERATIONS /Image Acquisition With types of dyeing of the specimen to observe, the Barrier Filters will be set automatically to the optical path. To change the Barrier Filter types, see to section 1-3-2-6, “Configuring the Filters” and change by [Optical System Configuration] window. Make the channels ready for image acquisition.
Page 151: Transmitted Image
APPLIED OPERATIONS /Image Acquisition 2-2-3-3 Transmitted Image Images obtained by transmitted light observation can also be acquired or observed simultaneously with images obtained by fluorescence observation. When observing fluorescence images simultaneously, set the dyeing method. From the page tabs on the bottom right of the [Acquire] panel, select the [Dyes] sub-panel.
Page 152 APPLIED OPERATIONS /Image Acquisition One Point! The [Assign dyes manually] check box can also be used to set the dyeing method to the desired channel. Check the [Assign dyes manually] check box in the [Dyes] sub-panel. Select the dyeing method in the [Available Dyes] list box and drag it directly to the field of the [Ch] check box.
Page 153 APPLIED OPERATIONS /Image Acquisition With types of dyeing of the specimen to observe, the Barrier Filters will be set automatically to the optical path. To change the Barrier Filter types, see section 1-3-2-6,”Configuring the Filters “ and change by [Optical system Configuration] window. Turn off the transmitted light bulb of the microscope.
Page 154 APPLIED OPERATIONS /Image Acquisition When observing a fluorescence image simultaneously, set the required channel ready for acquisition of fluorescence image. Make sure that the check box showing the dyeing method in the [Ch1]/ [Ch2]/[Ch3]/[Ch4] group box is check-marked to indicate that the corresponding channel is ready for image acquisition.
Page 155: Image Acquisition By Rotating It (Rotation Scan)
APPLIED OPERATIONS /Image Acquisition 2-2-4 Image Acquisition by Rotating It (Rotation Scan) Rotation scan enables image acquisition with tilting the field of viewed. NOTE Rotation Scan can not be used with Free Line Scan. Acquire an image in the XY observation mode. For the operating procedure, see section 2-2-1, “Image Acquisition in XY Observation Mode”.
Page 156: Image Acquisition Of Only The Rectangular Position (Clip Scan)
APPLIED OPERATIONS /Image Acquisition 2-2-5 Image Acquisition of Only the Rectangular Position (Clip Scan) The Clip Scan mode limits the image acquisition range to the range to be observed and acquires the image of only that range. Applying this mode in an image acquisition mode involving large amount of data, such as the XYZ, XYT and XYZT observation modes, makes it possible to acquire data of a long period of time in a small file size.
Page 157 APPLIED OPERATIONS /Image Acquisition Change the frame size. To change the frame size, click a point inside the frame with the mouse pointer. When square handles are displayed on the frame edges, place the mouse pointer on one of them and drag it. Handle Change the inclination angle of the frame.
Page 158 APPLIED OPERATIONS /Image Acquisition Acquire images by clicking the <XYZ>, <XYT> or <XYZT> button. After acquiring images, select <Normal> from the list displayed below the [Surface XY- Norm] option button in the [Mode] group box to set the scanning range to the original setting.
Page 159: Image Acquisition By Magnifying The Rectangular Position (Zoom-In Scan)2-75
APPLIED OPERATIONS /Image Acquisition 2-2-6 Image Acquisition by Magnifying the Rectangular Position (Zoom-In Scan) The Zoom-In Scan mode limits the scanning area to the range to be observed and acquires the image of that area by magnifying it. When the image is acquired in this mode, the number of pixels in the X direction becomes identical to the image size before start of this mode, and the magnification applied is determined according to the size of the limited range.
Page 160 APPLIED OPERATIONS /Image Acquisition Change the frame size. Place the mouse pointer inside the frame and click to display square handles around the frame. Then place the mouse pointer on one of these handles and drag it to change the frame size. (Here the X:Y ratio of the frame is not changed.) Handle Change the number of pixels in the Y direction.
Page 161 APPLIED OPERATIONS /Image Acquisition When using hardware that is compatible with rotation scan (FV5-IO3), specify the image rotation angle using the rotation arrow buttons on both sides of the circuit in the [Pan] group box in the [Acquire] panel. Dragging the blue handle along the circuit allows the Assuming that the angle at acquisition area to be tilted.
Page 162: High-Speed Image Acquisition
APPLIED OPERATIONS /Image Acquisition 2-2-7 High-Speed Image Acquisition An image can be acquired in 0.25 second. This image acquisition mode is valid under the following condition: • Max. 2 channels • Image size: 512 x 512 pixels • Zoom ratio of 2X or more, with setting in 2X steps Check one or both of the [Ch] check boxes in the [Ch] group box in the [Acquire] panel.
Page 163: Image Acquisition To Prevent Crosstalk Between Fluorescence (Sequential Scan)
APPLIED OPERATIONS /Image Acquisition 2-2-8 Image acquisition to prevent crosstalk between fluorescence (Sequential Scan) An image which is suppressed crosstalk can be acquired sequentially by combination of excitation laser and those image acquisition channel. With this image capturing method, the image of a multiple-dyed specimen can be obtained by sequentially acquiring image slice of each type of fluorescence.
Page 164 APPLIED OPERATIONS /Image Acquisition [Group] (which means each laser) and the < > or < > buttons appear on the lower part of each [CH] group box. Click to increase the acquired group Shows the group to Click to decrease the be acquired.
Page 165 APPLIED OPERATIONS /Image Acquisition <Off> button Select the laser type. Fig. 2-25 [Lasers] Sub-panel Among the laser <Off> buttons on the right of the [Laser Intensity] scales in the [Lasers] group box, click the <Off> button of the laser you want to use in transmitted observation. This should change the <Off>...
Page 166: Virtual Channel Function
APPLIED OPERATIONS /Image Acquisition 2-2-8-1 Virtual Channel Function The virtual channels refer to the simulated detection channels produced when a single detector switched in sequential scan. The virtual channels make it possible to perform sequential scan by switching the lasers and filters according to the preset condition by increasing the number of detection channels.
Page 167 APPLIED OPERATIONS /Image Acquisition In the [Mode] group box in the [Acquire] panel, click the [Surface XY-Norm] option button, select <Sequen> from the list below it and select the scan mode to be used from the displayed icons. NOTE Do not select <Line Aq> in the [Mode] group box in the [Acquire] panel because the virtual channels are not compatible with Line Sequential Scan.
Page 168 APPLIED OPERATIONS /Image Acquisition Right-click the mouse on the [Ch] group box on the upper part of the [Acquire] panel. When a pop-up menu appears, select the channel to be added. Select the channel to which you want to add a virtual channel.
Page 169 APPLIED OPERATIONS /Image Acquisition Select <SU Control> button of the [Settings] sub-panel in the [Acquire] panel. In the [Optical System Configuration] window, select the conditions such as laser and filter settings of each group. For details on the [Optical System Configuration] window, see section 1-3-2-6, “Setting the Filters”.
Page 170 APPLIED OPERATIONS /Image Acquisition Setting will disappear if <Normal> is selected at [Mode] group box of NOTE [Acquire] panel after setting virtual channel. It is useful that the setting is saved with use of <Save> button at [Acquisition Settings] group box on [Settings] sub panel of [Acquire] panel before selecting <Normal>.
Page 171 APPLIED OPERATIONS /Image Acquisition Click the <XY Repeat> or <Focus> button to perform repeated scanning in order to adjust the brightness of the channels. <XY Repeat> button Select a group before repeated scanning. The group selection cannot be <Focus> button changed during repeated scanning.
Page 172: Image Acquisition Of A Line At Desired Angle
APPLIED OPERATIONS /Image Acquisition 2-2-9 Image Acquisition of a Line at Desired Angle When the sample is not standing upright, the image of a line can be acquired by changing the angle. Acquire an image in the XY observation mode. For the operation procedure, see section 2-2-1, “Image Acquisition with XY Observation”.
Page 173: Display The Change Of Image Intensity (Point Scan)
APPLIED OPERATIONS /Image Acquisition 2-2-10 Display the change of image intensity (Point Scan) The change of fluorescence intensity according to time-lapse can be displayed graphically by irradiating the laser to certain point on the image. If you have FLUOVIEW TIME COURSE software, image acquisition can be started with buttons, keys or input of external trigger signals.
Page 174 APPLIED OPERATIONS /Image Acquisition The graph window showing the cross cursor for scanning and the intensity values appears in the [Live] panel. Cross cursor Graph window Move the cross cursor to the area you want to observe. To move the cross cursor, place the mouse pointer on it and drag the mouse.
Page 175 APPLIED OPERATIONS /Image Acquisition The speed recommended for point scan area as follows. 2 µ s(The scroll bar indicates “Fast” position. It is useful for the specimen changes rapidly.), 10 µ s, 100 µ s(The scroll bar indicates “Slow” position. It is useful for the specimen changes slowly.) Set the time for measurement.
Page 176 APPLIED OPERATIONS /Image Acquisition The image shown in the [Live] panel after image acquisition is the image that acquired at the coordinates specified in the [Live] panel and arranged in the X direction, from the top left to the bottom right. The image shown after image acquisition is the same a width as that in the X direction specified in the [Live] panel.
Page 177 APPLIED OPERATIONS /Image Acquisition When click the mouse on the X or Y Axis on the graph window, the [Editing] dialog box appears and the graph parameters or the graph display method can be modified. After image acquisition, select <Normal> under the [Surface XY] option button in the [Mode] group box in the [Acquire] panel to return from the image acquisition mode.
Page 178: Image Acquisition On Desired Line (Xz, Xt Or Xzt Observation)
APPLIED OPERATIONS /Image Acquisition 2-2-11 Image Acquisition on Desired Line (XZ, XT or XZT Observation) When the sample is in curved form, the image can be acquired by drawing a desired line on Acquire an image in the XY observation mode. For detailed operation, see section 2-2- 1, “Image Acquisition with XY observation”.
Page 179 APPLIED OPERATIONS /Image Acquisition Change the curved line shape. Place the mouse pointer within the box and right click to display the pop-up menu. Then select <Edit>. The cross shaped handles appear around the curve. Place the mouse pointer on either handle and drag it to change the line shape.
Page 180 APPLIED OPERATIONS /Image Acquisition When <Free> is selected at [Mode] group box on [Acquire] panel, the NOTE sampling speed cannot be changed. Set the interval time or the number of scans. The operating procedure is same as that in the XY observation mode. See section 2- 2-2, “XT Observation Mode”...
Page 181: Image Acquisition In The Laser Excitation Mode
APPLIED OPERATIONS /Image Acquisition 2-2-12 Image Acquisition in the Laser Excitation Mode When you use the FV system with AOTF (FV5-COMBA), the function described in this section is available. In the FV system with AOTF, it is possible to cut excitation of laser except the region where scanning is performed.
Page 182: Making Rex Mask File
APPLIED OPERATIONS /Image Acquisition NOTE In the REX and Bleach mode, image acquisition in the Focus mode, fast scan, and image acquisition in the Line mode (Normal, Slant, and Free) can not be performed. NOTE The specimen in the images carried in this section is not for FRAP, but for the user’s manual.
Page 183 APPLIED OPERATIONS /Image Acquisition In the list of buttons displayed as shown below, select the <Rectangular>, <Circle>, <Polyregion>, or <Free region> button. And drag on the image to specify the region of laser excitation. <Rectangular> button <Circle> button <Polyregion> button <Free Region>...
Page 184 APPLIED OPERATIONS /Image Acquisition From the page tabs on the bottom right of the [Acquire] panel, select the [Lasers] sub- panel. <Make REX mask> button Makes the REX mask file of the specified region. Fig. 2-26 [Lasers] sub-panel When the <Make REX mask> button is displayed in gray, click and select the specified region on the [Live] panel.
Page 185 APPLIED OPERATIONS /Image Acquisition Select the <Make REX mask> button. The [REX Live] panel (the REX mask file) is made in the image window. <Make REX mask> button Make the REX mask file which masks the region outside of the specified region.
Page 186 APPLIED OPERATIONS /Image Acquisition In order to adjust the laser intensity for every region, right-click the mouse on the white region on the image window where the REX mask file is displayed. The pop-up menu as shown below appears. Select [Set intensity] in the menu. The [Laser Intensity] dialog box as shown below appears.
Page 187: Example Of Frap Experiment
APPLIED OPERATIONS /Image Acquisition 2-2-12-2 Example of FRAP experiment The example of procedure of FRAP experiment using AOTF is described in this section. In order to perform photobleaching to a specimen, the value of laser intensity is raised to 100% to acquire an image since strong laser intensity is temporarily required. When 100% or more of laser intensity value is required, zoom magnification is gathered to acquire an image.
Page 188 APPLIED OPERATIONS /Image Acquisition Select the <Once> button in the [Acquire] panel and acquire an image. If necessary, set the range for image acquisition with clip scan, for example, after acquiring an image. 3 Making REX Mask File See section 2-2-12-1, “Making REX Mask File” for the procedure to make the REX mask file. If the setup of the region of laser excitation remains after making the REX mask file, the intensity change of the region can be observed in the TIME COURSE software (optional).
Page 189 APPLIED OPERATIONS /Image Acquisition 4 Selecting REX Mask File and Setting the Laser ON/OFF Set the REX mask file in the REX mode and Bleach mode, and the laser ON/OFF respectively. Select the [REX mode] option button in the [AOTF] group box in the [Lasers] sub-panel. [REX mode] option button A frame to specify the REX mask file appears on the right side of the [REX mode]...
Page 190 APPLIED OPERATIONS /Image Acquisition Select the [Bleach mode] option button in the [AOTF] group box of the [Laser] sub- panel. A frame to specify the REX mask file appears on the right side of the [Bleach mode] option button. Right-click the mouse inside the frame to display the pop-up menu as shown below.
Page 191 APPLIED OPERATIONS /Image Acquisition 5 Setting the XYT Observation mode Confirm that [XY-Norm] is displayed in the [Mode] group box in the [Acquire] panel. Select the [XYT] option button in the [Acquire] panel. OPERATION INSTRUCTIONS Page 2 - 1 0 7...
Page 192 APPLIED OPERATIONS /Image Acquisition From the page tabs on the bottom right of the [Acquire] panel, select the [Time Series] sub-panel. The panel as shown below appears. [Interval] text box Set the interval with the < > and< > buttons. Key entry is also acceptable. [N] text box Set the number of scans with the <...
Page 193 APPLIED OPERATIONS /Image Acquisition 7 Acquiring Data The experiment data before and after photobleaching of certain region can be obtained after performing an ordinary image acquisition in the first scan, an image acquisition in the Bleach mode in the second scan , and an ordinary image acquisition in the third scan. When the REX mode is selected in the third scan, the data of the region other than the specified region can not be obtained since the laser is irradiated only to the specified region.
Page 194 APPLIED OPERATIONS /Image Acquisition Start the second scan in 10 seconds after the first scan is performed. The laser of strong intensity is irradiated specified region. Select the [Disabled] or [REX mode] option button in the [AOTF] group box in the [Lasers] sub-panel.
