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Olimpus FLUOVIEW FV500 User Manual

Confocal laser scanning biological microscope
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User's Manual
FLUOVIEW
FV500
CONFOCAL LASER SCANNING
BIOLOGICAL MICROSCOPE
Ver.3.3
Petition
This user's manual is for the software to be run on Olympus FLUOVIEW FV500 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.
SYSTEM OVERVIEW
This user's manual is composed of two volumes including "
", "OPERATION
INSTRUCTIONS", "MAINTENANCE" and "TROUBLESHOOTING". Together with this manual, please
also read the "User's manual FLUOVIEW FV500" and the instruction manual of the microscope in order
to understand overall operation methods. To ensure the safety operation of laser system, we
recommend you to study the manual of each laser and the light source equipment besides this manual.
Retain this manual in an easily accessible place near a system for future reference.
AX9787

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Summary of Contents for Olimpus FLUOVIEW FV500

  • Page 1 Ver.3.3 Petition This user’s manual is for the software to be run on Olympus FLUOVIEW FV500 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 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 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: •...
  • 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 On This Volume This volume describes the outline of the FLUOVIEW FV500 system. Please read this volume so that you can understand the system before use.

  • Page 9: Table Of Contents

    CONTENTS 1 SYSTEM OVERVIEW 1-1 Principle ....................1-1 1-2 Features of FLUOVIEW FV500 ............1-2 1-3 Light Path Diagram ................1-3 1-4 System Configuration ..............1-4 1-4-1 System Component Units and Their Functions ........... 1-4 1-4-2 Block Diagram..................... 1-6 1-5 Software Functional Configuration ..........1-9 1-5-1 Function Panel and Display Panel...............

  • Page 11: System Overview

    Principle 1 SYSTEM OVERVIEW Olympus FLUOVIEW FV500 is a confocal scanning type laser reflected fluorescence microscope which uses confocal optics for high resolution, high contrast and drastically improved resolution in the light axis direction. It offers researchers features for sectioning, 3D construction and time-series observation as well as a variety of image processing and analysis functions.

  • Page 12: Features Of Fluoview Fv500

    SYSTEM OVERVIEW /Features of FLUOVIEW FV500 1-2 Features of FLUOVIEW FV500 1. The detector has 12-bit resolution and is capable of detecting very small changes in fluorescence inside cells. 2. The system has high resolution of 2048 x 2048 pixels. The output video signal is non-interlaced for clear images without flickering.

  • Page 13: Light Path Diagram

    SYSTEM OVERVIEW/ Light Path Diagram 1-3 Light Path Diagram Pinholes 1 to 4 Emission DM turrets 1 to 3 Confocal lenses 1 to 4 Emission filter turret To IR laser unit Mirror To UV laser unit To VIS laser combiner X/Y galvano Excitation Mirrors...

  • Page 14: System Configuration

    SYSTEM OVERVIEW /System Configuration 1-4 System Configuration 1-4-1 System Component Units and Their Functions Laser combiner Scan unit Unit for merging laser beams from the lasers Heart of a laser microscope, composed Transmitted light detector such as an Ar laser, Kr laser and HeNe laser of the light scanner and light detector.
  • Page 15 SYSTEM OVERVIEW/ System Configuration Microscope Upright microscope exclusively designed for FLUOVIEW FV500. Photo 1-2 Combination with IX Page...

  • Page 16: Block Diagram

    SYSTEM OVERVIEW /System Configuration 1-4-2 Block Diagram (6) 2.9m MONITOR Dsub15pin Dsub9pin (11) Dsub9pin U-MCB 50pin Dsub15pin (combination COMPUTER (5) 2.9m (7) 2.9m with AX only) (11) Dsub15pin Dsub15pin 50pin (7) 2.9m Dsub15pin BX-UCB FV5-PSU Dsub9pin (combination with Dsub25pin (4) 2.9m BX51,61 only) U-PS POWER...
  • Page 17 SYSTEM OVERVIEW/ System Configuration The block diagram in AOTF (FV-COMBA) use. (6) 2.9m MONITOR (11) Dsub9pin Dsub15pin 50pin Dsub9pin U-MCB 50pin Dsub15pin (combination COMPUTER (5) 2.9m (7) 2.9m with AX only) (11) Dsub15pin Dsub15pin 50pin FV5-LCU (7) 2.9m Dsub15pin BX-UCB Dsub9pin FV5-PSU (combination with...
  • Page 18 SYSTEM OVERVIEW /System Configuration Either the Kr laser or HeNe-G laser can be used. (1) Control cable (2) Interlock cable (3) Optical fiber cable (4) Analog signal cable (5) I/O cable (6) Galvano control cable (7) Galvano X/Y cable (8) Power/control cable (9) Power cable (10) Laser power cable (11) RS-232C cable...

  • Page 19: Software Functional Configuration

    SYSTEM OVERVIEW/ Software Functional Configuration 1-5 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 20: Panel Structure Of The Software

    SYSTEM OVERVIEW /Software Functional Configuration 1-5-2 Panel Structure of the Software This software cannot show the indexes of all function panels at a glance, but uses scrolling to display the desired page tab. Please use the following list of the panels for reference in scrolling.

  • Page 21: Icons Executed By Dragging & Dropping

    SYSTEM OVERVIEW/ Software Functional Configuration 1-5-3 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 22: Identification Of Images Depending On The Observation Methods