Page 195 APPLIED OPERATIONS /Image Acquisition Start the third scan in 10 seconds after the second scan is performed. The image of the specimen is fading only where the laser is irradiated. (The [Disabled] option button is selected.) Start the forth and subsequent scan in 10 seconds after the third scan is performed. The image of the photobleached specimen is acquired.
Page 196: Notes For Image Acquisition
APPLIED OPERATIONS /Image Acquisition 2-2-13 Notes for image acquisition 2-2-13-1 Memory of setting information for scanning region Frame with handle described in 2-2-5 Clip Scan, Frame with hand described in 2-2-6 Zoom- in Scan, Straight line with handle described in 2-2-9, Cross line cursor described in 2-2-10 Point Scan are all called Scanning Region Setting - ROI (Region Of Interest).
Page 197: Saving, Opening And Shredding Images
APPLIED OPERATIONS/ Saving, Opening and shredding Images 2-3 Saving, Opening and Shredding Images Use the [File I/O] panel to save, open or shred an image. Display the [File I/O] panel at the front. NOTE The file name has to be within 120 characters including the path for the file.
Page 198 APPLIED OPERATIONS /Saving, Opening and Shredding Images <Image Icons> Images are represented by icons which can also Image Icon Significance identify the observation modes used when acquiring XZ observation them. XZ observation, 2-channel mode The icon of the selected image (image in the image window) is displayed in the frame at the top of the Xt observation function panel such as the [File I/O] panel.
Page 199 APPLIED OPERATIONS /Saving, Opening and Shredding Images <[Files] List Box> The information displayed in the list box can be changed. <Details> button To display all information, click the <Details> button above the [Files] list box to broaden it. To return to the display of icons and file names only, click the <List> button. <List>...
Page 200: Saving Images
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-1 Saving Images Either a series of images or single image being displayed can be saved. 2-3-1-1 Saving Images As a Series The displayed images can be saved in a disk as a series image file. Display the image window showing the image to be saved at the front.
Page 201 APPLIED OPERATIONS /Saving, Opening and Shredding Images When it is required to change the save destination drive or directory, use the [Save in:] drop-down list. When it is required to change the saved file type, use the [Save as Type:] drop-down list.
Page 202: Saving A Display
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-1-2 Saving a Display The displayed images can be hardcopied and saved in a disk. This method is used when it is required to use a FLUOVIEW image in another application. Display the image window showing the image to be saved at the front. When there is more than one image to be saved, display the image to be saved using the <Display>...
Page 203 APPLIED OPERATIONS /Saving, Opening and Shredding Images When it is required to change the saved file type, use the [Save as Type:] drop-down list. See section 2-3-1-4, “File Types Available for Save” for details. Enter the file name in the [File Name:] text box. Click the <Save>...
Page 204: Saving Specified Area Of Image
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-1-3 Saving Specified Area of Image Only the specified part of an image can be saved as an image in a disk. Display the image window of the image containing the part to be saved in the front. When the image was acquired through multiple channels, you can select whether the image slices for multiple channels are saved together or the image slice of only 1 channel is saved.
Page 205 APPLIED OPERATIONS /Saving, Opening and Shredding Images Specify the area to be saved in the image in the image window. The area is displayed on the image with the handles on its frame. The area becomes the save target while these handles are displayed.
Page 206 APPLIED OPERATIONS /Saving, Opening and Shredding Images 12. A dialog box as shown below appears. Size of the specified area Fig. 2-32 [Saving a subset of Experiment’s Data] Dialog Box The [Saving a subset of Experiment’s Data] dialog box does not appear if no area is specified in the image on the image window.
Page 207: Saving Animation Images
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-1-4 Saving Animation Images Transform a created animation image into the AVI file format and save it in the file type which can display the animation image without this software. Create an animation image. Refer to “2-9-2 Animation”...
Page 208 APPLIED OPERATIONS /Saving, Opening and Shredding Images Click the <Animation> button in the [Save] group box. The [Save Experiment As] dialog box appears as shown below. <Save Animation>button Fig. 2-33 [Save Experiment As] Dialog Box When it is required to change the save destination drive or directory, use the [Save in : ] drop-down list.
Page 209: File Types Available For Save
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-1-5 File Types Available for Save The file type used for saving image in a file can be selected by the user. See section 2-3-1- 1, “Saving Images As a Series” for the operation method. Click the <Experiment>...
Page 210 APPLIED OPERATIONS /Saving, Opening and Shredding Images NOTE If you want to save the image with the comment drawn on it as an image, select the file type according to the usage of the image as described below. Fluoview Multi Tiff:Select when the image will be analyzed or processed after save.
Page 211 APPLIED OPERATIONS /Saving, Opening and Shredding Images Saving a channel under Saving merged images Saving extended image Comment Saving side-by-side displayed condition (Example: When Ch1, Ch2 drawn on Z/T series switch button (Example: When only Ch3 and Ch3 are displayed) images Over and Under images...
Page 212 APPLIED OPERATIONS /Saving, Opening and Shredding Images Saving a channel under Saving merged images Saving extended image Comment Saving side-by-side displayed condition (Example: When Ch1, Ch2 drawn on Z/T series switch button (Example: When only Ch3 and Ch3 are displayed) images Over and Under images...
Page 213 APPLIED OPERATIONS /Saving, Opening and Shredding Images Saving a channel under Saving merged images Saving extended image Comment Saving side-by-side displayed condition (Example: When Ch1, Ch2 drawn on Z/T series switch button (Example: When only Ch3 and Ch3 are displayed) images Over and Under images...
Page 214 APPLIED OPERATIONS /Saving, Opening and Shredding Images Saving a channel under Saving merged images Saving extended image Comment Saving side-by-side displayed condition (Example: When Ch1, Ch2 drawn on Z/T series switch button (Example: When only Ch3 and Ch3 are displayed) images Over and Under images...
Page 215 APPLIED OPERATIONS /Saving, Opening and Shredding Images Saving a channel under Saving merged images Saving extended image Comment Saving side-by-side displayed condition (Example: When Ch1, Ch2 drawn on Z/T series switch button (Example: When only Ch3 and Ch3 are displayed) images Over and Under images...
Page 216: Opening Previously Saved Images
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-2 Opening Previously Saved Images Image files saved in the disk can be opened as follows. If the image file name that you want to open is not displayed in the [Files] list box, change the drive and/or directory to those containing the desired file using the [Drive] drop-down list and/or [Directory] list box.
Page 217: Shredding Images
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-3 Shredding Images Shredding an image refers to removing it from the objects of processing including display. Shredding does not actually deletes the image saved in the disk. Click the <Experiment List> button in the toolbar at the bottom of the [File I/O] panel. The [Experiments in Memory] dialog box appears as shown below.
Page 218 APPLIED OPERATIONS /Saving, Opening and Shredding Images Click the <Done> button in the [Experiments in Memory] dialog box to close it. Multiple images displayed can be shredded simultaneously. Display the [Experiments in Memory] dialog box while showing multiple images in the image window. Pressing down the Shift key, select multiple image files to be...
Page 219: Saving Comment Together With Image
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-4 Saving Comment Together with Image Click the <Experiment List> button in the toolbar at the bottom of the [File I/O] panel. The [Experiments in Memory] dialog box appears as shown below. <Experiment List> button Fig.
Page 220 APPLIED OPERATIONS /Saving, Opening and Shredding Images In the [Experiments in Memory] dialog box, select the file name of the image to be saved with comment and click the <Comments> button. The [Image Comments] dialog box appears as shown below. Fig.
Page 221 APPLIED OPERATIONS /Saving, Opening and Shredding Images Click the <OK> button. Click the <Done> button in the [Experiments in Memory] dialog box to close it. Display the [File I/O] panel at the front. Click the <Experiment> button in the [Save] group box. See section 2-3-1, “Saving Images”...
Page 222: Checking The Image Information/Acquisition Parameters
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-5 Checking the Image Information/Acquisition Parameters Click the <Experiment List> button in the toolbar at the bottom of the [File I/O] panel. The [Experiments in Memory] dialog box appears as shown below. <Experiment List> button Fig.
Page 223 APPLIED OPERATIONS /Saving, Opening and Shredding Images Display the [Image Attributes] panel at the front. The panel as shown below appears, where the information on the selected image is shown. [CH] Shows the number of image Displays number of Z [X]/[Y] acquisition channels.
Page 224 APPLIED OPERATIONS /Saving, Opening and Shredding Images NOTE The objective setting can be changed even after having acquired the image. If the objective is not set with the [Acquire] panel before image acquisition, the analysis and measurement results of the image may be erroneous.
Page 225 APPLIED OPERATIONS /Saving, Opening and Shredding Images Select the information items to be checked from the list box on the right. The kind of information displayed when each item is selected is shown below. After checking the information, click the <OK> button. Click the <Done>...
Page 226: Saving The Image Information/Observation Condition
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-6 Saving the Image Information/Observation Condition The image information and observation condition can be saved as ASCII text file in a disk. Display the image window of the image to be saved at the front position. Select the <Experiment List>...
Page 227 APPLIED OPERATIONS /Saving, Opening and Shredding Images In the list in the [Experiments in Memory] dialog box, select the file name including the image whose image information and observation condition are to be saved. And click the <Comments> button. The [Image Comments] dialog box as shown below appears. <Properties>...
Page 228 APPLIED OPERATIONS /Saving, Opening and Shredding Images Select the <Save> button. The [Save As ASCII Text] dialog box as shown below appears. Fig. 2-43 [Save As ASCII Text] dialog box In order to change the save destination drive or directory, use the [Save in: ] drop-down list.
Page 229: Saving/Reading The Region File
APPLIED OPERATIONS /Saving, Opening and Shredding Images 2-3-7 Saving/Reading the Region File The information on the shape, position, and color of certain region can be saved in a file and they can also be read. 2-3-7-1 Saving the Region File Display the image window of the image including the region to be saved at the front position.
Page 230 APPLIED OPERATIONS /Saving, Opening and Shredding Images Select the image file name including the region to be saved in the [Experiments in Memory] dialog box and click the <Comments> button. The [Image Comments] dialog box as shown below appears. <Annotations/ROIs> button in the [Export to File] group box Saves the region into a file.
Page 231: Reading The Region File
APPLIED OPERATIONS /Saving, Opening and Shredding Images Select “FLUOVIEW ROI (*.roi)” in the [Files of type] drop-down list. Enter the file name into the [File name] text box and click the <Save> button. NOTE When the text file having the same name as the entered name is already exists, the dialog box appears to ask you to overwrite the existing file or not.
Page 232 APPLIED OPERATIONS /Saving, Opening and Shredding Images In the list in the [Experiments in Memory] dialog box, select the image file name including the region to be read and click the <Comments> button. The [Image Comments] dialog box as shown below appears. <Annotations/ROIs>...
Page 233 APPLIED OPERATIONS /Saving, Opening and Shredding Images Select the drive or folder where the file to be read is saved in the [Look in] drop-down list. In the list below the [Look in] drop-down list, double-click the folder or sub-folder where the data to be read is saved to open.
Page 234: Protocol Processor
APPLIED OPERATIONS / Protocol processor 2-4 Protocol processor The Protocol Processor makes FLUOVIEW image acquisition flexible, especially time series image acquisition. The function enables to create and modify the experiment procedure such as image acquisition condition with interval setting, laser excitation area and its power setting. The protocol can save and modify to be suitable to new experiment.
Page 235: Starting The Protocol Processor
APPLIED OPERATIONS /Protocol processor 2-4-1 Starting the Protocol Processor Starts the Protocol Processor by clicking <PAPP> button in [Time Series] sub-panel of [Acquire] panel. <PAPP> button Starts the Protocol Processor. The [Programmable Acquisition Protocol Processor] window appears as shown below. (It is referred to as the [PAPP] window in this manual.) [PAPP] window appears where is the last time in use.
Page 236: Editing The Protocol
APPLIED OPERATIONS / Protocol processor : <Get FV Status> button: Acquires each setting value of FLUOVIEW software parameters and registers to PAPP window. The track which the parameters to be registered has to be highlighted. : <Set to FV> button: Sets PAPP parameters to FLUOVIEW software parameters.. The track which the parameters to be registered has to be highlighted.
Page 237: Description Of Setting Items
APPLIED OPERATIONS /Protocol processor 2-4-2-1 Description of Setting Items The [PAPP] window has the setting items as described in the following. <Get Init Values> button <Path> button Click to acquire the current [Image File Name] text box Specify the folder for settings in the [Acquire] Enter the file name in which the image saving...
Page 238 APPLIED OPERATIONS / Protocol processor Only available Laser type and PMT channel column is used with [Laser Intensity] and [PMT Voltage]. The values in each cell are applied to the [Acquire] panel at the beginning of the protocol. When “Init” is specified in a cell, the value set in the [Init Val] box is applied. The blank cell applies the value set to the track immediately above.
Page 239 APPLIED OPERATIONS /Protocol processor Normal : Starts image acquisition when protocol comes the Track. Trigger : Starts image acquisition after receiving external trigger input. Wait : Starts image acquisition after wait time describes the track, the unit is second. Condition: See the following table in case that [New mode] or [Series mode] is selected in [Mode].
Page 240 APPLIED OPERATIONS / Protocol processor Pan Status: Enter Pan value, refer to [Pan] [Zoom]group box in [Acquire] panel. X Pan, Y Pan Zoom Angle FLUOVIEW software [Pan][Zoom] Group Box Pan value can not enter the following condition. · Mode is set to any of XY, XYT, XYZ or XYZT. ·...
Page 241 APPLIED OPERATIONS /Protocol processor Sub Mode: Sub Mode is for scan mode setting of New mode appears by click on Mode area. A certain scan mode may not set because of hardware configuration. See 2-4-10 “Restrictions at [PAPP] window input time” for restriction of scan mode setting.
Page 242 APPLIED OPERATIONS / Protocol processor Scan speed: Sets image acquisition speed from “Fast”, “Middle”, “Slow” and “Init” in the pull-down menu. The above speed is the same with those of [Scan Speed] group box in [Acquire] panel. In case that [Fast] or [Fast Clip] is selected in [Sub mode], [Scan Speed] is automatically set to [High Speed].
Page 243 APPLIED OPERATIONS /Protocol processor One Point! It is possible to designate folder in [Save File Name] column. For example, if [R001 Image001] is entered, folder – [R001] is created under path of [Image File Name:] and image file is saved under the name of [Image001]. Trigger Out: Select the trigger output from “00”, “01”, “10”...
Page 244: Command List
APPLIED OPERATIONS / Protocol processor 2-4-2-2 Command List The list of macro commands is shown below. When “Command” is selected in column [Mode] in the [PAPP] window, enter the macro command to be carried out for the track. The macro command to be entered can be selected from the drop-down list.
Page 245 APPLIED OPERATIONS /Protocol processor Command Entry form Description designated is to be Valid, select ON and, in case that Invalid is to be set, select OFF. <E.g> In case that channel 1 is to be Valid, enter [EnCh1, ON]. Filter [NORMAL Filter [NORMAL/KALMAN/PEAK]: It defines type of /KALMAN/PEAK]...