    SYSTEM OVERVIEW /Software Functional Configuration 1-5-4 Identification of Images Depending on the Observation Methods On many occasions, FLUOVIEW displays Image Icon Significance image icons to allow identification of the XZ observation observation method used when each image is acquired. (See table on the left.) XZ observation, 2-channel mode When the [File I/O], [Tile], [Process], [Analyze] Xt observation...
  • Page 23 On This Volume This volume describes the operating procedures of the FLUOVIEW FV500 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 25 CONTENTS 1 Getting Started FLUOVIEW 1-1 Basic Operations................1-1 1-1-1 Microscope....................1-1 1-1-2 General Mouse Operation Procedures ............1-13 1-1-3 Names of Major Panel and Window Controls and Their Functions.... 1-14 1-2 Outline of LSM Observation Procedures ........1-16 1-2-1 Turning Power On ..................1-18 1-2-2 Starting the Software.................
  • Page 26 CONTENTS 1-2-8-10 Stopping Repeated Scanning ................ 1-54 1-2-9 Acquiring Image ..................1-55 1-2-10 Saving Image ..................1-55 1-2-11 Exiting from the Software ................ 1-57 1-2-12 Turning Power Off ................... 1-58 1-3 Online Help ..................1-60 1-3-1 Function Help .................... 1-60 1-3-2 Microscope Help..................1-61 1-3-2-1 Configuring the Microscope................
  • Page 27 CONTENTS 2-2-2-6 XYZT Observation Mode................. 2-54 2-2-3 Differences in Image Acquisition Method Between Fluorescent and Transmitted Images .................. 2-60 2-2-3-1 Monochrome Image ..................2-60 2-2-3-2 Dual-Fluorochrome Image ................2-63 2-2-3-3 Transmitted Image ..................2-66 2-2-4 Acquiring Multiple Images (XYZ, XYT or XYZT Observation)....2-70 2-2-5 High-Speed Image Acquisition ..............
  • Page 28 CONTENTS 2-4-2-2 LUT Graph Editing by Gamma Correction............. 2-139 2-4-3 Switching the Displayed Channels (Ch1 – Ch5) ........2-140 2-4-4 Displaying Images of Multiple Channels Simultaneously (Side By Side Views, Over And Under Views, Single View) ............2-141 2-4-4-1 Displaying Images Separately Per Channel (Side By Side Views, Over And Under Views).........................
  • Page 29 CONTENTS 2-5-3-7 Image OR Image ................... 2-181 2-5-3-8 Image XOR Image ..................2-182 2-6 Image Analysis ................2-183 2-6-1 Checking the Intensity of a Specific Part ..........2-184 2-6-1-1 Intensity Values on a Line (Line Profile) ............2-184 2-6-1-2 Intensity Values on a Planar Region (Bird’s Eye View) ......... 2-187 2-6-2 Checking the Intensity Distribution of a Specific Part ......
  • Page 30 CONTENTS 2-13-1 Displaying the Image Intensity............... 2-230 2-11-3 Displaying the X-coordinate/Y-coordinate of the Image......2-231 2-11-4 Drawing a Figure in Image ..............2-232 2-11-5 Drawing a Scale in Image..............2-233 2-11-6 Drawing an Arrow in Image ..............2-235 2-11-7 Drawing Color Bars in Image..............2-236 2-11-8 Deleting Comment.................
  • Page 31 CONTENTS Appendix D-1 Mirror Cube Data Input(CUBE)........D-2 Appendix D-2 Objective Data Input(OB.)..........D-3 Appendix D-3 Transmitted light DIC prism Setting (UCD)....D-5 Appendix D-4 Setting PC Communications ........D-7 Appendix E User Registration Appendix F Formatting of Magnetic Optical Disk Appendix G Converting Analysis Data into a Chart Using EXCEL Appendix H USER REGISTRATION OF FV500...
  • Page 32 CONTENTS Appendix J-3 Other ................J-5 Appendix J-3-1 Channel Number................J-5 Appendix J-3-2 Objective Power................J-5 Appendix J-3-3 Date of Image Capturing.............. J-5 Appendix J-3-4 Time of Image Capturing ............. J-5 Appendix J-3-5 Image File Name ................. J-5...

  • Page 33: 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. For detailed microscope operation procedures, refer to the instruction manuals of your microscope.
  • Page 34 Getting Started FLUOVIEW/ Basic Operations (1) Light path selector button Select the light path between the visual observation and laser microscopy. • Select the < > button for visual observation. • Select the <1> button for TV or photomicrography. • Select the <2>...
  • Page 35 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.) (5) Filters These filters are used to adjust the transmitted light.
  • Page 36 Getting Started FLUOVIEW/ Basic Operations Combination with AX70 or AX70WI (1) Light path selector button (3) Analyzer FV5-ANA (4) Transmitted light (2) Universal vertical DIC slider illuminator mirror FV5-DICT cube housing AX-URBC (6) Universal condenser U-UCDB (5) Filters (7) Hand switch Filter lever (ND) U-HSTR Filter levers (IF550, LBD)
  • Page 37 Getting Started FLUOVIEW/ Basic Operations (3) Analyzer FV5-ANA Polarizing plate for use in differential interference observation and polarized light observation. • Set to the pushed-in position to engage the FV5-ANA in the light path for visual transmitted light differential interference observation or transmitted polarized light observation.
  • Page 38 Getting Started FLUOVIEW/ Basic Operations (6) Universal condenser U-UCDB Condenser for transmitted lighting. In addition, the rotary turret for the transmitted light DIC prism and the polarizing plate for differential interference observation (polarizer) are also provided. • To perform differential interference observation, engage the transmitted light DIC prism matching the objective in use in the light path.
  • Page 39 Getting Started FLUOVIEW/ Basic Operations Combination with IX (6) Filters (5) Condenser (4) Transmitted light DIC slider (2) Cube turret FV5-DICT (optional) Left side view (1) Light path selector (7) Intermediate magnification knob (3) Analyzer FV5-ANI Page...
  • Page 40 Getting Started FLUOVIEW/ Basic Operations (1) Light path selector Select the light path between laser microscopic observation, direct observation and photography observation. • Refer to the following table and set the dial switch to the position corresponding to the required light path. <<Light intensity of each light path>>...
  • Page 41 Getting Started FLUOVIEW/ Basic Operations NOTE With transmitted light differential interference observation using an immersion objective, set the microscope’s field diaphragm so that it inscribes the field of view. Otherwise the contrast may degrade. (This applies to both visual observation and laser differential interference observation.) (5) Condenser, polarizing plate Condenser for transmitted lighting.
  • Page 42 Getting Started FLUOVIEW/ Basic Operations Combination of IXBP (Bottom port) (6) Filters (5) Condenser (4) Transmitted light DIC slider FV5-DICT (optional) Left side view (1) Light path selector (2) Cube turret (3) Analyzer FV5-ANI (1) Light path selector knob Switch the light path between laser microscope observation, visual observation and side port.
  • Page 43 Getting Started FLUOVIEW/ Basic Operations (2) Cube turret Switch the cubes for fluorescence observation by rotating this turret. • For visual fluorescence observation, engage the specified cube in the light path. • For laser microscopy or for visual transmitted light observation, rotate the turret so that the index indicates .
  • Page 44 Getting Started FLUOVIEW/ Basic Operations (5) Condenser and polarizer Condenser for transmitted light. The rotary turret for the transmitted light DIC slider and the polarizer for DIC observation are also provided here. • For DIC observation, engage the transmitted light DIC slider (optional) matching the objective in use in the light path.

  • Page 45: 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 46: 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 by taking the [Acquire] panel and [Microscope Configuration] window as examples.
  • Page 47 Getting Started FLUOVIEW/ Basic Operations Check box Clicking this box enables or disables the indicated item. The item is enabled when the check box is checked (x). Title bar Shows the title of the window. The title bar of a window that is active is displayed in a different color from that of other windows.