Page 246 APPLIED OPERATIONS / Protocol processor Command Entry form Description protocol. Offset ch value Offset Sets the Offset value of channel. <E.g.> Enter “Offset 2, 5” to set 5 to the offset value of channel 2. PasteROI PasteROI Pastes ROI to the next track. Note: Before the paste, Normal scan is executed one time.
Page 247 APPLIED OPERATIONS /Protocol processor Command Entry form Description combination. Be careful that other combination than the following cannot be entered. (256, 256) (320, 240) (512, 512) (640, 480) (800, 600) (1024, 768) (1024, 1024) (2048, 2048). Stop&Save Stop&Save Saves all the images acquired before the Stop&Save command is executed.
Page 248 APPLIED OPERATIONS / Protocol processor NOTE The images acquired in different sizes cannot be saved together. Execute the Stop&Save command to save the image acquired before changing the image size. One Point! Multiple image files saved using the Stop&Save command can be combined into a single time-lapse image file.
Page 249: Protocol Repetition Processing
APPLIED OPERATIONS /Protocol processor 2-4-2-3 Protocol Repetition Processing A protocol can be executed repeatedly for the specified number of times using a variable. Use the For and Next commands to execute repetition. The track enclosed between For and Next can be repeated for the number of times specified using a variable.
Page 250 APPLIED OPERATIONS / Protocol processor One Point! It is possible to use variables in [For] statement in other cell of protocol processor. <E.g> It is possible to change laser intensity value every image acquiring time. When laser intensity of argon laser is defined as variable N, it creates protocols that acquire image with interval of step 10 by N equal to 10 to 100.
Page 251: Input Supporting Function
APPLIED OPERATIONS /Protocol processor 2-4-2-4 Input supporting function [Track] window is available as input supporting function for each track. [Track] window appears by double-clicking No of [PAPP] window. • All of parameters that can be set for the Track can modify. •...
Page 252 APPLIED OPERATIONS / Protocol processor One Point! It is possible to select [Set Init Val] and [Get FV Status] from [Edit] menu. When [Select All] is selected from [Edit] menu, checks will enter all check boxes of group boxes. When [Invert Selection] is selected from [Edit] menu, state of checks in all check boxes of group boxes will be inverted.
Page 253: Protocol Procedure Supporting Function
APPLIED OPERATIONS /Protocol processor 2-4-2-5 Protocol procedure supporting function [Protocol Creator] window helps to make procedure, especially time-series, easily.. Track No. Drag & Drop Interval time for each track End time of each track Setting value display text box When [Protocol Creator] that exists in [Tool] menu bar of [PAPP] window is selected, [Protocol Create] window appears.
Page 254: Saving The Protocol
APPLIED OPERATIONS / Protocol processor By pressing <More> button, the detail information is displayed. By pressing <Less> button, close the information display.. NOTE During [Protocol Creator] window is displayed, [PAPP] window can not be operated. 2-4-3 Saving the Protocol The Protocol Processor creates and saves protocol files in the CSV file format. The CSV file format applies the text format delimiting the items with comma in a database.
Page 255 APPLIED OPERATIONS /Protocol processor Select file type, .pap or .csv, with [Save as type:] dropdown list. Click the <Save> button. NOTE If a CSV file having the same file name as the file name entered above already exists, a dialog box appears to ask if you want to overwrite the existing file.
Page 256: Executing The Protocol
APPLIED OPERATIONS / Protocol processor 2-4-4 Executing the Protocol The created protocol can be executed to acquire time series images. In the [PAPP] window, click the <Start> button. <Start> button The number of rows in the [No.] list in the [PAPP] window becomes 2, and the window display is reduced.
Page 257 APPLIED OPERATIONS /Protocol processor To stop the protocol without executing it till the end, click the <End> button. <End> button When clicked while the protocol is executed, the <End> button performs the same function as the <Series Done> button in the [Acquire] panel. One Point! The protocol can be processed using the items under the [Run] menu in the [PAPP] window.
Page 258: Loading A Protocol
APPLIED OPERATIONS / Protocol processor 2-4-5 Loading a Protocol The Protocol Processor creates and saves protocol files in the CSV file format. The following procedure loads a CSV file (extension .pap or .csv) for editing the protocol in it. Start the Protocol Processor software and display the [PAPP] window. For details, see section 2-4-1, “Starting the Protocol Processor”.
Page 259: Loading A Protocol Of Previous Format
APPLIED OPERATIONS /Protocol processor 2-4-6 Loading a protocol of previous format FLUOVIEW software Ver4.2 inform the protocol data conversion when the software reads previous version of the data. The rule of the conversion is explained below. Rule A • Before Ver4.2, Frame = XYT or XYZT is converted to XYT or XYZT in count of Z slice parameter.
Page 260 APPLIED OPERATIONS / Protocol processor Fig. 2-51 Multi-Point Time Lapse Protocol Rule C • For and Command in Series appears with indented + ( ). • Combined For – Next structure becomes single layer. Fig. 2-52 Protocol Using [For] Statement OPERATION INSTRUCTIONS Page 2 - 1 7 6...
Page 261: Com Communication Function
APPLIED OPERATIONS /Protocol processor 2-4-7 COM Communication function PAPP can communicate with RS-232C to the other PC. The data for sending and receiving should described with Macro commands. For detail of Macro command, see 2-4-2 [2 Table of commands]. Select COM port of PC. •...
Page 262: Example Of Assembling A Protocol
APPLIED OPERATIONS / Protocol processor 2-4-8 Example of assembling a protocol This section describes an example of assembling a protocol. 2-4-8-1 Example of Setting Procedure of FRAP Observation An example of editing a protocol is described below assuming the FRAP observation. Protocol Repeat 30 times Image acquisition:...
Page 263 APPLIED OPERATIONS /Protocol processor Setting the observation condition Before setting and starting the FRAP observation using the Protocol Processor, execute a repeated scan and set the condition for the observation. Assuming FITC to Channel 2 and image size to 320X240 pixels, the following figure shows an example of image acquisition.
Page 264 APPLIED OPERATIONS / Protocol processor Displaying the Real-Time Graph (TIEMPO option software required) Specify the region in the image to display the real-time graph. For specifying method, refer to section 2-4-3, “Specifying the Regions Where Intensity Graph is Displayed in Real Time”. Display the [Tiempo] sub-panel in the [Acquire] panel to check the [Show live plot] check box.
Page 265 APPLIED OPERATIONS /Protocol processor Editing the Protocol Start and edit the protocol after setting of the observation condition has been completed. For starting method, refer to section 2-4-1, “Starting the Protocol Processor”. Fig. 2-55 Panel Displaying the [PAPP] window Set the [PAPP] window as shown below; Fig.
Page 266 APPLIED OPERATIONS / Protocol processor Set each Track as sown below; Setting Track 1 In XYT observation, set the zoom ratio to 2X and acquire an image before bleaching. Select [New mode]-“XYT” in [Mode] of [Track]. Select “Normal” in [Start by] of [Track]. Enter “1”...
Page 267 APPLIED OPERATIONS /Protocol processor Setting Track 3 In XYT observation, return the zoom ratio to 2X and acquire 30 images every 5 seconds. (Observe the change in fluorescence image with time after bleaching.) Select “Series mode” in [Mode] of [Track]. Select “Normal”...
Page 268 APPLIED OPERATIONS / Protocol processor One Point! The protocol to perform the following operations can be assembled after setting the observation conditions. Specifying the region for bleaching in the laser irradiation mode using REX mask files. In Track 4, select a REX mask file in [Mask] of [REX]. Then in the laser irradiation mode specified by the REX mask file selected, images can be acquired.
Page 269 APPLIED OPERATIONS /Protocol processor Saving the Protocol Save the edited protocol data. The protocol can also be saved after being executed. Select [Save] or [Save as] in the [File] menu in the [PAPP] window. Save using the dialog box displayed. For details, see section 2-4-3, “Saving the Protocol“.
Page 270 APPLIED OPERATIONS / Protocol processor Exiting the Protocol Terminate the protocol after acquiring the images. The images saved. Fig. 2-58 Panel When Exiting the Protocol OPERATION INSTRUCTIONS Page 2 - 1 8 6...
Page 271: Example Of Setting Procedure Of Xyt Observation For Hours
APPLIED OPERATIONS /Protocol processor 2-4-8-2 Example of Setting Procedure of XYT Observation for hours (Protocol using repetition processing) An example of editing a protocol using the repetition processing is described below assuming the XYT observation including the GFP recovery observation for hours. Protocol 7 repeats Acquisition of...
Page 272 APPLIED OPERATIONS / Protocol processor One Point! The variable in the [For] statement can also be used by another cell in the protocol processor. <Setting example> It is also possible to change the laser intensity value at every image acquisition. In this case, assume that the laser intensity of the Ar laser is variable N and create a protocol for acquiring image as N varies from 10 to 100 in steps of 10.
Page 273 APPLIED OPERATIONS /Protocol processor Setting the Observation Condition Before setting and starting the XYT observation for hours using the Protocol Processor, execute a repeated scan and set the condition for the observation. Assuming FITC to Channel 2 and image size to 320X240 pixels, the following figure shows an example of image acquisition.
Page 274 APPLIED OPERATIONS / Protocol processor Editing the Protocol Start and edit the protocol after setting of the observation condition has been completed. For starting method, refer to section 2-4-1, “Starting the Protocol Processor”. Fig. 2-60 Panel Displaying the [PAPP] window Set the [PAPP] window as shown below;...
Page 275 APPLIED OPERATIONS /Protocol processor Set each Track as sown below; Setting Track 1 Acquire nine images in XYT observation. Select “New mode”-“XYT” in [Mode] of [Track]. Select “Normal” in [Start by] of [Track]. Enter “9” to [Condition] of [Track]. Select “Middle” in [Scan Speed]. Enter the laser intensity value for image acquisition to [Laser Intensity].
Page 276 APPLIED OPERATIONS / Protocol processor Setting Track 3 Acquire nine images at one-hour interval in each Track in XYT observation. Select “New mode”-“XYT” in [Mode] of [Track]. Enter “Wait 3600” in [Stand by] of [Track]. (Enter the value by the second.) Enter “9”...
Page 277 APPLIED OPERATIONS /Protocol processor One Point! XYZT observation protocol becomes available when “New mode”-“XYZT” is selected instead of selecting “New mode” – XYT on [Mode] of [Track]. Saving the Protocol Save the protocol edited. The protocol can also be saved after being executed. Select [Save] or [Save as] in the [File] menu in the [PAPP] window.
Page 278 APPLIED OPERATIONS / Protocol processor Exiting the Protocol After the initial image has been acquired and saved, “saving the image acquired in Track 4 in Track 5” is repeated for 7 times, after which the protocol terminates. The images saved Fig.
Page 279: Pop-Up Menu
APPLIED OPERATIONS /Protocol processor 2-4-9 Pop-up Menu Right-clicking the mouse in the cells in the [PAPP] window displays the pop-up menu to edit the protocol as sown below; Removes the effect of the previous operation. Repeats the previous operation. Copies the value in the selected cell. Cuts the value in the selected cell.
Page 280: Restrictions Of [Papp] Setting
APPLIED OPERATIONS / Protocol processor 2-4-10 Restrictions of [PAPP] setting 2-4-10-1 Restrictions with [Mode] and [Sub Mode] There are restrictions at Mode and its Sub Mode setting in [PAPP]. The following table shows the restriction. Scan Status Row Mode Sub Mode X Pos Y Pos X Size...
Page 281 APPLIED OPERATIONS /Protocol processor Scan Status Row Mode Sub Mode X Pos Y Pos X Size Y Size XYZT Normal Clip ZoomIn Seq_Normal Seq_Clip Seq_ZoomIn LineSeq_Normal LineSeq_Clip LineSeq_ZoomIn Normal Clip ZoomIn FreeLine Fast FastClip LineSeq_Normal LineSeq_Clip LineSeq_ZoomIn Normal Clip ZoomIn FreeLine LineSeq_Normal LineSeq_Clip...
Page 282: Changing The Image Display Method
APPLIED OPERATIONS /Changing the Image Display Method 2-5 Changing the Image Display Method The method of displaying an image acquired by observation or opened from a file can be changed as described below. 2-5-1 Displaying an Image in Simulated Colors Display the image window of the image to be colored at the front.
Page 283 APPLIED OPERATIONS /Changing the Image Display Method One Point! The [Color Tool] dialog box can also be displayed by a mouse operation. Display the image to be colored at the front of the image window, and right-click a point in the image. A pop-up menu as shown below is displayed.
Page 284: Editing The Lut (Look Up Table)
APPLIED OPERATIONS /Changing the Image Display Method 2-5-2 Editing the LUT (Look Up Table) An original LUT can be created by editing an existing LUT. 2-5-2-1 LUT Graph Editing According to Colors Display the image window of the image that you want to edit the LUT. Click the <LUT>...
Page 285 APPLIED OPERATIONS /Changing the Image Display Method Click the <Graph display> button at the top right of the [Color Tool] dialog box again. Finally, click the <OK> button to exit from the LUT editing. One Point! The [Color Tool] dialog box can also be displayed by a mouse operation. Display the image to be colored at the front of the image window, and right-click a point in the image.
Page 286: Lut Graph Editing By Gamma Correction
APPLIED OPERATIONS /Changing the Image Display Method 2-5-2-2 LUT Graph Editing by Gamma Correction The intensity data of an image can be reallocated to make it easier to view. Display the image window of the image to be subjected to LUT change. <LUT>...
Page 287: Switching The Displayed Channels (Ch1 - Ch5)
APPLIED OPERATIONS /Changing the Image Display Method If it is required to save the LUT in a file, click the <Save LUT> button in the [Color LUT Tool] group box. To load a previously saved LUT, click the <Load LUT> button. 2-5-3 Switching the Displayed Channels (Ch1 –...
Page 288: Displaying Images Of Multiple Channels Simultaneously
APPLIED OPERATIONS /Changing the Image Display Method Display the image window of for the image obtained from multiple channels at the front. Click the image to display the <Display channel switch> buttons on the bottom left of the image. <Display channel switch> Select the channels which should be displayed by pushing <Display channel switch>...
Page 289: Displaying Images Separately Per Channel
APPLIED OPERATIONS /Changing the Image Display Method 2-5-4-1 Displaying Images Separately Per Channel (Side By Side Views, Over And Under Views) Display the image window of one of the images to be displayed side by side at the front. Click the <Display switching> button at the top of the image window. The list of buttons as shown below appears.
Page 290: Displaying Merged Image Of Multiple Channels (Single View)
APPLIED OPERATIONS /Changing the Image Display Method 2-5-4-2 Displaying Merged Image of Multiple Channels (Single View) Display the image window of the merged image of multiple channels at the front. Click the <Display switching> button at the top of the image window. The list of buttons <Display switching>...
Page 291: Changing The Number Of Divided Images
APPLIED OPERATIONS /Changing the Image Display Method 2-5-5 Changing the Number of Divided Images The number of images viewed simultaneously can be changed. Increased image is only to be displayed. The image increased in Add View is not subjected to these operations described below. 2-5-5-1 Increasing the Number of Divided Images Display the image window of the image to be changed at the front.