  • Page 48: 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. • Turn the system power ON. Change the ND filter with a filter (Sec 1-2-1) with higher transmittance • Start the FLUOVIEW software.
  • Page 49 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures • Transmitted light observation procedure Start the system • Turn the system power ON. (Sec 1-2-1) • Start the FLUOVIEW software. Change the ND filter with a higher transmittance filter. -2-2) Sec 1 (Sec 1-2-7) Set the observation condition.

  • Page 50: 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. As the computer, monitor, power unit and hard-copy unit (optional) are connected to the switched power outlet unit, their power is turned ON or OFF simultaneously according to the power ON/OFF of the computer.
  • Page 51 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 2.2. Kr laser: (1) Set the power switch to ON. (2) Turn the key to the ON position. (The laser fan will start.) (3) Turn the key further in the clockwise direction. (The key will return to the ON position.) After the key is set to ON, it takes tens of HeNe (Green/Red) laser supply seconds before the laser oscillation starts.
  • Page 52 Getting started FLUOVIEW/ Outline of LSM Observation Procedures (2) Make sure that the output control is set to the maximum position. (3) Warm up for about 30 minutes until the oscillation stabilizes. (4) Open the shutter lever of the laser tube. 2.5.
  • Page 53 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures • Turning the computer (PC) ON When the power outlet unit is used, turning the computer power ON also turns ON the power supplies to the microscope, monitor, power supply unit, hard-copy unit and transmitted light unit (when the transmitted light detector is used).

  • Page 54: Starting The Software

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-2 Starting the Software Before starting up this software, wait for more than 2 minutes after NOTE turning the microscope and power supply unit ON. When executing a function that does not need image acquisition such as analysis of image data previously saved in a file, it is enough to turn only the computer and hard-copy unit ON.
  • Page 55 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 3. The following window appears when the FLUOVIEW software starts. Fig. 1-3 Window at Start-up 1-23 Page...

  • Page 56: Focusing On The Specimen

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-3 Focusing on the Specimen 1-2-3-1 Combination with BX 1. From the page tabs on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. Fig. 1-4 [Optics] Sub-panel 2. Select the <BI> button in the [Light Path] group box. The <BI>...
  • Page 57 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures When the Z motor is in use, clear the check mark in the [Locked] check box in the [Z Stage] sub-panel in the [Acquire] panel (see section 2-2-1-4-7 of this volume), then focus on the specimen by operating the fine focus adjustment knob of the microscope.

  • Page 58: Combination With Ax

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-3-2 Combination with AX 1. From the page tabs on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. Fig. 1-5 [Optics] Sub-panel 2. Select the <BI> button in the [Light Path] group box. The <BI>...
  • Page 59 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures specimen float during oil-immersed NOTE observation. In this case, prepare an optional clip for immersion objective and attach it as shown on the left. NOTE To observe a TV image when the system is combined with a BX or AX microscope, click the <TV>...

  • Page 60: Combination With Ix

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-3-3 Combination with IX 1. Turn the light path selector (1) on the right side of the microscope to 2. Engage the optimum cube for specimen dye by operating the cube turret (2). 3.

  • Page 61: Combination Of Ixbp (Bottom Port)

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-3-4 Combination of IXBP (Bottom Port) Turn the light path selector (1) on the right side of microscope to From the index tabs on the bottom right of the [Acquire] panel, select the [Optics] sub-panel.
  • Page 62 Getting started FLUOVIEW/ Outline of LSM Observation Procedures Look into the eyepiece and bring the specimen into focus. Be sure to adjust the eyepiece diopter correctly (refer to the instruction manual for the IX50/70). When the Z-motor is in use, clear the check mark in the [Locked] check box in the [Z Stage] sub-panel in the [Acquire] panel (see section 2-2- 1-4-7 of this volume) before bringing the specimen into focus using the focus adjustment knobs of the microscope.

  • Page 63: Setting The Lsm Light Path

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-4 Setting the LSM Light Path 1-2-4-1 Combination with Upright Microscope (BX) From the page tabs on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. Fig. 1-6 [Optics] Sub-panel Select the <LSM>...
  • Page 64 Getting started FLUOVIEW/ Outline of LSM Observation Procedures When only fluorescence observation is required, disengage the U-DICTHR/WI- DICTHRA transmitted DIC (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 U- DICTHR/WI-DICTHRA and the optimum transmitted light DIC for the objective in the light path by operating the universal condenser (5).

  • Page 65: Combination With Inverted Microscope (Bx50Wi)

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-4-2 Combination with Inverted Microscope (BX50WI) From the page tabs on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. Fig. 1-7 [Optics] Sub-panel Select the <LSM> button in the [Light Path] group box. The <LSM>...
  • Page 66 Getting started FLUOVIEW/ Outline of LSM Observation Procedures (3) Analyzer FV5-ANU (optional) (2) Cube turret (4) Transmitted light DIC slider FV5-DICT (optional) (6) Universal condenser (5) Filters ND25 1-34 Page...

  • Page 67: Combination With Upright Microscope (Ax)

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-4-3 Combination with Upright Microscope (AX) From the page tabs on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. Fig. 1-8 [Optics] Sub-panel Select the <LSM> button in the [Light Path] group box. The <LSM>...
  • Page 68 Getting started FLUOVIEW/ Outline of LSM Observation Procedures (2) Analyzer FV5-ANA (3) Transmitted light DIC slider (1) Universal vertical FV5-DICT illuminator mirror cube housing AX-URBC (5)Universal condenser (4) Filters Filter lever (ND) Filter levers (IF550, LBD) Hand switch U-HSTR 1-36 Page...

  • Page 69: Combination With Upright Microscope (Ax70A)

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-4-4 Combination with Upright Microscope (AX70A) 1. From the page tabs on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. Fig. 1-9 [Optics] Sub-panel Select the <LSM> button in the [Light Path] group box. The <LSM>...
  • Page 70 Getting started FLUOVIEW/ Outline of LSM Observation Procedures (1) Analyzer FV5-ANA (2) Transmitted DIC slider FV5-DICT 1-38 Page...

  • Page 71: Combination With Inverted Microscope (Ix)

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-4-5 Combination with Inverted Microscope (IX) Turn the light path selector (1) to Set the intermediate magnification knob (7) to 1X. (The 1.5X position cannot be used.) Rotate the cube turret of the reflected light fluorescence unit to When the FV5-ANI analyzer (3) is in use, disengage it from the light path by setting the switch to the pulled-out position.
  • Page 72 Getting started FLUOVIEW/ Outline of LSM Observation Procedures (6) Filters (5) Condenser (4) Transmitted DIC slider FV5-DICT Left side view (1) Light path selector (2) Cube turret (3) Analyzer FV5-ANI 1-40 Page...