Page 292 APPLIED OPERATIONS /Changing the Image Display Method Select [Views], then select [Add a view] in the sub-menu. A view is added to the rightmost position on the image window. Fig. 2-71 Image window After View Addition Up to 6 views can be displayed at once. OPERATION INSTRUCTIONS Page 2 - 2 0 8...
Page 293: Decreasing The Number Of Divided Images
APPLIED OPERATIONS /Changing the Image Display Method 2-5-5-2 Decreasing the Number of Divided Images Display the image window of the image to be changed at the front. Fig. 2-72 Image window Right-click the image. A pop-up menu as shown below appears. Select [Views], then select [Remove a last view] in the sub-menu.
Page 294: Switching The Display Method Of Multiple Images
APPLIED OPERATIONS /Changing the Image Display Method A view is removed from the image window. Fig 2-73 Image window after View Removal 2-5-6 Switching the Display Method of Multiple Images With images composed of multiple slices, such as time-lapse images or images acquired by changing the multiple sections, the image to be displayed at the front position can be switched or the images can be displayed successively.
Page 295 APPLIED OPERATIONS /Changing the Image Display Method To switch the image to the image of another multiple sections, click the <Z/T series switch> button then, from the displayed list of buttons, click the <XYZ series> button. To switch the image to the image of another moment in the elapsed time, click the <Z/T series switch>...
Page 296: Displaying Multiple Image Slices Together
APPLIED OPERATIONS /Changing the Image Display Method 2-5-7 Displaying Multiple Image Slices Together With images composed of multiple slices, such as time-lapse images or images acquired by changing the multiple sections, the image slices can be displayed together for simultaneous viewing.
Page 297: Displaying Multiple Images Per Channel
APPLIED OPERATIONS /Changing the Image Display Method One Point! [Tile] panel moved to [Display Options] dialog box. Select <Change to tiled or multiplane view> button on tool bar and bring [Display Options] dialog box to appear. <Change to tiled or multiplane view >...
Page 298 APPLIED OPERATIONS /Changing the Image Display Method Fig. 2-75 Panel Displaying Images Per Channel OPERATION INSTRUCTIONS Page 2 - 2 1 4...
Page 299: Displaying Images Of Two Channels Together
APPLIED OPERATIONS /Changing the Image Display Method 2-5-7-2 Displaying Images of Two Channels Together Images acquired in a multi-channel mode can be displayed together for simultaneous view. The operation method is identical to the method for displaying images per channel except for the following point.
Page 300: Displaying Time-Lapse Images
APPLIED OPERATIONS /Changing the Image Display Method 2-5-7-3 Displaying Time-Lapse Images Multiple images acquired over time can be displayed side by side for simultaneous view. Display the image window of one of the time-lapse images to be displayed together. Set the number of images to be displayed together by using the < >...
Page 301: Displaying Multiple Multiple Sections Images
APPLIED OPERATIONS /Changing the Image Display Method 2-5-7-4 Displaying Multiple Multiple sections Images Images acquired from different multiple sections can be displayed together for simultaneous view. The operation method is identical to the method for displaying time-lapse images except for the following point.
Page 302 APPLIED OPERATIONS /Changing the Image Display Method Set the number of images to be displayed together by using the < > or < > buttons in the [Columns] and [Rows] text boxes. How the images will be arranged can be confirmed in the gray box at the upper part of the [Tiling] group box.
Page 303 APPLIED OPERATIONS /Changing the Image Display Method Perform steps 5 and 6 above for each of the displayed images. Fig. 2-79 Panel Showing 2-Channel Mode Images in Different Display Methods Fig. 2-79 shows a panel where the display methods are varied as shown below. Z(2) Z(1) OPERATION INSTRUCTIONS...
Page 304: Re-Arranging Images Using The Same Display Method
APPLIED OPERATIONS /Changing the Image Display Method 2-5-7-6 Re-arranging Images Using the Same Display Method All of the images displayed together in a image window can be rearranged simultaneously based on the same display method (channels, magnification, scroller position). Click one of the images displayed together. Two sets of buttons appear above and below the clicked image.
Page 305 APPLIED OPERATIONS /Changing the Image Display Method Click the <Experiment List> button in the toolbar. The [Experiments in Memory] dialog box appears as shown below. <Experiment List> button Fig. 2-80 [Experiments in Memory] Dialog Box From the [Experiments in Memory] dialog box, select the file name of the second image to be displayed and drag it into the frame at the top right of the [Tile] panel.
Page 306 APPLIED OPERATIONS /Changing the Image Display Method When displaying images acquired in a multi-channel mode, select the display method from the [Display Type] drop-down list. Click the <New Page> button. A new image window appears showing the two images one above the other. The image of the file displayed in the frame at the top left of the [Tile] panel is displayed on the upper part of the image window, and that of the file displayed in the frame at the top right is displayed on the lower part.
Page 307: Magnifying/Reducing An Image
APPLIED OPERATIONS /Changing the Image Display Method 2-5-8 Magnifying/Reducing an Image The image can be magnified or reduced using the buttons displayed at the top of the image window. Magnification or reduction up to 3:1 or 1:3 the original image is possible. Display the image window of the image to be magnified or reduced.
Page 308 APPLIED OPERATIONS /Changing the Image Display Method Fig. 2-82 Panel Showing a 2:1 Magnified Image OPERATION INSTRUCTIONS Page 2 - 2 2 4...
Page 309: Image Processing
APPLIED OPERATIONS /Image Processing 2-6 Image Processing Images can be processed using the [Process] panel. Display the [Process] panel at the front. 2-6-1 Filtering Use the [Filters] sub-panel in the [Process] panel to apply filtering to images. Display the [Filters] sub-panel in the [Process] panel at the front. Icon of the image being displayed (Image to be processed by filtering).
Page 310: Contour Enhancement
APPLIED OPERATIONS /Image Processing 2-6-1-1 Contour Enhancement When an image is blurred by the boundaries between image grains becoming unclear, it can be sharpened by applying contour enhancement. Five types of filters are provided for use in the contour enhancement. 1 Laplacian filter This filter enhances the contours of the image grains.
Page 311 APPLIED OPERATIONS /Image Processing Fig. 2-84 Panel Displaying the Laplacian Filtered Image 2 Sobel filter This filter enhances the contours of the image grains. If the image contains noise, the noise is also enhanced. It has two filter formats, X and Y, as shown below. The format providing the larger value after filtering is used.
Page 312 APPLIED OPERATIONS /Image Processing 3 High-pass X filter The HIGH-PASS X filter passes the high-frequency structures in the X-direction of image. In this way, it can extract details by detecting positions with large variation. This processing is useful for making structures clear or extract the edges. The filter format is as shown below. The operation method is identical to Laplacian filtering except for the following point.
Page 313: Noise Reduction
APPLIED OPERATIONS /Image Processing 5 Prewitt filter This filter enhances the contours of image grains in a similar way to the Sobel filter, but more strongly than it. It has two filter formats, X and Y, as shown below. The format providing the larger value after filtering is used.
Page 314 APPLIED OPERATIONS /Image Processing 2 Median filter The Median filter reduces noise in image while leaving the edges intact. However, it may be ineffective in case noise is concentrated in some positions or the image is very noisy. The operation method is identical to Laplacian filtering except for the following point. See section 2-6-1-1-1, “Laplacian filter”.
Page 315: Image Sharpening
APPLIED OPERATIONS /Image Processing 2-6-1-3 Image Sharpening 1 Sharpen filter The sharpen filter turns blurred image into a clear image. The filter format is as shown below. The operation method is identical to Laplacian filtering except for the following point. See section 2-6-1-1-1, “Laplacian filter”.
Page 316: Dic Correcting Dic Level Irregularities
APPLIED OPERATIONS /Image Processing 2-6-1-4 DIC Correcting DIC Level Irregularities DIC level irregularities refers to uneven brightness of the image background which may be observed when a transmitted image is acquired in IDC observation. The DIC level irregularities can be corrected to make the image easier to view. Display the [Filters] sub-panel in the [Process] panel.
Page 317 APPLIED OPERATIONS /Image Processing Click the <DIC leveling> button. The [Level] panel is newly created in the image window and shows an image after the correction of the DIC level irregularities of the background. The background irregularities are solved. Fig. 2-86 [Level] Panel After DIC Level Correction If the DIC level irregularities cannot be reduced, repeat steps 1 to 3 above by varying the correction factor.
Page 318: Contrast Conversion
APPLIED OPERATIONS /Image Processing 2-6-2 Contrast Conversion The LUT intensity can be mapped (re-assigned) while observing a histogram. Mapping (re-assignment) results in changing the image contrast. An image acquired by observation contains intensity information in values from 0 to 4095, but the intensity information used in actual display takes values from 0 to 255 by assigning the original values from 0 to 4095 to values from 0 to 255 usually.
Page 319 APPLIED OPERATIONS /Image Processing 4. When the image was acquired in the multi-channel mode, select whether the LUT <Display channel switch> intensity mapping (re-assignment) is applied to multiple channels simultaneously or to a buttons single channel. To select the target channel(s), use the <Display channel switch> buttons. The histogram of the selected channel(s) is displayed.
Page 320 APPLIED OPERATIONS /Image Processing Enclose the histogram section of interest using the scale in the [Master View] field. The magnified view of the region selected by the scale is shown in the [Selected View] field. The vertical chart axis represents the degree and the horizontal axis represents the mapped intensity values.
Page 321: Mathematical Operations Between Images
APPLIED OPERATIONS /Image Processing 2-6-3 Mathematical Operations Between Images Arithmetic or logical operations can be applied between two different images or between an image and a constant. 2-6-3-1 Image Addition Addition of an image to an image (constant to an image) is possible as described below. Select <Process-Math>...
Page 322 APPLIED OPERATIONS /Image Processing Right click on the frame at the top left of [Math] sub-panel, and select the file name of first image in displayed pop up menu. Before the dragging and dropping, the frame at the top left shows the icon of the image file displayed in the image window.
Page 323 APPLIED OPERATIONS /Image Processing To add an image to an image: Click the <Add 2 Images> button in the [Multi Image Operations] group box. A new image window showing [Image+Image] in the page tab appears, displaying the image obtained by the addition operation. To add a constant to an image: Click the <Add to Image>...
Page 324: Image Subtraction
APPLIED OPERATIONS /Image Processing 2-6-3-2 Image Subtraction Subtraction of an image from an image (constant from an image) is possible as described below. The operation method is identical to Image + Image (Image + Constant) except for the following point. See section 2-6-3-1, “Image Addtion”. To subtract an image from an image: Click the <Subtract 2 Images>...
Page 325: Image Division
APPLIED OPERATIONS /Image Processing 2-6-3-4 Image Division Division of an image by an image (image by a constant) is possible as described below. The operation method is identical to Image + Image (Image + Constant) except for the following point. See section 2-6-3-1, “Image Addition”. To divide an image by an image: Click the <Divide 2 Images>...
Page 326: Not Image
APPLIED OPERATIONS /Image Processing 2-6-3-5 NOT Image The NOT operation of an image allows the bright and dark areas of the image to be reversed. Display the [Math] sub-panel of the [Process] panel at the front. Sets the image to be subjected to operation.
Page 327 APPLIED OPERATIONS /Image Processing 3. Click the <NOT Image> button. A new image window showing [NOT] in the page tab <NOT Image> appears, showing the image obtained by the operation. button Fig. 2-93 [NOT] Panel OPERATION INSTRUCTIONS 2 - 2 4 3 Page...
Page 328: Image And Image
APPLIED OPERATIONS /Image Processing 2-6-3-6 Image AND Image Two different images can be ANDed. Select <Process-Math> button on tool bar and bring [Math] sub panel to appear. <Process-Math> button Sets the image to be subjected to Sets the second image operation.
Page 329: Image Or Image
APPLIED OPERATIONS /Image Processing Click the <Image AND Image> button. A new image window showing [Image AND <Image AND Image> Image] in the page tab appears, showing the image obtained by the operation. button Fig. 2-95 [Image AND Image] Panel 2-6-3-7 Image OR Image Two different images can be ORed.
Page 330: Brightness Overlap Level Between 2 Channels (Colocalization)
APPLIED OPERATIONS /Image Processing 2-6-4 Brightness overlap level between 2 channels (Colocalization) This function enables to observe overlap intensity of two channel of image. Acquires or opens images which more than two channels, and displays the images on image window. On the image window, displays and overlaps 2 channel image that to be observed.
Page 331: Annotation Mode
APPLIED OPERATIONS /Image Processing 2-6-4-1 Annotation Mode 1 None Mode None Mode is the default of [Colocalization Processor] dialog box. The mode can be changed by option button in [Annotation Mode] Group Box. The items described for this mode can be used in other modes. [Data Selection] Group Box <Disabled>...
Page 332 APPLIED OPERATIONS /Image Processing 2 Thresholds Mode Specifies Threshold level on [Histogram] graph, then measures by use of the threshold level. [Histogram] Graph Threshold line vertical horizontal red line) appears. The value can be adjust by dragging those lines. [Annotation mode] Group [Annotation] Text Box When <Thresholds>...
Page 333 APPLIED OPERATIONS /Image Processing [Histogram] graph Explanation of [Annotation] Text Box Display in Text Box Explanation Threshold Annotation Thresholds Mode Total samples in histogram: Total numbers of image pixels to be processed (Dye-name1)[Thresh: Name of Dye method. Threshold value in X- axis.
Page 334 APPLIED OPERATIONS /Image Processing 3 Min-Max Bounds Mode Specifies the rectangular on [Histogram] graph, and measures colocalization by use of the rectangular. In addition, distribution level of brightness inside rectangular is displayed on image window. Color is the same as color of rectangular selected. [Histogram] Graph Rectangle is displayed in graph.
Page 335 APPLIED OPERATIONS /Image Processing Explanation of [Annotation] Text Box Display in Text Box Explanation Min-Max Annotation Min-Max Bounds Mode Total samples in histogram Total numbers of image pixels to be processed (Dye-name1) limits: X-axis range of rectangular Minimum: Minimum value of X-axis rectangular Maximum: Maximum value of X-axis rectangular (Dye-name2) limits:...
Page 336 APPLIED OPERATIONS /Image Processing 4 Regions Mode Specifies the arbitrary region in [Histogram], and measures colocalization within the region. In addition, distribution level of brightness in draw pictorial figure is displayed on image window. Color is the same as color of draw pictorial figure selected. [Histogram] Graph You can draw a pictorial figure on [Histogram], using graphic...
Page 337 APPLIED OPERATIONS /Image Processing Explanation of [Annotation] Text Box (In case that 3 pictorial figures exist. Items (1) will be increased according to number of pictorial figures.). Display in Text Box Explanation Regions Annotation Regions Mode Total samples in histogram Total numbers of image pixels to be processed RGB:...
Page 338: Colocalization For Series Image Data Set
APPLIED OPERATIONS /Image Processing 2-6-4-2 Colocalization for series image data set Colocalization is also available for series image data set, i.e XYZ image data set. [Data Selection] Group Box <Current slice> Option Button The cocalization is processed for the image in a series data set which is displayed in image window.