  • Page 73: Combination Of Ixbp (Bottom Port)

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-4-6 Combination of IXBP (Bottom Port) From the index tabs on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. Fig. 1-5 [Optics] Sub-panel Click the <LSM> button in the [Light Path] group box. The <LSM>...
  • Page 74 Getting started FLUOVIEW/ Outline of LSM Observation Procedures (6) Filter (5) Condenser (4) Transmitted light DIC slider FV5-DICT (optional) Left side view (1) Light path selector knob (2) Cube turret (3) Analyzer FV5-ANI 1-42 Page...

  • Page 75: Selecting The Dyeing Method

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-5 Selecting the Dyeing Method From the page tabs on the bottom right of the [Acquire] panel, select the [Dyes] sub-panel. Fig. 1-10 [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 76 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 77 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 78: Selecting The Filters

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-6 Selecting 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, see section 1-3-2-4, “Configuring the Filters” in this volume and follow instructions in the [Optical System Configuration] window. The following table shows the possible combinations of the barrier and excitation filters.
  • Page 79 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures Spectral Spectral Spectral Excitation Filter Barrier Filter 1 Barrier Filter 2 Barrier Filter 3 Barrier Filter 4 Laser Combination Filter 1 Filter 2 Filter 3 Ar,HeNe(G),HeNe(R (1)BS2/8 (1)Mirror (1)Mirror (1)BA505 (1)BA565 (1)BA660 (2)DM351/488 (2)Glass (2)Glass...

  • Page 80: Selecting The Nd Filters

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-7 Selecting the ND Filters • When the laser combiner is used 1. Display the [Acquire] panel. In the [Laser Intensity] group box of the [Lasers] sub-panel, set the ND filter scale of each laser according to the levels of specimen brightness, fluorescence crosstalk and fading.

  • Page 81: 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 82: Setting The Zoom Ratio To 1X

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-8-2 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”. 1-2-8-3 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 83: Setting The Xy Observation Mode

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures The focus mode makes it possible to increase the line skipped scan speed. From the page tabs on the bottom right of the [Acquire] panel, select the [Scan] sub-panel. Select either option button in the [Focus Mode] group box. [Focus Mode] group box [X2] option button Acquires image at twice the...

  • Page 84: Repeated Scanning Operation

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-8-6 Repeated Scanning Operation Select the <XY Repeat> button. The acquired image will be displayed in the [Live] panel. <XY Repeat> button Use the <FOCUS> button to acquire image at an even higher speed. If the specimen is already being scanned, stop scanning with the <STOP SCAN>...

  • Page 85: Setting The Area To Be Observed

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures Check the [Locked] check box in the [Z Stage] sub-panel. Do not turn the fine focus adjustment knob while the [Locked] check box is checked, for this may damage the Z motor. 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 86: Setting A Lower Scan Speed

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-8-9 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 87: Acquiring Image

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-9 Acquiring Image Select the <Once> button. The acquired image will be displayed in the [Live] panel. <Once> button Fig. 1-14 Image Acquired in the [Live] Panel 1-2-10 Saving Image Display the [File I/O] panel. 1-55 Page...
  • Page 88 Getting started FLUOVIEW/ Outline of LSM Observation Procedures 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 <Display channel switch> buttons the images is saved.

  • Page 89: Exiting From The Software

    Getting Started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-11 Exiting from the Software Click the <Exit> button in the toolbar at the bottom of the screen. This will allows you to exit the software. The [Shut Down FLUOVIEW?] dialog box as shown below appears.

  • Page 90: Turning Power Off

    Getting started FLUOVIEW/ Outline of LSM Observation Procedures 1-2-12 Turning Power Off 1. Turn off the power supply to the units (reflected light power supply, switched power outlet unit). 2. Turn the laser power OFF. • Ar laser: Turn the key to the OFF position, then set the power switch to OFF. (For details, refer to the instruction manual of your laser unit.) •...
  • Page 91 Getting Started FLUOVIEW/ Outline of LSM Observation Procedures • IR laser: Refer to the instruction manual of the IR laser (manufactured by MELLES GRIOT). • HeCd laser: Turn the <OFF> button of remote interface module OFF. Wait till “LASER OFF” message appears on digital display. (For about 5 minutes to cool it down.) Turn the key switch of remote interface module to the OFF position.

  • Page 92: 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 93: 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. 1-3-2 Microscope Help The microscope, scan unit and laser types can be set up from the FLUOVIEW software, by selecting the dyeing method and following the displayed guidance information.
  • Page 94 Getting started FLUOVIEW/ Online Help 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 changed filter. And the dyeing method is shown in the [Ch] group box.
  • Page 95 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 96: Configuring The Microscope

    Getting started FLUOVIEW/ Online Help 1-3-2-1 Configuring the Microscope Display the [Acquire] panel. [Scan]/[Z Stage]/[Time Series]/[Optics]/[Lasers] sub-panels These are used to set the information required for image acquisition. 1-64 Page...
  • Page 97 Getting Started FLUOVIEW/ Online Help From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. <Scope Control> button Displays useful information for the system setup. Fig. 1-18 [Optics] Sub-panel Select the <Scope Control> button at the bottom of the panel. The window as shown below will appear (in case of a combination with the IX).

  • Page 98: Configuring The Microscope (Combination With Bx51, Bx61)

    Getting started FLUOVIEW/ Online Help Select the <BI> button in the [Observation] group box, and select the microscopy from the option buttons below it. The points where system setting is to be changed <BI> button for microscope observation will blink in red. Change the system configuration (setup of cube turret, etc.) by following the guidance given by the red blinking light.
  • Page 99 Getting Started FLUOVIEW/ Online Help Select the <Scope Control> button on the bottom of the [Optics] sub-panel. The [BX Control] window as shown below appears. [Shutter] group box [EPI lamp] Clicking inside the box switches the EPI Indicates the EPI lamp. shutter to be closed/opened, [Filter Turret] group The shutter is opened.
  • Page 100 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 when the objective is selected and changed in the [Nosepiece] group box.
  • Page 101 Getting Started FLUOVIEW/ Online Help Selecting the <Save Setting> button displays the [Save As] dialog box as shown below. To change the save destination drive or directory, use the [Save in: ] drop-down list. Enter the setting file name into the [File name] text box. Select the <Save>...