Page 339: Image Measurement
APPLIED OPERATIONS /Image Processing 2-6-4-3 Image measurement When option button of [Image Measures] of [Analysis Mode] Group Box is selected, the dialog as shown below appears. [Analysis Mode] Group Box [Image Measurements] Group Box It displays results of measurement in text box below.
Page 340 APPLIED OPERATIONS /Image Processing · Pearson’s Coefficient : In case that the value to be acquired is defined as ∑ − ⋅ − ∑ ∑ − ⋅ − However, legends used for image measurement are as follows: λ λ : Brightness of wavelength -th pixel, : Brightness of wavelength -th pixel...
Page 341 APPLIED OPERATIONS /Image Processing In case that the value to be acquired is defined as ∑ ∈ ∑ i ∈ However, should be -th pixel that belongs to aggregation A that is greater than threhold value. OPERATION INSTRUCTIONS 2 - 2 5 7 Page...
Page 342: Appending Image (Append)
APPLIED OPERATIONS /Image Processing 2-6-5 Appending image (Append) 2-6-5-1 Appending two images Two images (A and B) acquired in XY or XYZ observation can be appended along the Z- or T-direction or as animation. XY image Appending in XYT image T-direction TIME XY image...
Page 343 APPLIED OPERATIONS /Image Processing Display the [Experiment Editor] sub-panel of the [Process] panel at the front. Fig. 2-102 [Experiment Editor] Sub-panel In the toolbar at the bottom of the [Process] panel, click the <Experiment List> button. The [Experiments in Memory] dialog box appears as shown below. <Experiment List>...
Page 344 APPLIED OPERATIONS /Image Processing The icon of the image file being displayed at the front of the image window (A) is shown in the frame at the top left of the [Process] panel. In the [Experiments in Memory] dialog box, select the file name of the second image (B) and drag it to the frame at the top right of the [Process] panel.
Page 345 APPLIED OPERATIONS /Image Processing Depending on the type of the image to be created by appending, various option buttons appears under the <Append> button in the [Experiment Editor] sub-panel. Option buttons depending on the image mode. · make new dim ..Create an image of new observation mode. (Select the type in the [Append on New Dimension] dialog box.) ·...
Page 346: Appending Image From Several Image Data Set
APPLIED OPERATIONS /Image Processing 2-6-5-2 Appending image from several image data set An image data set can be made from appending several image data set. NOTE There are the following restrictions when you append image files. ·Two image files must be the same image size. ·Two image files must be the same channel number.
Page 347 APPLIED OPERATIONS /Image Processing [Append on New dimension] dialog box appears as shown below. File area Image Info area [Order] Text Box Folder area <Clear> Button <Append> Button [Created file] <Brows> [Resolution] [Dimension] Text Box Button Text Box Group Box [Comments] [Sort] Text Box...
Page 348 APPLIED OPERATIONS /Image Processing Select Series data set type that will be created in [Dimension] Group Box. The option buttons work as the followings. ·on T......Appends file in T direction. ·on Z......Appends file in Z direction. ·on An ..... Appends file as animation. Time information of each file stays as its original, in case of T direction appending.
Page 349 APPLIED OPERATIONS /Image Processing Enter folder and file name into [Create file] text box. By pressing <Browse…> button, folder appears for easy folder selection. Click <Append> button, then new appended image created and stored. Pressing <Clear> button clears the file assignment. [Resolution] text box works as follows.
Page 350 APPLIED OPERATIONS /Image Processing One Point! Plural numbers of images can be appended without starting FLUOVIEW software. 1. Select <Start> button at bottom of Windows screen. 2. In case of Windows XP, select [All Programs] – [Accessories] - [Windows Explorer] command from the [start] menu.
Page 351: Extract Image (Crop)
APPLIED OPERATIONS /Image Processing 2-6-6 Extract image (Crop) New image data with selected slice image from an image data set can be made by this function. The following is an example for Image extraction from an XYZT image data set. Display the image to be extract in image window.
Page 352 APPLIED OPERATIONS /Image Processing Select image slices by pressing on [Extract] dialog box. The following method of slice selection is also available. ·All of image slices can be select by pressing <select all> button. ·All of row or column slices can be selected by pressing [No. Display]. ·Image slices with a certain time interval and/or Z step can be selected by pressing <Set>...
Page 353 APPLIED OPERATIONS /Image Processing One Point! When arbitrary portion of XYT image is extracted and plural numbers of [No. Display] columns at T columns are selected, the dialog box as shown below will appear in case that number of units at Z portion of [No. Display] on all T columns are not the same number of units.
Page 354 APPLIED OPERATIONS /Image Processing One Point! It is possible to cut out the image and extract it by specifying arbitrary region. Image cut out is done as follows. <Annotate> button 1. Select [Annotate] button in tool bar. <Rectangular> button <Circle> button <Polyregion>...
Page 355 APPLIED OPERATIONS /Image Processing One Point! Slice extract function works even if FLUOVIEW software is not running. However, clipping function works only with FLUOVIEW software. 1.Press Windows <Start> button. 2.In case of Windows XP, select [All Programs] – [Accessories] - [Windows Explorer] command from the [start] menu.
Page 357 APPLIED OPERATIONS /Image Processing Combinations of images with which appending are possible XYZT Pt-Pt XY-AN XY-AN XY-AN XYZT XYZT XYZT XYZT XYZT XT-AN Pt-Pt Pt-Pt-AN XY-AN XY-AN XY-AN Point-scanned image Pt..Animation image AN ..OPERATION INSTRUCTIONS 2 - 2 7 3 Page...
Page 358: Image Analysis
APPLIED OPERATIONS /Image Analysis 2-7 Image Analysis Images can be analyzed using the [Analyze] panel. Display the [Analyze] panel at the front. Displays the icon of the image being <Begin Analysis> button displayed (image to be subjected to analysis). Starts analysis. [Measurement Results] box Shows the measurement data of the specified line or region.
Page 359: Checking The Intensity Of A Specific Part
APPLIED OPERATIONS /Image Analysis 2-7-1 Checking the Intensity of a Specific Part 2-7-1-1 Intensity Values on a Line (Line Profile) The intensity values on a line in an image can be displayed graphically. Display the [Single] sub-panel at the front. Display the image window of the image to be subjected to the intensity checking at the front.
Page 360 APPLIED OPERATIONS/ Image Analysis To specify a free line: On the image, drag the mouse pointer along the line to be <Free Line>button checked. The line is displayed on the image together with the handles on it. The intensity profile can be displayed while Handle the handles are displayed.
Page 361 APPLIED OPERATIONS /Image Analysis Double-click the [Intensity Profile] button. The [Enhanced Profile] window appears as shown below. <Properties> button Displays the [Editing] dialog box for use in detailed setting of the chart or change of the chart display. See section 2-15, “Changing the Chart Display Method”...
Page 362: Intensity Values On A Planar Region (Bird's Eye View)
APPLIED OPERATIONS/ Image Analysis 2-7-1-2 Intensity Values on a Planar Region (Bird’s Eye View) The intensity values on a region in an image can be displayed graphically. Display the [Single] sub-panel at the front. Display the image window of the image to be subjected to the intensity checking at the front.
Page 363 APPLIED OPERATIONS /Image Analysis Specify the region to be checked in the image in the image window. They can be specified as described below. To specify a rectangle: On the image, drag the mouse pointer along the diagonal line of the desired rectangle, from the top left corner to the bottom right corner.
Page 364 APPLIED OPERATIONS/ Image Analysis The checked region can be moved, deleted or changed of size or color. This is possible with the same method as entering comment in the image. For details, see sections 2-12-6, 2-12-7, 2-12-8 and 2-12-9 in section 2- 12, “Entering Comment in Image”.
Page 365 APPLIED OPERATIONS /Image Analysis When the image was acquired in the multi-channel mode, the channel(s) to be subjected to the bird’s eye view display can be selected using the [Channel] option buttons. Shows: Measurement results including; · Perimeter · Area ·...
Page 366 APPLIED OPERATIONS/ Image Analysis 10. Double-click the [Intensity Profile] button. The [Intensity Map] window appears as shown below. [Angle] scale Sets the angle in the horizontal direction. The result can be confirmed with the small bird’s eye view in the frame on the [Tilt] scale Sets the angle in the vertical direction.
Page 367: Checking The Intensity Distribution Of A Specific Part
APPLIED OPERATIONS /Image Analysis 2-7-2 Checking the Intensity Distribution of a Specific Part 2-7-2-1 Intensity Distribution on a Line (Histogram) The histogram on a line in an image can be displayed. The histogram is displayed in the [Region Histogram] box in the [Single] sub-panel. The operation method is identical to displaying the intensity profile on a line.
Page 368: Intensity Distribution On A Planar Region (Histogram)
APPLIED OPERATIONS/ Image Analysis The magnification or scrolling of the graph can be canceled by dragging the left button of the mouse from the bottom left to the right of the magnified graph. When the mouse pointer is placed on a graph line while the Ctrl or Alt key is held depressed, the coordinates can be displayed.
Page 369 APPLIED OPERATIONS /Image Analysis When a desired area is specified by dragging mouse left button over the graph, from upper left to lower right, the specified area can be magnified. When the right button of the mouse is dragged on the graph, the graph can be scrolled.
Page 370: Image Measurement
APPLIED OPERATIONS/ Image Analysis 2-7-3 Image Measurement 2-7-3-1 Length Measurement The length between two points in an image or the perimeter of a region in an image can be measured. The [Measurement Results] box in the [Single] sub-panel shows the measurement results such as the length between 2 points (Length) or perimeter of the region (Perimeter) and the horizontal and vertical lengths (X/Y, Z or T) of the region, and the statistic result of each channel such as the total (Total), average (Average) and standard deviation (Std Dev).
Page 371: Measuring The Change In Mean Value Of Intensity
APPLIED OPERATIONS /Image Analysis 2-7-3-3 Measuring the Change in Mean Value of Intensity The mean value of the intensity in a region specified in an image can be measured and displayed graphically. Select <Analysis-Series> button on tool bar and bring [Series] sub-panel. Display the image window of the image that you want to check the intensity at the front.
Page 372 APPLIED OPERATIONS/ Image Analysis When the image is composed of multiple image slices, the range of image slices to be subjected to the operation can be set using the <Set start position> button <Set start position> and <set end position> buttons above the images. First display the image slice to start the operation using the <Display>...
Page 373 APPLIED OPERATIONS /Image Analysis Click the <Annotate> button in the toolbar. A list of buttons appear as shown below. <Annotate> button <Rectangular > button < Circle > button <Polyregion > button < Free Region > button From the displayed buttons, click the <Rectangular> button, <Circle> button, <Polyregion>...
Page 374 APPLIED OPERATIONS/ Image Analysis Click the mouse. A pop-up menu as shown below appears. Select [Properties] from the menu. The [Properties] dialog box as shown below appears. Display the [Color] panel at the front. Fig 2-113 [Properties] Dialog Box 10. Select the desired color from the [Color Palette] list box. 11.
Page 375 APPLIED OPERATIONS /Image Analysis 14. Click the <Begin Analysis> button. The mean value of the specified regions will be displayed graphically in the [Mean Intensity] box. NOTE The colors of the chart lines corresponding to the colors assigned to the regions. NOTE When the image was acquired in the multi-channel mode, the channel number is displayed to the right of each chart line.
Page 376 APPLIED OPERATIONS/ Image Analysis 15. Double-click the [Mean Intensity] box. The [Average Intensity Trace] window appears as shown below <Properties> button Displays the [Editing] dialog box for use in detailed setting of the chart or change of the chart display. See section 2-15, “Changing the Chart Display Method”...
Page 377: Measuring The Change In Integrated Intensity
APPLIED OPERATIONS /Image Analysis 2-7-3-4 Measuring the Change in Integrated Intensity The total value of the intensity in a region specified in an image can be measured and displayed graphically. The operation results are displayed graphically in the [Integrated Intensity] box in the [Series] sub-panel.
Page 378 APPLIED OPERATIONS/ Image Analysis The magnification or scrolling of the graph can be canceled by dragging the left button of the mouse from the bottom left to the right of the magnified graph. When the mouse pointer is placed on a graph line while the Ctrl or Alt key is held depressed, the coordinates can be displayed.
Page 379: Building An Image From A Different Viewpoint
APPLIED OPERATIONS /Building an Image from a Different Viewpoint 2-8 Building an Image from a Different Viewpoint 2-8-1 Building Extended Focus Image from XYZ Image 2-8-1-1 Display Switching to Built Image An extended focus image can be built from XYZ (multiple sections) images and the display can be switched to show the built image.
Page 380 APPLIED OPERATIONS /Building an Image from a Different Viewpoint (Image No. 0) (Image No. 1) (Image No.2) (Image No. 3) Fig. 2-117 Four Images Used in Building Extended-Focus Image Fig. 2-118 Panel Showing an Extended-Focus Image OPERATION INSTRUCTIONS Page 2 - 2 9 6...
Page 381 APPLIED OPERATIONS /Building an Image from a Different Viewpoint Among the multiple image slices composing the XYZ image, the range of image slices to be used in extend image building can be specified. 1. Display the image slice to be set as the start image by using the <Frame advance> or <Successive display>...
Page 382: Turning Built Image Into Single Image
APPLIED OPERATIONS /Building an Image from a Different Viewpoint 2-8-1-2 Turning Built Image into Single Image From XYZ (multiple sections) image, an extended-focus image can be built as a separate image from the original image. Use the [Visualize] panel to build the image. First display the [Visualize] panel.
Page 383 APPLIED OPERATIONS /Building an Image from a Different Viewpoint Click the <Begin Visualize> button to start the image building. When it completes, the built image is displayed in the [Extended] panel. Fig. 2-120 Panel Showing Extended-Focus Image OPERATION INSTRUCTIONS Page 2 - 2 9 9...
Page 384: Turning Built Image Into Time Series Image
APPLIED OPERATIONS /Building an Image from a Different Viewpoint 2-8-1-3 Turning Built Image into time series image From XYZT image, an extended-focus image can be built as a separate time series image from the original image. Use the [Visualize] panel to build the image. First display the [Visualize] panel.
Page 385: Building Line Images To Be Viewed In Z Direction
APPLIED OPERATIONS /Building an Image from a Different Viewpoint 2-8-2 Building line images to be viewed in Z direction The images acquired by cutting the XYZ image off vertically and horizontally can be displayed with the information on each line. Each line images is created as a separate image from the original one.
Page 386 APPLIED OPERATIONS /Building an Image from a Different Viewpoint If you change the slice to be displayed with the <Display> button, [Slice] and [Z:] aren’t effected. <Display> button [Slice] and [Z:] show current slice and Z position according to moving the red or brown line indicating XZ or YZ-direction on the XY-direction image displayed left above.
Page 387 APPLIED OPERATIONS /Building an Image from a Different Viewpoint Select <Accept this view as new experiment> button on [Visualize] panel. <Accept this view as In case of Ver5.0, select <Make experiment from the X-Z view> button or new experiment> button <Make experiment from the Y-Z view>...
Page 388 APPLIED OPERATIONS /Building an Image from a Different Viewpoint One Point! When building 3D image in Ver5.0 or subsequent version, pay attention to the following points. When using XZ direction as reference, 3D image including images of YZ (upper right) and XY (lower left) can be built. When using YZ direction as reference, 3D image including images of XY (upper right) and XZ (lower left) can be built.
Page 389: Viewing 3D Image
APPLIED OPERATIONS /Viewing 3D Image 2-9 Viewing 3D Image Use the [Visualize] panel to view an image three-dimensionally. First display the [Visualize] panel. [Begin Visualize] button Starts building the images for 3D image window display. Shows the image. The file name of the image is [Method] group box shown in the page tab of the panel.
Page 390 APPLIED OPERATIONS /Viewing 3D Image Fig 2-126 Example of Intensity Projection Fig 2-127 Example of Topographic Projection [Threshold range Low / High] scale Sets the range of intensity values to be used in image building. [Depth Weight] scale Sets the weighting in the depth direction. By increasing this setting, it is possible to provide the image with a perspective by darkening the far objects and brightening near objects.
Page 391: Successive Display Of Images
APPLIED OPERATIONS /Viewing 3D Image 2-9-1 Successive Display of Images Images composed of multiple image slices acquired by varying the multiple sections (XYZ observation, XYZT observation) can be displayed successively using the buttons at the top of the image window as shown below. 1.
Page 392: Changing The Successive Display Speed
APPLIED OPERATIONS /Viewing 3D Image 3. Click the <Set start position> button. (If the start position is not set, image No. 0 becomes the start image automatically.) 4. Display the image slice to end the successive display by using the <Frame advance> button at the top of the image window.
Page 393: Changing The Successive Image Display Position
APPLIED OPERATIONS /Viewing 3D Image 4. Select the option button of the speed to be varied. 5. Set the desired display speed in the scale on the right. 6. Click the <OK> button to close the [Animation speed] dialog box. 2-9-1-2 Changing the successive image display position The successive display position of multiple image slices in an image can be changed using a bottom displayed at the top of the image window.
Page 394: Animation
APPLIED OPERATIONS /Viewing 3D Image 2-9-2 Animation Images composed of multiple image slices acquired by varying the multiple sections (XYZ observation, XYZT observation) can be built into animation image, which can be displayed in 3D by rotating images. Display the image window of the image composed of multiple image slices. When the images were acquired in the multi-channel mode, select whether animation is built from images of more than one channel or from an image of only one channel.
Page 395 APPLIED OPERATIONS /Viewing 3D Image 10. Set the number of images to be rotating using the [Number of views] scale. 11. Click the <Begin Visualize> button to start building the animation. When the building completes, the built image is displayed in the [3D Animation] panel. The status bar shows the progress of building processing.
Page 396: Building Stereo 3D Images
APPLIED OPERATIONS /Viewing 3D Image 2-9-3 Building Stereo 3D Images Images composed of multiple image slices acquired by varying the multiple sections (XYZ observation) can be built into a pair of stereo 3D images. These images can be viewed three-dimensionally by watching them with two eyes for a while. The operation is similar with animation.
Page 397 APPLIED OPERATIONS /Viewing 3D Image Fig. 2-130 Panel Showing Stereo 3D Images OPERATION INSTRUCTIONS Page 2 - 3 1 3...
Page 398: Building A 3D Image To Be Viewed Through Color (Red/Green) Eyeglasses2-314
APPLIED OPERATIONS /Viewing 3D Image 2-9-4 Building a 3D Image to be Viewed Through Color (Red/Green) Eyeglasses Images composed of multiple image slices acquired by varying the multiple sections (XYZ observation) can be built into an image which looks 3D when viewed through a pair of color (red/green) eyeglasses.
Page 399 APPLIED OPERATIONS /Viewing 3D Image Fig. 2-131 Panel Showing 3D Image to be Viewed Through Color Eyeglasses OPERATION INSTRUCTIONS Page 2 - 3 1 5...
Page 400: Viewing Images Following The Progress Of Time
APPLIED OPERATIONS /Viewing Images Following the Progress of Time 2-10 Viewing Images Following the Progress of Time Images composed of multiple image slices (XYT, XYZT or XZT observation) can be displayed following the time lapse to show the change over time. 2-10-1 Displaying Images Together The change over time can be viewed at a glance by displaying multiple image slices together.
Page 401 APPLIED OPERATIONS /Viewing Images Following the Progress of Time <Loop> button <Z/T series switch> button Successive display or frame-by-frame display is repeated <Set start position> button in the same direction. When successive display or frame-by-frame display is <Loop/Bound switch>button required, set the image with which the display should start.
Page 402 APPLIED OPERATIONS /Viewing Images Following the Progress of Time 4. Click the <Set start position> button. (If the start position is not set, image No. 0 becomes the start image automatically.) 5. Display the image slice to end the successive display by using the <Display> button at the top of the image window.
Page 403: Transferring Data To Another Application
Quits the [Enhanced Profile Plot] window and returns to the [Analyze] panel. Fig. 2-132 [Enhanced Profile Plot] Window Microsoft Excel is not included in the FLUOVIEW FV1000 system. Please purchase it separately. OPERATION INSTRUCTIONS Page 2 - 3 1 9...
Page 404 APPLIED OPERATIONS /Transferring Data to Another Application 2. Click the <Save> button. When the [Save As] dialog box appears as shown below, set the file name and click the <OK> button to save the analysis data. Fig. 2-133 [Save As] Dialog Box 3.
Page 405 APPLIED OPERATIONS /Transferring Data to Another Application 7. Click the <Next> button. When the dialog as shown below appears, check the [Tab] check box in the [Delimiters] group box, then select [[none]] from the [Text Qualifier:] drop-down list. Fig. 2-135 Dialog Box When the File is Opened by Excel 2 / 3 8.
Page 406: Transferring The Plot Image Of Analysis Data To Another Application
APPLIED OPERATIONS /Transferring Data to Another Application For detailed operation procedures of Excel, refer to the [Excel manuals]. 2-11-2 Transferring the Plot Image of Analysis Data to Another Application The plot image of analysis data can be transferred to an application handling images, such as Paint.
Page 407: Transferring Image Data To Another Application (Paint, Etc.)
APPLIED OPERATIONS /Transferring Data to Another Application Click the <Copy> button to copy the plot image to the clipboard. Exit from FLUOVIEW or display the [Start] menu by pressing the Windows key or the Ctrl keys. Select [Programs]-[Accessories] and issue the [Paint] command. From the [Edit] menu of Paint, select the [Paste] command and paste the plot image which has been copied to the clipboard in step 3.
Page 408: Entering Comment In Image
APPLIED OPERATIONS /Entering Comment in Image 2-12 Entering Comment in Image Comment can be entered in an image for use in presentation or slide creation. Use the <Annotate> button in the toolbar. Click the <Annotate> button. A list of buttons appears as shown below.
Page 409 APPLIED OPERATIONS /Entering Comment in Image From the drop-down list in the dialog box, select one of the labels. Desired characters can also be entered. Click the character in the drop-down list of the dialog box. Delete the character by pressing the Delete Back Space Enter characters from the keyboard.
Page 410: Displaying The Image Intensity
APPLIED OPERATIONS /Entering Comment in Image 2-12-2 Displaying the Image Intensity The intensity of any pixel of an image can be displayed without using the [Analyze] panel. Display the image window of the image that you want to display the intensity at the front.
Page 411: Displaying The X-Coordinate/Y-Coordinate Of The Image
APPLIED OPERATIONS /Entering Comment in Image 2-12-3 Displaying the X-coordinate/Y-coordinate of the Image The X-coordinate position or the Y-coordinate position of any pixel of an image can be displayed. Display the image window of the image that you want to display the X-coordinate position or the Y-coordinate position at the front.
Page 412: Drawing A Figure In Image
APPLIED OPERATIONS /Entering Comment in Image 2-12-4 Drawing a Figure in Image This facility is used to draw figures in the image. FLUOVIEW provides seven figure drawing modes. Display the image window of the image in which you want to draw figures. Select one of the following command buttons and draw a figure in the image using the mouse.
Page 413: Drawing A Scale In Image
APPLIED OPERATIONS /Entering Comment in Image 2-12-5 Drawing a Scale in Image A scale can be drawn between two points in an image. Display the image window of the image in which you want to draw a scale. Click the <Scale> button in the displayed list of buttons. Click the image position you want to draw a scale.
Page 414: Drawing An Arrow In Image
APPLIED OPERATIONS /Entering Comment in Image 2-12-6 Drawing an Arrow in Image This facility is used to draw an arrow for indicating a point in interest in image or adding explanation in it. Display the image window of the image you want to draw an arrow. Click the <Arrow>...
Page 415: Drawing Color Bars In Image
APPLIED OPERATIONS /Entering Comment in Image 2-12-7 Drawing Color Bars in Image This facility is used to draw color bars in an image. Display the image window of the image you want to draw color bars. Click the <Color Bar> button in the displayed list of buttons. Draw color bars in the image by dragging the mouse pointer along the desired position <Color Bar>...
Page 416: Deleting Comment
APPLIED OPERATIONS /Entering Comment in Image 2-12-8 Deleting Comment Click the mouse on the comment to be deleted to make the comment active (i.e. handles displayed around the comment). Handle Click the right button of the mouse. A pop-up menu as shown below appears. Fig.
Page 417: Moving Comment
APPLIED OPERATIONS /Entering Comment in Image 2-12-9 Moving Comment Click the moues on the comment to be moved to make it active (i.e. handles displayed around it). Click the right button of the mouse. A pop-up menu as shown in Fig. 2-142 appears. Select [Move] from the menu.
Page 418: Changing The Comment Size
APPLIED OPERATIONS /Entering Comment in Image 2-12-10 Changing the Comment Size Click the mouse on the comment to be resized to make it active (i.e. handles displayed around it). Click the right button of the mouse. A pop-up menu as shown in Fig. 2-142 appears. Select [Size] from the menu.
Page 419: Changing The Comment Color
APPLIED OPERATIONS /Entering Comment in Image 2-12-11 Changing the Comment Color Click the mouse on the comment to be changed of color to make the comment active (i.e. handles displayed around it). Click the right button of the mouse. A pop-up menu as shown in Fig. 2-142 appears. Select [Properties] from the menu.
Page 420: Changing The Comment Font
APPLIED OPERATIONS /Entering Comment in Image 2-12-12 Changing the Comment Font Click the mouse on the comment to be changed of font to make the comment active (i.e. handles displayed around it). Click the right button of the mouse. A pop-up menu as shown in Fig. 2-142 appears. Select [Properties] from the menu.
Page 421: Image Output At Printer
APPLIED OPERATIONS /Image Output at Printer 2-13 Image Output at Printer Display the image window of the image to be output at the printer. Click the <Print> button in the toolbar at the bottom left of the panel. A dialog box as shown below appears.
Page 422: Merger/Extraction Of Image Channels
APPLIED OPERATIONS /Merger/Extraction of Image Channels 2-14 Merger/Extraction of Image Channels 2-14-1 Setting the Range of Multiple Image Slices When merging or extracting channels in images composed of multiple image slices, such as time-lapse images and images acquired by varying the cross-sections, it is possible to select only some of image slices as the target of channel merger or extraction.
Page 423: Merging Image Channels
APPLIED OPERATIONS /Merger/Extraction of Image Channels Display the image slice to start the range at the front using the <Display> button at the top of the image window. Click the <Set start position> button. (If the start position is not set, image No. 0 becomes the start image automatically.) Display the image slice to end the range at the front using the <Display>...
Page 424 APPLIED OPERATIONS /Merger/Extraction of Image Channels Display the [Experiment Editor] sub-panel in the [Process] panel. Fig. 2-146 [Experiment Editor] Sub-panel The [Experiments in Memory] dialog box appears as shown below. Fig. 2-147 [Experiment Editor] Group Box OPERATION INSTRUCTIONS Page 2 - 3 4 0...
Page 425 APPLIED OPERATIONS /Merger/Extraction of Image Channels The frame at the top left of the [Experiments in Memory] dialog box shows the icon of the image file displayed at the front of the image window. From the file list in the [Experiments in Memory] dialog box, select the file name of the first image and drag it into the frame at the top left.
Page 426 APPLIED OPERATIONS /Merger/Extraction of Image Channels When the mouse pointer is approached to the icon-side end of a line connected to [Merger], the color of the line end turns into yellow. Clicking the line in this condition switches the channel between the selected and deselected status alternately.
Page 427 APPLIED OPERATIONS /Merger/Extraction of Image Channels Click the <Merge> button in the [Experiments in Memory] dialog box. A new image window showing [Merge] in the page tab appears and the images including newly merged channel(s) are displayed in the panel. Fig.
Page 428: Extracting Channels From Image
APPLIED OPERATIONS /Merger/Extraction of Image Channels 2-14-3 Extracting Channels from Image Desired channels can be extracted from an image. Use this facility to extract only the images of required channels from an image acquired from more than one channel. Open the image file of the image to extract channels in advance. When the image has been saved in more than one image file, it is possible to use only some of the slices by setting a slice range.
Page 429 APPLIED OPERATIONS /Merger/Extraction of Image Channels The [Experiments in Memory] dialog box appears as shown below. Fig. 2-150 [Experiment Editor] Group Box The frame at the top left of the [Experiments in Memory] dialog box shows the icon of the image file displayed at the front of the image window. OPERATION INSTRUCTIONS Page 2 - 3 4 5...
Page 430 APPLIED OPERATIONS /Merger/Extraction of Image Channels From the file list in the [Experiments in Memory] dialog box, select the file name of the image from which to extract channels and drag it into the frame at the top left. The icon of the image is displayed in the frame at the top left.
Page 431 APPLIED OPERATIONS /Merger/Extraction of Image Channels Among the channel lines connected to [Extract], deselect the unnecessary channels as described in the TIP on the previous page so that only the necessary channels are selected. Click the <Extract> button in the [Experiments in Memory] dialog box. A new image window showing [Extract] in the page tab appears and the image of the extracted channel(s) is displayed in the panel.
Page 432: Changing The Chart Display Method
APPLIED OPERATIONS /Changing the Chart Display Method 2-15 Changing the Chart Display Method When the processed analysis data chart in the [Analyze] panel is double-clicked, the [Enhanced Profile Plot] dialog box appears. By clicking the <Properties> button, the [Editing] dialog box can be displayed, allowing the detailed chart settings and chart display method to be changed.
Page 433 APPLIED OPERATIONS /Changing the Chart Display Method [TeeChart Gallery] sub-panel Displayed when the <Add> or <Change> button is clicked. Used to set the chart type. The chart types that cannot be set cannot be selected. Enables or disables the 3D display.
Page 434 APPLIED OPERATIONS /Changing the Chart Display Method [Axis] sub-panel Used to set the coordinate axes of the chart. Displays the chart axes by Enables or disables the display selecting the optimum scale of chart axes. automatically. Sets maximum Selects the chart axes to be set minimum values of the chart in the [Scales], [Title], [Labels], axes either automatically or...