  • Page 102: Configuring The Microscope (Ax70A)

    Getting started FLUOVIEW/ Online Help 1-3-2-3 Configuring the Microscope (AX70A) When using the combination with the AX70A, the microscope and scan unit can be configured from the FLUOVIEW software. Use the following procedure for this. 1. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Optics] sub-panel.
  • Page 103 Getting Started FLUOVIEW/ Online Help 2. Click the <Scope Control> button at the bottom of the panel. The window as shown below will appear. [LVT] group box [Cube] group box Switches the light path. Select the excitation filter from the drop-down list. Click <BI>...

  • Page 104: Configuring The Filters

    Getting started FLUOVIEW/ Online Help During image acquisition, the <XY Repeat> and <Once> buttons are set as follows provided that the setting is not altered by the operator. • [Observation mode] group box: The <DIC> button is selected. • [Cube] group box: Set to OUT.
  • Page 105 Getting Started FLUOVIEW/ Online Help [Laser Unit] group box Shows the type of laser to be used. (With the laser combiner operation, the laser 2. Click the <SU Control> button at the bottom of the panel. The window as type is set and displayed automatically.) When the <On>...

  • Page 106: 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. 1. Display the [Acquire] panel. [Ch] group box Sets whether the image of each channel is to be acquired or not, the PMT voltage, Gain and Offset.
  • Page 107 Getting Started FLUOVIEW/ Online Help 2. 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 108 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 109 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 111: 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 112 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 113: 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 114: 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 115: 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. The procedures for the subsequent operations such as image processing are not in question here. For the detailed operation method of each item in the procedure, see the section specified in parentheses (( )).
  • Page 116 APPLIED OPERATIONS /General Operation Procedure Example 3) To acquire an image and compared it with Example 4) To open an image in a file, improve its a 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 117: 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 Do not move FLOUVIEW FV500 Menu while acquiring an image. Page...

  • Page 118: 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 119: 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. <Focus> button <Once> button The repetition images Acquires an image in the currently can be acquired at a high speed. selected observation mode and display Use this button to find an optimum image.
  • Page 120 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. [Assign dyes manually] check box Checking this enables the manual Place the pointer on the setting. Dragging the dyeing method in icon displayed in the the list directly to the [Ch] group box [Selected Dyes], and the...
  • Page 121 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 122 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 123 APPLIED OPERATIONS /Image Acquisition From the panel page tabs shown on the bottom right of the [Acquire] panel, select the [Optics] sub-panel. <Scope Control> button Displays useful information for the system setup. Fig. 2-4 [Optics] Sub-panel Select the <Scope Control> button at the bottom of the panel. The window as shown below will appear (in case of a combination with the IX).
  • Page 124 APPLIED OPERATIONS /Image Acquisition Select the <BI> button in the [Observation] group box, and select the microscopy from the option buttons below it. The points where system setting is to be changed <BI> button for microscope observation will blink in red. Change the system configuration (setup of cube turret, etc.) by following the guidance given by the red blinking light.

  • Page 125: 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 [Optics] sub-panel.
  • Page 126 APPLIED OPERATIONS /Image Acquisition Click the <SU Control> 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 127: Setting The Nd Filters

    APPLIED OPERATIONS /Image Acquisition 2-2-1-3 Setting the ND Filters • When the laser combiner is used Display the [Acquire] panel. In the [Lasers] group box of the [Lasers] sub-panel, set the ND filter scale of each laser according to the levels of specimen brightness, fluorescence crosstalk and fading.

  • Page 128: 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 With a combination using a microscope other than the AX70A, the measurement results will be inaccurate if the objective magnification set here does not match the actual magnification of the objective in use.
  • Page 129 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 130 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 131 APPLIED OPERATIONS /Image Acquisition 5 Setting the XY Observation Mode In the [Scan 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>...
  • Page 132 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. Do not turn the fine focus adjustment knob while the [Locked] check box is checked, for this may damage the Z motor.
  • Page 133 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. The 4 buttons represent directions, and clicking a button moves the acquired Clicking a point in the scale area to...
  • Page 134 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 135 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 136: 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 137: 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 138 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 [Scan] sub-panel. [Filter] group box Select the accumulation mode. Two accumulation modes, [Kalman] and [Accumulate To Peak] are available.
  • Page 139 APPLIED OPERATIONS /Image Acquisition When [Accumulate To Peak] 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 FV300 system with AOTF (FV5-COMBA).

  • Page 140: Saving The Acquired Image In File

    APPLIED OPERATIONS /Image Acquisition Enter the accumulation count in the text box and select the [Line Kalman] option button displayed. Enter the accumulation count. [Line Kalman] option button The accumulation count can be set up a maximum of 63 times. When 0 is set as the number of times of accumulation, an ordinary image acquisition is to be performed.

  • Page 141: 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 142 APPLIED OPERATIONS /Image Acquisition Set the dyeing method. Set the range of the multiple sections (Section 2-2-1-1 in [OPERATION]) to be observed (the Z-direction scanning range), Configure the microscope and scan unit. (Section 2-2-2-1-1 in [OPERATION]) (Sections 2-2-1-1 & 2-2-1-2 in [OPERATION]) Set a lower scan speed.
  • Page 143 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 Stage] sub-panel.
  • Page 144 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 145 APPLIED OPERATIONS /Image Acquisition 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. The number of steps has already been set and displayed in the [Step Size] text box. This number can be changed using the <...

  • Page 146: 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 147 APPLIED OPERATIONS /Image Acquisition 1 Setting the observation mode In the [Scan 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.

  • Page 148: 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 149 APPLIED OPERATIONS /Image Acquisition Set the dyeing method. Set the range of the multiple sections (Section 2-2-1-1 in [OPERATION]) to be observed (the Z-direction scanning range), (Section 2-2-2-3-1 in [OPERATION]) Configure the microscope and scan unit. (Sections 2-2-1-1 & 2-2-1-2 in [OPERATION]) Set a lower scan speed.
  • Page 150 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 151 APPLIED OPERATIONS /Image Acquisition Check the [Locked] check box in the [Z Stage] sub-panel. Do not turn the fine focus adjustment knob while the [Locked] check box is checked, for this may damage the Z motor. 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>...
  • Page 152 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 153 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 154 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 155: 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 156 APPLIED OPERATIONS /Image Acquisition Set the dyeing method. (Section 2-2-1-1 in [OPERATION]) Configure the microscope and scan unit. (Sections 2-2-1-1 & 2-2-1-2 in [OPERATION]) Set the range of the cross-section to be observed (the Z-direction scanning range), (Section 2-2-2-4-1 in [OPERATION]) Set the objective magnification.
  • Page 157 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 158 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 159 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 [Step Size] text box by the system so that the depth scale of the acquired image is identical to the horizontal scale.
  • Page 160 APPLIED OPERATIONS /Image Acquisition 5 Appending image An additional image can be appended to an image acquired in the XYZ observation mode. 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 161: 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 162 APPLIED OPERATIONS /Image Acquisition 1 Setting the observation mode In the [Scan 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 163 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 164: 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 165 APPLIED OPERATIONS /Image Acquisition Set the dyeing method. (Section 2-2-1-1 in [OPERATION]) Set the range of the cross-section to Configure the microscope and scan unit. be observed (the Z-direction (Sections 2-2-1-1 & 2-2-1-2 in [OPERATION]) scanning range), (Section 2-2-2-6-1 in [OPERATION]) Set the objective magnification.
  • Page 166 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 167 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 168 APPLIED OPERATIONS /Image Acquisition 3 Setting the observation mode In the [Scan Mode] group box in the [Acquire] panel, select the [Surface] option button. 2. In the [Acquire] panel, select the XYZT observation mode option button. 4 Setting the interval time From the page tabs on the bottom right of the [Acquire] panel, select the [Time Series] sub-panel.
  • Page 169 APPLIED OPERATIONS /Image Acquisition 5 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. 6 Acquiring image Click the <XYZT> button in the [Acquire] panel. The acquired image will be displayed in the [Live] panel.