Page 435 APPLIED OPERATIONS /Changing the Chart Display Method [Labels] sub-panel Used to set the label of the coordinate axis. Enables or disables the label display on a point of crossing with another Enables or disables the label axis. display. Sets the label character Not used with the present font.
Page 436 APPLIED OPERATIONS /Changing the Chart Display Method [Position] sub-panel Used to set the coordinate axis display position. Sets the coordinate axis display position with respect to the chart. Sets the coordinate axis display range with reference to the start point. Sets the coordinate axis display range with reference to the end...
Page 437 APPLIED OPERATIONS /Changing the Chart Display Method [Legend] sub-panel Sets the items to be displayed Used to set the chart legend display. in the legend. Usually set [Automatic]. Enables or disables the legend display. Sets the legend display style. Sets the legend background This setting is effective when color.
Page 438 APPLIED OPERATIONS /Changing the Chart Display Method [Paging] sub-panel Used to paginate a chart for detailed viewing. Sets the number of horizontal axis points displayed per page. Enables or disables the change in scaling in the last page. Sets the current page number and total number of pages.
Page 439 APPLIED OPERATIONS /Changing the Chart Display Method [3D] sub-panel Change the zoom ratio. Used to set the 3D display. Rotates the chart in the horizontal direction with Switches between the 3D respect to the bottom side. and 2D chart display. Rotates the chart in the vertical direction with respect Sets the 3D chart display...
Page 440: Series] Panel
APPLIED OPERATIONS /Changing the Chart Display Method 2-15-2 [Series] Panel [Format] sub-panel Used to set the display method of a series of images. The set items are variable depending on the type of the chart. The following descriptions take a Line 3D chart and Point 3D chart as examples. This is the [Format] sub-panel when the Line 3D chart is selected.
Page 441 APPLIED OPERATIONS /Changing the Chart Display Method [Point] sub-panel Used to set the inflection points in the chart. Enables or disables the Sets the width of inflection inflection point display. points. Enables or disables the 3D Sets the height of inflection inflection point display.
Page 442 APPLIED OPERATIONS /Changing the Chart Display Method [Marks] sub-panel Used to set the items to be displayed along the coordinate points. Enables or disables the transparent background Enables or disables the item display. display in the chart. Enables or disables the item display of lines Sets the background color of outside the coordinate...
Page 443: Pop-Up Menus
APPLIED OPERATIONS /Pop-up Menus 2-16 Pop-up Menus Selection of function windows and image windows and other frequently-used FLUOVIEW functions (full-screen display, printer output, image save, LUT setting, comment setting) can be controlled by clicking the right button of the mouse, without selecting specific page tabs or buttons. Pop-up menu of comment When the right button of mouse is clicked on an comment in image, a pop-up menu appears to allow editing or deleting the specified comment.
Page 444 APPLIED OPERATIONS /Pop-up Menus Pop-up menu of image When the right button of mouse is clicked on the image, a pop-up menu appears to allow selection of image operations (full-screen display, printer output, image save, LUT setting, number of image divisions, comment editing).
Page 445 APPLIED OPERATIONS /Pop-up Menus Pop-up menu of the [Experiments in Memory] dialog box When the right button of mouse is clicked in the frame for opening an image file in the [File I/O], [Tile] or [Process] panel, a pop-up menu appears. This makes it possible to select the file to be opened when performing tile display, inter-image operations, image channel merger/extraction, etc.
Page 447: Appendix A List Of Hot Keys
Appendix A List of Hot Keys Appendix A List of Hot Keys Frequently-used FLUOVIEW functions (scanning, panel switching) can be controlled from the keyboard without using the mouse. Image acquisition-related keys Image acquisition channel selection Target Operation Switches the status of the Ch1 check box (Ch1 scanned/not scanned). Ctrl + Ctrl + Switches the status of the Ch2 check box (Ch2 scanned/not scanned).
Page 448 Appendix A List of Hot Keys Image acquisition (scanning) Target Operation Displays the on-line help screen. Performs repeated scanning. [XY Repeat] Starts scanning according to the current scan mode. [Scan Once] Performs focus scanning. [Focus] Acquires another image after completing series image acquisition. [Append Next] Completes image acquisition after completing series image acquisition.
Page 449 Appendix A List of Hot Keys Z stage setting Target Operation Page Up Moves the Z stage up. (Fine adjustment) Moves the Z stage up. (Coarse adjustment) Shift Page Up Down Moves the Z stage down. (Fine adjustment) Page Page Down Moves the Z stage down. (Coarse adjustment) Shift Sets the Z stage to the Stop Z position.
Page 450 Appendix A List of Hot Keys [Hi-Lo]LUT switchover key Target operation Ctrl + It alternatively switches over between the previously-assigned LUT and [Hi-Lo] LUT on the channel, whose PMT adjustment window ([PMT], [Gain] and [Offset]) is opening on the [Acquire] panel during the acquisition period.
Page 451 Appendix A List of Hot Keys [Process] sub-panels Target Operation Selects the [Math] sub-panel. Selects the [Filter] sub-panel. Selects the [Histogram] sub-panel. Selects the [Mask] sub panel. (It is an option panel with TIEMPO available). Selects the [Experiment Editor] sub panel. [Analysis] sub-panels Target Operation Selects the [Single] sub-panel.
Page 453: Appendix B Glossary
Appendix B Glossary Appendix B Glossary Clipboard The place which relays data when an operation such as “Copy”, “Cut” and “Paste” is executed. AOTF AOTF represents Acoustic Optical Tunable Filter. Command button AOTF is the sound optical element having optical A figure in the shape of button in the window.
Page 454 Appendix B Glossary Disk drive Group The storage device storing the files. Some disk drives Refers to the applications registered in the program such as the hard disk drive and floppy disk drive are manager. When a group is opened on the program capable of both input and output, and some such as manager, more than one icon is displayed in the the CD-ROM drive is designed for read only.
Page 455 Appendix B Glossary Piezo- Abbreviation piezoelectric, which phenomenon of generating electricity when a force is applied. This phenomenon is utilized in electric spark Macro generators for use with gas appliances, etc. Record of a series of operations. When a macro is executed, the series of operations defined for it are Pixel executed.
Page 456 Appendix B Glossary Simulated colors Colors used to display the image data acquired by observation on a display. Original simulated colors can be created by editing the LUT. Status bar The line showing information at the bottom of a window. It shows the information on the operation or the description of the function selected with the mouse pointer.
Page 457: Appendix C User Registration Of Fv1000
Appendix C USER REGISTRATION OF FV1000 FLUOVIEW FV1000 can store the system setup information (PMT Voltage, Gain, Offset, etc.) on a per-user basis. To make this possible, you have to register yourself as a user and log in personally when starting up the FV1000 software.
Page 458 Appendix C USER REGISTRATION OF FV1000 /User Registration Click the [Users] icon in [Hardware Settings] to display the [FLUOVIEW Users] panel in the front position. [Users] icon <Delete a user> button <Add a user and Log in> button <Reset user to Factory Defaults> button Deletes the user selected in the list box.
Page 459 Appendix C USER REGISTRATION OF FV1000 /User Registration Enter the user name in the [User Name] text box and click the <OK> button. The newly set user name is added in the list box. The registered user name is shown here. The user name is added.
Page 460: Appendix C-2 Logging Into The Fv1000
Appendix C USER REGISTRATION OF FV1000 /Logging into the FV1000 Appendix C-2 Logging into the FV1000 After the user name has been registered and the FV1000 started, a dialog box for entering the user name appears. Enter the user name to log in the FV1000. Double-click the [FLUOVIEW] icon on the desktop.
Page 461: Appendix C-3 Deleting A User
Appendix C USER REGISTRATION OF FV1000 /Deleting a User Appendix C-3 Deleting a User Double-click the [FLUOVIEW] icon on the desktop. The dialog box for entering the user name appears as shown below. [FLUOVIEW Setup] icon Enter “Administrator” in the [User Name:] text box and click the <OK> button. The [FLUOVIEW Setup] dialog box appears as shown below.
Page 462 Appendix C USER REGISTRATION OF FV1000 /Deleting a User Click the [Users] icon in [Hardware Settings] to display the [FLUOVIEW Users] panel at the front. [Users] icon <Add a user and Log in> button <Delete a user> button Used to add a new user. <Reset user to Factory Defaults>...
Page 463 Appendix C USER REGISTRATION OF FV1000 /Deleting a User Click the <Yes> button if you want to delete the user or the <No> button if you do not. The deleted user name disappears from the list. NOTE The user is deleted at the moment the <OK> button is clicked. Click the <Save New Setup>...
Page 465: Appendix D Change Of Default Folder For [File I/O] Panel
Appendix D Change of Default Folder for [File I/O] Panel FLUOVIEW FV1000 usually opens the default folder for the [File I/O] panel (C: FLUOVIEW IMAGES) to save acquired images or load saved images. The default folder for saving an image can be changed or a desired folder can be specified directly when loading a saved image.
Page 466 Appendix D Change of Default Folder for [File I/O] Panel Click the [Start] button to open the [Start] menu. Then select commands [Settings] - [Control Panel] - [System]. The [System Properties] dialog box appears as shown below. Select the [Advanced] sub-panel. OPERATION INSTRUCTIONS Page D - 2...
Page 467 Appendix D Change of Default Folder for [File I/O] Panel Select the <Environment Variables> button. The dialog box appears as shown below. User name logging in Windows XP or Windows 2000. OPERATION INSTRUCTIONS Page D - 3...
Page 468 Appendix D Change of Default Folder for [File I/O] Panel Select [FLUOVIEWIMAGES] under [Variable] in the [User variables for Administrator:] list box, and select <Edit> button. “Administrator” of [User Variables for Administrator:] is variable depending on the user name logging in Windows XP or Windows 2000. In the [Variable value:] text box, enter the path name of the default folder to be changed by delimiting the drive and folder names using backslashes ( ).
Page 469 Appendix D Change of Default Folder for [File I/O] Panel Click the <OK> button Change is reflected. <OK> button Saves the change and closes the dialog box. <Cancel> button Closes the dialog box without saving the change. OPERATION INSTRUCTIONS Page D - 5...
Page 470 Appendix D Change of Default Folder for [File I/O] Panel Click the <OK> button to close the [Environment Variables] dialog box. Now, the default folder for the [File I/O] panel will be changed from the next time you start FLUOVIEW. When the default folder is changed to C: IMAGES New default folder C IMAGES...
Page 471: Appendix E List Of Functions In The [Active Overlays] Dialog Box
Appendix E List of Functions in the [Active Overlays] Dialog Box /Coordinate Position Data Appendix E List of Functions in the [Active Overlays] Dialog Box Active Overlays is a kind of overlay function displayed on an image. Active Overlays does not simply show the entered characters, but searches the image data related to the keyword specified in <...
Page 472: Appendix E-1-2 Y-Coordinate
Appendix E List of Functions in the [Active Overlays] Dialog Box /Coordinate Position Data Appendix E-1-2 Y-Coordinate The Y-coordinate position with respect to the top left corner of the screen is displayed. «Syntax» <y[ [hotspot] [raw/calibrated] value [units]]> Arguments inside [ ] can be omitted. If [raw/calibrated] is omitted, the same setting as when [calibrated] is specified will be applied.
Page 473: Appendix E-1-3 Other
Appendix E List of Functions in the [Active Overlays] Dialog Box /Coordinate Position Data Appendix E-1-3 Other Positions can also be displayed by entering the following keywords in the syntax in place of the X- and Y-coordinate positions described in sections J-1-1 and J-1-2. 1 Z Position With cross-section related images such as XYZ images, the Z position with respect to the first image can be displayed.
Page 474: Appendix E-2 Intensity Data
Appendix E List of Functions in the [Active Overlays] Dialog Box /Intensity Data Appendix E-2 Intensity Data The intensity value can be displayed. When images are overlapped, the intensity value of each image is accompanied with the channel number placed after it. «Syntax»...
Page 475: Appendix E-3 Other
Appendix E List of Functions in the [Active Overlays] Dialog Box Appendix E-3 Other The following image data can be displayed in addition to the X- and Y-coordinate positions and intensity value. Appendix E-3-1 Channel Number The channel number can be displayed. When images are overlapped, the displayed channel numbers are connected by the “+”...
Page 477: Appendix F Hand Switch And Microscope Frame Function Allocation
Appendix F Hand Switch and Microscope Frame Function Allocation/ Hand Switch Functions Appendix F Hand Switch and Microscope Frame Function Allocation When the U-HSTR2 hand switch, the BXBX61/IX81 microscope frames, and U-FH focus adjustment knob are shipped from the factory, the following functions have been allocated to their control buttons. These function allocations are valid only when the FLUOVIEW software is NOTE started.
Page 478 Appendix F Hand Switch and Microscope Frame Function Allocation/ Hand Switch Functions Appendix F-1-2 IX Button Function Reflected light shutter OPEN/CLOSE switching Not used Not used Filter wheel position down Filter wheel position up Condenser position down Condenser position up Cube position down Cube position up 1 –...
Page 479: Appendix F-2 Microscope Frame Functions
Appendix F Hand Switch and Microscope Frame Function Allocation/ Microscope Frame Functions Appendix F-2 Microscope Frame Functions Appendix F-2-1 BX Left side Fig. Appendix F-2 BX Microscope Frame Button Function Transmitted light voltage up Transmitted light voltage down Z stage moves far from an objective lens. Z stage nears to an objective lens.
Page 480 Appendix F Hand Switch and Microscope Frame Function Allocation/ Microscope Frame Functions Appendix F-2-2 IX Front Left side Fig. Appendix F-3 IX81 Microscope Frame Button Function BI/Side or Back port light path switching Objective lens nears to Z stage. Objective lens moves far from Z stage. Not used Transmitted light voltage up Transmitted light voltage down...
Page 481: Appendix F-2-3 Focus Adjustment Knob
Appendix F Hand Switch and Microscope Frame Function Allocation/ Microscope Frame Functions Appendix F-2-3 Focus Adjustment Knob 1 BX Fig. Appendix F-4 Focus Adjustment knob(BX) Button Function Z stage moves far from an objective lens. Z stage nears to an objective lens. Stage escape/return switching Focus fine/coarse adjustment switching Not used...
Page 483: Maintenance
On This Volume This volume describes the user maintenance procedures of the FLUOVIEW FV1000 system. Please read this volume so that you can use the system for an extended period of time.
Page 485 CONTENTS 1 Software Setup 1-1 Re-setup or Updating of the Software ...........1-1 1-2 New Setup of the Software .............1-3 1-3 Setting the System Configuration..........1-5 1-3-1 Overall Setting of FLUOVIEW ............... 1-5 1-3-2 Setting the [Microscope Control Panel] ............1-15 1-4 Adding the dyeing method ............1-22 1-5 Adding the filters ................1-25 1-5-1 Adding Filters ....................
Page 487: Software Setup
Software Setup/ Re-setup or Updating of the Software 1 Software Setup The FLUOVIEW software has been set up before it is delivered to the user. A CD-ROM containing the software program is provided with FLUOVIEW. This CD-ROM is intended for use when re-setup is required due to a fault or when the user wants to set up the software newly.
Page 488 Software Setup/ Re-setup or Updating of the Software [Update FV Software] dialog box is displayed and starts the current settings. For save, carry out according to a wizard. Continuously, deletion of software is started. For deletion, carry out according to a wizard. Continuously, installation of the software of a new version is started.
Page 489: New Setup Of The Software
Software Setup/ New Setup of the Software 1-2 New Setup of the Software The following procedure allows you to set up the FLUOVIEW software in a computer you use. Load the FLUOVIEW setup CD-ROM in the CD-ROM drive. The dialog box as shown below will appear. If the dialog box doesn’t appear, run the 'Install.exe' file that is present in the root directory of the CD-ROM.
Page 490 Software Setup/ New Setup of the Software When the [Choose Destination Location] dialog box appears, confirm the setup destination drive name and directory and select the <Next> button. When the setup has completed, the [Setup Complete] dialog box appears. Select the [Yes, I want to restart my computer now.] option button and press the <Finish>...
Page 491: Setting The System Configuration
The [FLUOVIEW User Login] dialog box appears as shown below. [FLUOVIEW Setup] icon Enter the user name in the [User Name : ] text box and click the <OK> button to log into FLUOVIEW FV1000. Refer to Appendix H-2 “Logging into the FV1000” for details. MAINTENANCE...
Page 492 Software Setup/ Setting the System Configuration The dialog as shown below appears for use in saving the system configuration. First, select the type of microscope and set the extra (intermediate attachment) magnification to 1.0. [Excitation DM Turret] check box Check box appears when selecting “2CH”.
Page 493 Software Setup/ Setting the System Configuration Click the [Z Stage] icon in [User Settings] to display the [Z Stage] panel in the front position, and check and set the presence of motor controller, positioning of the motor controller, backlash and moving amount of the Z motor. Set the backlash of motor operation in the [Z Stage] sub-panel.
Page 494 Software Setup/ Setting the System Configuration Click the [Objectives] icon in [User Settings] to display the [Objectives] panel in the front position. In this panel, select the items to be included in the list of objectives used by the FLUOVIEW application. (Each user should set the objectives that the user wants to use with FLUOVIEW.) List of all objectives available at present...
Page 495 Software Setup/ Setting the System Configuration Click the [Fluorescence] icon in [User Settings] to display the [Fluorescent Deys/Colors] panel in the front position. In this panel, select the items to be included in the list of dyes used by the FLUOVIEW application.
Page 496 Software Setup/ Setting the System Configuration <To change the button settings> Press the button to indicate that you want to change the setting. When the dialog box as shown below appears, specify the button name and LUT file name and select the <OK>...
Page 497 Software Setup/ Setting the System Configuration Click the [Hardware] icon in [Hardware Settings] to display the [Hardware] panel in the front position. In this panel, enter the values for delay timing for TD unit and enter the maximum duration for continuous bi-directional scanning when setting fast scan mode. [Delay Timing for TD Unit] group box This data should usually be left in the default values and does not have to be changed.
Page 498 Software Setup/ Setting the System Configuration 10. Click the [Lasers] icon in [Hardware Settings] to display the [Lasers] panel in the front of position. Set the lasers to be used and set its default intensity. [Default Laser Intensity] scale Set the initial ND filter value at the start [Lasers Installed] check box of the FLUOVIEW application.
Page 499 Software Setup/ Setting the System Configuration 12. The [Registration] panel have been set at the factory and do not need to be changed here. 13. After completing the setup, select the <Save New Setup> button on the bottom left of the panel.
Page 500: Setting The [Microscope Control Panel]
Software Setup/ Setting the System Configuration 1-3-2 Setting the [Microscope Control Panel] The <Microscope Setting> button as shown below appears when selecting “IX71” and checking the [Automatic] check box or selecting “BX61, 62”, “BX61,62WI”, or “IX81” in the [Microscope] group box in the [Microscope] panel in the [FLUOVIEW Setup] dialog box. <Microscope Setup>...
Page 501 Software Setup/ Setting the System Configuration [Shutter] group box Sets up to use the shutter for transmitted observation. [Filter Turret] group box Sets up the name and color of the filter turret for transmitted observation. [Condenser Turret] group box Sets up the name and color of the universal condenser.
Page 502 Software Setup/ Setting the System Configuration The example of setting procedure in the [Microscope Setup] window. Changing the Number of Buttons Section 1-3-1-1 Setting the Button Section 1-3-1-2 Editing the name of the buttons in the [Mirror Unit], [Condenser Turret] and [Filter Turret] group boxes Setting the Name Editing in the [Nosepiece] group box •...
Page 503 Software Setup/ Setting the System Configuration 1-3-2-1 Setting the Number of Buttons Right-click the mouse on the area outside of the buttons in the group box where the number of buttons to be displayed is changed. The pop-up menu as shown below appears.
Page 504 Software Setup/ Setting the System Configuration Select “Edit”. The [Edit] dialog box as shown below appears. [Magnification] text box [Recommended Confocal Pinhole] text box Enter the magnification of Enter the number of the confocal pinhole with keyboard. the objective with keyboard.
Page 505 Software Setup/ Setting the System Configuration 2 Setting the color of the button engaged into the light path In [BX Control Panel] window on the software, specify the color of the button of the cube, the objective, and the condenser engaged into the light path. Right-click the mouse on the area outside of the buttons in the [Mirror Unit], [Nosepiece], [Condenser Turret], or [Filter Turret] group box.
Page 506 Software Setup/ Setting the System Configuration 1-3-2-4 Finishing the setting Finishing the setting and closing the [Microscope Setup] window. <Quit w/o Saving> button <Save New Setup> button Click the < Save New Setup> button to save the setup and close the window. Or click the <Quit w/o Saving>...
Page 507: Adding The Dyeing Method
The [FLUOVIEW User Login] dialog box as shown below appears. [FLUOVIEW Setup] icon Enter user name into the [User Name: ] text box and click the <OK> button to log into FLUOVIEW FV1000. When using the system for one user, the [FLUOVIEW User Login] dialog box does not appear.
Page 508 Software Setup/ Adding the dyeing method Display the [Fluorescent Dyes/Colors] panel at the front position in the [FLUOVIEW Setup] dialog box. Double-click here to add the dyeing method. Double-click the “Double click here to make a new Dye” in the [Dyes on your Samples] list.
Page 509 Software Setup/ Adding the dyeing method Enter the value of the excitation wavelength into the [WaveLength 1] text box in the [Excitation Wave Length] group box (e.g. 442). Enter the value of the emission wavelength into the [WaveLength 1] text box in the [Emission Wave Length] group box (e.g.
Page 510: Adding The Filters
End the setup. 1-5-1 Adding Filters For the method of adding filters to the scanning unit, please consult your local Olympus representative for assistance. In this example, let us add “DM440” to excitation dichroic mirror turret No. 5 and ”BA465- 495”...
Page 511: Setting The Scanning Unit
[FLUOVIEW Setup] icon Enter “Administrator” in the [User Name:] text box and click the <OK> button to log in the FLUOVIEW FV1000. The [FLUOVIEW User Login] dialog box is not displayed if the system is used in a single-user configuration.
Page 512 Software Setup/ Adding the filters In the fifth field from the top of [Excitation DM] in the [DMs & Filters in SU] dialog box, select the excitation dichroic mirror name (DM440 in this example) from the drop-down list. The excitation diachronic mirror name (DM440 in this example) can also be typed in from the keyboard.
Page 513: Adding A Dyeing Method
Enter the user name in the [User Name:] text box and click the <OK> button to log in the FLUOVIEW FV1000. The [FLUOVIEW User Login] dialog box is not displayed if the system is used in a single-user configuration.
Page 514 Software Setup/ Adding the filters In the [Dyes on your Samples] list, double-click “Double click here to make a new Dye”. The [Edit Dye] dialog box appears. [Excitation Wave Length] group box Enter the excitation [Name of the Dye] text box wavelength.
Page 515: Associating A Detection Channel And Filter To The Dyeing Method
Software Setup/ Adding the filters 1-5-4 Associating a Detection Channel and Filter to the Dyeing Method The detection channel and filter can be associated to a dyeing method using the [Optical System Configuration Setup] dialog box. Display [FLUOVIEW Setup] dialog. See section 1-3-1 of this manual to display [FLUOVIEW Setup] dialog.
Page 516 Software Setup/ Adding the filters Click [Edit Dye Assign Database] button. [Optical System Configuration Setup] dialog box appears as shown below. [Beam Splitter] drop-down list [Barrier Filter] drop-down list Select the beam splitter for each channel. Select the barrier filter for each channel. Select the blank when the selection is not Select the blank when the selection is not required.
Page 517 Software Setup/ Adding the filters Associations between dyeing methods and filters are saved per dyeing method combination, and applied when the combination is selected in the [Dyes] sub- panel of the FLUOVIEW software. To change the excitation dichroic mirror, use the [Excitation DM] drop-down list. When it is not required to specify excitation dichroic mirror, select the blank.
Page 518 Software Setup/ Adding the filters To change the dyeing method detection channel, set the [Dye] drop-down list of the previous channel to blank and select the dyeing method in the [Dye] drop-down list for the new channel.When it is not required to specify the detection channel, select the blank.
Page 519: Maintenance Of Major System Units
Maintenance of Major System Units /Laser Scanning Microscope 2 Maint enance of Major System Units This section describes the maintenance of the Microscope. 2-1 Laser Sca nning Microscope According to IEC60825 “Safety of Laser Product” and IEC60825(EN60825), this product is a CLASS 3B laser product.
Page 520: Uv-Ar Laser
Maintenance of Major System Units/ UV-Ar Laser The definitions of the term “maintenance” by CDRH 21CFR are quoted below for reference. CDRH “Maintenance” means performance of those adjustments or procedures specified in user information provided by the manufacturer with the laser product which are to be performed by the user for the purpose of assuring the intended performance of the product.
Page 521: Checking, Refilling And Replacing Water In Chiller
Maintenance of Major System Units/ UV-Ar Laser 2-2-2 Checking, R efilling and Replacing Water in Chiller Checking Chiller is a water-cooled system. After long hours of use, check the water level and, if water is insufficient, refill distilled water to the specified level. If the water in the tank is polluted, change the distilled water.
Page 522 Maintenance of Major System Units/ UV-Ar Laser Remove the water tank cover and check the level and pollution of water in it. Water tank Distilled water Specified level MAINTENANCE Page 2 - 4...
Page 523 Maintenance of Major System Units/ UV-Ar Laser Refill and Replacement If the water level in the water tank is below the specified level, refill distilled water until the specified level. If the water is polluted by algae or moth, replace the water to ensure cooling. Replacement procedure Set [COOL DOWN CYCLE] to “O-OFF”.
Page 525: Setting The Confocal Aperture
Setting the Confocal Aperture 3 Setting the Confocal Aperture The confocal aperture position is determined by the combination of the excitation dichroic mirror (hereinafter “excitation DM”) and spectral beam splitter (hereinafter “spectral BS”). If the confocal aperture position has not be factory set for the combination of excitation DM and spectral BS that you selected, it may become impossible to acquire correct images.
Page 526: Starting The Fluoview Software
Setting the Confocal Aperture/ Starting the FLUOVIEW Software 3-1 Starting the FLUOVIEW Software Start the FLUOVIEW software. For details, refer to Sections 1-1, “Turning the power On” and 1-2, “Starting Software” Volume [PREPARATION OPERATION] FV1000 User’s Manual. [HARDWARE GUIDE] of the enable saving confocal...
Page 527: Displaying The [C.a. Adjustment] Dialog Box
Setting the Confocal Aperture/ Displaying the [C.A. Adjustment] Dialog Box 3-2 Displaying the [C.A. Adjustment] Dialog Box Open the [C.A. Adjustment] dialog box of the FLUOVIEW software. Select the tab for the [Settings] subpanel from the bottom right of the [Acquire] panel. <Scan Unit>...
Page 528 Setting the Confocal Aperture/ Displaying the [C.A. Adjustment] Dialog Box Click the <Scan Unit> button. A window as shown below opens. Fig. 3-2 [Optical System Configuration] Window Click the <C.A. Adjustment> button. A dialog box as shown below opens. [Current] text box Adjust the confocal aperture position.
Page 529: Confocal Aperture Setting
Setting the Confocal Aperture/ Confocal Aperture Setting NOTE If the message below appears by clicking the <C.A. Adjustment> button, exit from FLUOVIEW software execute “FVCAReader.exe.” in the “FLUOVIEW” folder under drive C. 3-3 Confocal Aperture Setting Engage the 10X or 20X objective in the light path and place the mirror specimen on the stage.
Page 530 Setting the Confocal Aperture/ Confocal Aperture Setting Click the <SU Control> button. When the [Optical System Configuration] window is displayed, set the excitation DM and the spectral BS of the channel you want to set the confocal aperture. (For the display, see step 2.) NOTE Set the spectral BSs of the channels closer to the excitation DM than the adjusted channel (on the left side of the adjusted channel in...
Page 531 Setting the Confocal Aperture/ Confocal Aperture Setting Rotate the focus adjustment knob of the microscope to bring the mirror surface into focus. The focal position is the position where the image is bright and displays scratches on the mirror surface most clearly. While checking the repeated scanning image on the image window, adjust the confocal aperture to the position that provides the brightest image by using the arrow buttons on the center of the [Control] group box in the [C.A.
Page 532 Setting the Confocal Aperture/ Confocal Aperture Setting 14. Perform the confocal aperture adjustment in step 8. The correct confocal aperture position is the position with which the bright part comes on the center of the screen. The bright part may sometimes move across the stop edges of the image window during the focal position adjustment.
Page 533: Troubleshooting
On This Volume This volume describes the treatment against possible troubles. In case of trouble, please read volume before calling for service. If the normal operation cannot still be restored, please contact your local Olympus representative.
Page 535 CONTENTS 1 TROUBLESHOOTING GUIDE 1. Message – “Scanning Unit hardware not responding. Acquisition Not Enabled” appears................1-1 2. Message – “Microscope hardware not responding. Acquisition Not Enabled” appears................1-1 3. Message – “Scan hardware not responding. Initializing as FLUOVIEW Review Station” appears (In case that Review Station is used). .1-1 4.
Page 537 If you cannot solve the problem after checking the entire list, please contact your local Olympus representative for assistance. Before contacting Olympus, please fill the “Inquiry Table” at the end of this volume and inform Olympus of its contents. Phenomenon...
Page 538 TROUBLESHOOTING GUIDE Phenomenon Cause Treatment Manual Ref. Pages 9. FLUOVIEW software Something wrong with the 1.Terminate FLUOVIEW “Maintenance cannot be booted up. ‘ini’ file. software and FLUOVIEW Section 1-3” Setup. 2.Delete [GBSTATE.ini] file in [FLUOVIEW System] folder in HDD where FLUOVIEW soft ware is installed, Then set it up again by FLUOVIEW Setup...
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Olympus FLUOVIEW FV1000 User Manual

Fluoview Manual Configuration

Getting Started Fluoview 1-1

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