  • Page 170: Differences In Image Acquisition Method Between Fluorescent And Transmitted Images

    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 (Ch1/Ch2/Ch3/Ch4). The subsequent description deals with the differences in operation between the monochrome dyeing and dual-fluorochrome dyeing.
  • Page 171 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 172 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 1-3-2-4, “Configuring the Filters” and change by [Optical System Configuration] window. Make the channel ready for image acquisition.

  • Page 173: 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 174 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 175 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 1-3-2-4, “Configuring the Filters” and change by [Optical System Configuration] window. Make the channels ready for image acquisition.

  • Page 176: 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 177 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 178 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 1-3-2-4,”Configuring the Filters “ and change by [Optical system Configuration] window. Turn off the transmitted light bulb of the microscope.
  • Page 179 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 180: Acquiring Multiple Images (Xyz, Xyt Or Xyzt Observation)

    APPLIED OPERATIONS /Image Acquisition 2-2-4 Acquiring Multiple Images (XYZ, XYT or XYZT Observation) Acquisition of a large number of image slices takes a very long time. In such a case, it is possible to acquire the images of only the required areas in a shorter time by narrowing the observation range and scanning range.

  • Page 181: High-Speed Image Acquisition

    APPLIED OPERATIONS /Image Acquisition 2-2-5 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 182: Acquiring Finer Image (Sequential Scan)

    APPLIED OPERATIONS /Image Acquisition 2-2-6 Acquiring Finer Image (Sequential Scan) An image without containing superimposition of fluorescence can be obtained by acquiring the image slice of each laser excitation or channel sequentially. 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 183 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 Click to decrease the Shows the group to acquired group No.
  • Page 184 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.

  • Page 185: Image Acquisition Of Rectangular Position At Desired Angle

    APPLIED OPERATIONS /Image Acquisition 2-2-7 Image Acquisition of Rectangular Position at Desired Angle When the sample is not standing upright, the image of a rectangular position can be acquired by changing the angle. Acquire an image in the XY observation mode. See section 2-2-1, “Image Acquisition with XY observation for operation procedure.
  • Page 186 APPLIED OPERATIONS /Image Acquisition Change the inclination angle of the frame. Click the mouse right button inside the frame to display the pop-up menu, and select [Rotate] in it. The inclination angle of the frame can be varied according to the mouse pointer movement.

  • Page 187: Image Acquisition Of A Line At Desired Angle

    APPLIED OPERATIONS /Image Acquisition 2-2-8 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 188: Display The Change Of Image Intensity (Point Scan)

    APPLIED OPERATIONS /Image Acquisition The inclination angle of the line can be varied according to the mouse pointer movement. Place the moue pointer on an inclining position and click the mouse left button to fix the line inclination angle. Acquire the image with the buttons such as the <XYZ>, <XYT>, <XYZT> buttons. After acquiring the image, select <Normal>...
  • Page 189 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 190 APPLIED OPERATIONS /Image Acquisition The speed recommended for point scan are as follows. 2µs(The scroll bar indicates “Fast” position. It is useful for the specimen changes rapidly.), 10µs, 100µs(The scroll bar indicates “Slow” position. It is useful for the specimen changes slowly.) Set the time for measurement.
  • Page 191 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 192 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 [Scan Mode] group box in the [Acquire] panel to return from the image acquisition mode.

  • Page 193: Image Acquisition On Desired Line (Xz, Xt Or Xzt Observation)

    APPLIED OPERATIONS /Image Acquisition 2-2-10 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 it. Acquire an image in the XY observation mode. For detailed operation, see section 2-2-1, “Image Acquisition with XY observation”.
  • Page 194 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 195 APPLIED OPERATIONS /Image Acquisition 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” for details. Up to 8000 times, images can be acquired. Set the number of scans in the [N] text box in the [Time Series] sub-panel.

  • Page 196: Image Acquisition In The Laser Excitation Mode

    APPLIED OPERATIONS /Image Acquisition 2-2-11 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 197: 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 198 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 199 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 200 APPLIED OPERATIONS /Image Acquisition Select the <Make REX mask> button. The [REX Live] panel (the REX mask file) is made in the [Display] panel. <Make REX mask> button Make the REX mask file which masks the region outside of the specified region.
  • Page 201 APPLIED OPERATIONS /Image Acquisition In order to adjust the laser intensity for every region, right-click the mouse on the white region on the [Display] panel 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 202: Example Of Frap Experiment

    APPLIED OPERATIONS /Image Acquisition 2-2-11-2 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 203 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 2-2-11-1, “Making REX Mask File” for the procedure to make the REX mask file.
  • Page 204 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.
  • Page 205 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 206 APPLIED OPERATIONS /Image Acquisition 5 Setting the XYT Observation mode Confirm that [XY-Norm] is displayed in the [Scan Mode] group box in the [Acquire] panel. Select the [XYT] option button in the [Acquire] panel. 2-96 Page...
  • Page 207 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 208 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 209 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 210 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 211: 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. [Display] panel [Open] Displays the image. The image file To open a file, select the desired file in name is shown in the panel page tab.
  • Page 212 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 XZ observation acquiring them. XZ observation, 2-channel mode The icon of the selected image (image in the [Display] panel) is displayed in the frame at the Xt observation top of the function panel such as the [File I/O]...
  • Page 213 APPLIED OPERATIONS /Saving, Opening and Shredding Images <[Files] List Box> The enter view of this list box cannot be displayed. Scroll the entries when viewing this list box. Name: Image file names. Icon: Icons showing the image observation modes. Image sizes in the X-direction. Image sizes in the Y-direction.

  • Page 214: 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 [Display] panel showing the image to be saved at the front.
  • Page 215 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 216: 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 [Display] panel 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 <Display>...
  • Page 217 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 218: 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 [Display] panel 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 219 APPLIED OPERATIONS /Saving, Opening and Shredding Images Specify the area to be saved in the image in the [Display] panel. 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 220 APPLIED OPERATIONS /Saving, Opening and Shredding Images 12. A dialog box as shown below appears. Size of the specified area Fig. 2-33 [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 [Display] panel.

  • Page 221: 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 “2-8-2 Animation”...

  • Page 222: File Types Available For Save

    APPLIED OPERATIONS /Saving, Opening and Shredding Images Two saving methods can be selected. AVI compressed : Saves the AVI file being compressed. AVI Uncompressed : Saves the AVI file not being compressed. Enter the file name in the [File Name: ] text box. Click the <Save>...
  • Page 223 APPLIED OPERATIONS /Saving, Opening and Shredding Images In the file types above, characters inside ( ) indicate the extension when a file is saved. NOTE When saving a display, the file format can be selected from Single TIF(s) 8-bit *.tif), Single TIF(s) 24-bit (*.tif), Bitmap 8-bit (*.bmp) and Bitmap 24-bit (*.bmp).

  • Page 229: 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 230 APPLIED OPERATIONS /Saving, Opening and Shredding Images In the [Experiments in Memory] dialog box, select the file name of the image to be shredded and click the <Shredder> button. <Shredder> button The file can also be shredded by placing the mouse pointer on it and dragging it to the <Shredder>...

  • Page 231: 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 232 APPLIED OPERATIONS /Saving, Opening and Shredding Images Enter comment from the keyboard. NOTE During modification of previously entered comment, if it is required to restore the originally entered comment, click the <Restore Comments> button. However, once the <Save Comments To Image> button is pressed, the original comment cannot be restored.

  • Page 233: 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 234 APPLIED OPERATIONS /Saving, Opening and Shredding Images The [X Size], [Y Size] and [Z Size] text boxes can also be used to change the lengths per image pixel or the number of steps in the Z-direction. These values are used in the scale display and many other analysis operations. When opening and analyzing an image file creased with another application on FLUOVIEW, enter the lengths per image pixel and the number of steps in the Z-direction if these values are known, then click the...
  • Page 235 APPLIED OPERATIONS /Saving, Opening and Shredding Images Range of intensity values mapped for each channel. LUT status. Acquisition parameters. Version information. Comment. Changes contents of Image Field. Fig. 2-40 [Image Fields] Panel 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.
  • Page 236 APPLIED OPERATIONS /Saving, Opening and Shredding Images One Point! The [Image Comments] dialog box can also be displayed by a mouse operation. Display the image to be saved at the front of the [Display] panel, and right-click a point in the image. A pop-up menu as shown below is displayed.

  • Page 237: 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 [Display] panel of the image to be saved at the front position. Select the <Experiment List>...
  • Page 238 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.
  • Page 239 APPLIED OPERATIONS /Saving, Opening and Shredding Images Select the <Save> button. The [Save As ASCII Text] dialog box as shown below appears. Fig. 2-44 [Save As ASCII Text] dialog box In order to change the save destination drive or directory, use the [Save in: ] drop- down list.

  • Page 240: 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 [Display] panel of the image including the region to be saved at the front position.
  • Page 241 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 242: Reading The Region File

    APPLIED OPERATIONS /Saving, Opening and Shredding Images 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 243 APPLIED OPERATIONS /Saving, Opening and Shredding Images <Annotations/ROIs> button in the [Export to File] group box Saves the region into a file. <Annotations/ROIs> button in the [Import from File] group box Reads the file saving the region. Fig. 2-49 [Image Comments] dialog box Select the <Annotations/ROIs>...
  • Page 244 APPLIED OPERATIONS /Saving, Opening and Shredding Images Select the file to be read and click the <Open> button. When the image size of the file including the region to be read and that of the specified file differ, the [Careful] dialog box as shown below appears. Clicking the <OK>...

  • Page 245: Changing The Image Display Method

    APPLIED OPERATIONS /Changing the Image Display Method 2-4 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. 2-4-1 Displaying an Image in Simulated Colors Display the [Display] panel of the image to be colored at the front.
  • Page 246 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 [Display] panel, and right-click a point in the image. A pop-up menu as shown below is displayed.

  • Page 247: Editing The Lut (Look Up Table)

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-2 Editing the LUT (Look Up Table) An original LUT can be created by editing an existing LUT. 2-4-2-1 LUT Graph Editing According to Colors Display the [Display] panel of the image that you want to edit the LUT. Click the <LUT>...
  • Page 248 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 [Display] panel, and right-click a point in the image.

  • Page 249: Lut Graph Editing By Gamma Correction

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-2-2 LUT Graph Editing by Gamma Correction The intensity data of an image can be reallocated to make it easier to view. 1. Display the [Display] panel of the image to be subjected to LUT change. <LUT>...

  • Page 250: Switching The Displayed Channels (Ch1 - Ch5)

    APPLIED OPERATIONS /Changing the Image Display Method 6. 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. 2-4-3 Switching the Displayed Channels (Ch1 –...

  • Page 251: Displaying Images Of Multiple Channels Simultaneously

    APPLIED OPERATIONS /Changing the Image Display Method Display the [Display] panel 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 <Display channel switch> of the image. buttons Select the channels which should be displayed by pushing <Display channel switch>...

  • Page 252: Displaying Images Separately Per Channel

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-4-1 Displaying Images Separately Per Channel (Side By Side Views, Over And Under Views) Display the [Display] panel of one of the images to be displayed side by side at the front. Click the <Display switching> button at the top of the [Display] panel. The list of buttons as shown below appears.

  • Page 253: Displaying Merged Image Of Multiple Channels (Single View)

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-4-2 Displaying Merged Image of Multiple Channels (Single View) Display the [Display] panel of the merged image of multiple channels at the front. Click the <Display switching> button at the top of the [Display] panel. The list of <Display switching>...

  • Page 254: Changing The Number Of Divided Images

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-5 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. 2-4-5-1 Increasing the Number of Divided Images Display the [Display] panel of the image to be changed at the front.
  • Page 255 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 [Display] panel. Fig. 2-58 [Display] Panel After View Addition Up to 6 views can be displayed at once. 2-145 Page...

  • Page 256: Decreasing The Number Of Divided Images

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-5-2 Decreasing the Number of Divided Images Display the [Display] panel of the image to be changed at the front. Fig. 2-59 [Display] panel 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 257 APPLIED OPERATIONS /Changing the Image Display Method A view is removed from the [Display] panel. Fig 2-60 [Display] panel after View Removal 2-147 Page...

  • Page 258: Switching The Display Method Of Multiple Images

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-6 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 259: Displaying Multiple Image Slices Together

    APPLIED OPERATIONS /Changing the Image Display Method Click and hold the <Display> button for successive display. To stop it, click the <Display> button again. For frame-by-frame display, simply click the <Display> button. 2-4-7 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 260: Displaying Multiple Images Per Channel

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-7-1 Displaying Multiple Images Per Channel Display the [Display] panel of one the images which are to be displayed together. The icon of the image is displayed in the frame at the top left of the [Tile] panel and the acquisition parameters used in image acquisition are displayed in the [Experiment] panel.
  • Page 261 APPLIED OPERATIONS /Changing the Image Display Method Fig. 2-62 Panel Displaying Images Per Channel 2-151 Page...

  • Page 262: Displaying Images Of Two Channels Together

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-7-2 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 263: Displaying Time-Lapse Images

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-7-3 Displaying Time-Lapse Images Multiple images acquired over time can be displayed side by side for simultaneous view. Display the [Display] panel of one of the time-lapse images to be displayed together. Set the number of images to be displayed together by using the < >...

  • Page 264: Displaying Multiple Multiple Sections Images

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-7-4 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. See section 2-4-7-3, “Displaying Time-Lapse Images”. Select [Z] from the [Tile Over] drop-down list.
  • Page 265 APPLIED OPERATIONS /Changing the Image Display Method Click the <New Page> button. A new [Display] panel appears showing the same images as the displayed image. The number of the displayed images is as set in step 2 above. NOTE Use the <Retile> button when it is required to re-arrange the images in the currently displayed [Display] panel.

  • Page 266: Re-Arranging Images Using The Same Display Method

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-7-6 Re-arranging Images Using the Same Display Method All of the images displayed together in a [Display] panel 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 267 APPLIED OPERATIONS /Changing the Image Display Method Click the <Experiment List> button in the toolbar at the bottom of the [Tile] panel. The [Experiments in Memory] dialog box appears as shown below. <Experiment List> button Fig. 2-67 [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 268 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 [Display] panel 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 [Display] panel, and that of the file displayed in the frame at the top right is displayed on the lower part.

  • Page 269: Displaying An Image In Full Screen

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-8 Displaying an Image in Full Screen Only the image itself can be displayed to fill the screen by erasing all other display components such as the toolbar, panel and status bar. This feature is useful for taking pictures using an analog printer for creation of a slide.
  • Page 270 APPLIED OPERATIONS /Changing the Image Display Method One Point! The image can also be displayed full screen by the mouse operation on the image. Display the [Display] panel of the image to be displayed full screen, and click the mouse right button on the image. A pop-up menu as shown below appears.

  • Page 271: Magnifying/Reducing An Image

    APPLIED OPERATIONS /Changing the Image Display Method 2-4-9 Magnifying/Reducing an Image The image can be magnified or reduced using the buttons displayed at the top of the [Display] panel. Magnification or reduction up to 3:1 or 1:3 the original image is possible. Display the [Display] panel of the image to be magnified or reduced.

  • Page 272: Image Processing

    APPLIED OPERATIONS /Image Processing 2-5 Image Processing Images can be processed using the [Process] panel. Display the [Process] panel at the front. 2-5-1 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 273: Contour Enhancement

    APPLIED OPERATIONS /Image Processing 2-5-1-1 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 274 APPLIED OPERATIONS /Image Processing Fig. 2-72 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 275 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.

  • Page 276: 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 277: Image Sharpening

    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 2-5-1-1-1, “Laplacian filter”.

  • Page 278: Dic Correcting Dic Level Irregularities

    APPLIED OPERATIONS /Image Processing 2-5-1-4 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 279 APPLIED OPERATIONS /Image Processing Click the <DIC leveling> button. The [Level] panel is newly created in the [Display panel] and shows an image after the correction of the DIC level irregularities of the background. The background irregularities are solved. Fig. 2-74 [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 280: Contrast Conversion

    APPLIED OPERATIONS /Image Processing 2-5-2 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 281 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 buttons to a single channel. To select the target channel(s), use the <Display channel switch> buttons. The histogram of the selected channel(s) is displayed.
  • Page 282 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 283: Mathematical Operations Between Images

    APPLIED OPERATIONS /Image Processing 2-5-3 Mathematical Operations Between Images Arithmetic or logical operations can be applied between two different images or between an image and a constant. 2-5-3-1 Image Addition Addition of an image to an image (constant to an image) is possible as described below. Display the [Math] sub-panel of the [Process] panel.
  • Page 284 APPLIED OPERATIONS /Image Processing Click the <Experiment List> button in the toolbar at the bottom of the [Process] panel. The [Experiments in Memory] dialog box appears as shown below. <Experiment List> button Fig. 2-79 [Experiments in Memory] Dialog Box From the [Experiments in Memory] dialog box, select the file name of the first image and drag it to the frame at the top left of the [Process] panel.
  • Page 285 APPLIED OPERATIONS /Image Processing Enter the constant for use in operation in the [Scalar Value] text box in the [Scalar Operations] group box. (This step is required only for operation between an image and a constant.) To add an image to an image: Click the <Add 2 Images>...

  • Page 286: Image Subtraction

    APPLIED OPERATIONS /Image Processing 2-5-3-2 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 2-5-3-1, “Image Addtion”. To subtract an image from an image: Click the <Subtract 2 Images>...

  • Page 287: Not Image

    APPLIED OPERATIONS /Image Processing To divide an image by an image: Click the <Divide 2 Images> button in the [Multi Image Operations] group box. A <Divide 2 Images> new [Display] panel showing [Image/Image] in the page tab appears, displaying the button image obtained by the division operation.
  • Page 288 APPLIED OPERATIONS /Image Processing 3. Click the <NOT Image> button. A new [Display] panel showing [ImageNot] in the <NOT Image> page tab appears, showing the image obtained by the operation. button Fig. 2-82 [ImageNot] Panel 2-178 Page...

  • Page 289: Image And Image

    APPLIED OPERATIONS /Image Processing 2-5-3-6 Image AND Image Two different images can be ANDed. Display the [Logical] sub-panel of the [Process] panel at the front. Sets the second image of the operation. The icon of the image is Sets the image to be subjected displayed here.
  • Page 290 APPLIED OPERATIONS /Image Processing 3. From the [Experiments in Memory] dialog box, select the file name of the first image and drag it to the frame at the top left of the [Process] panel. The icon of the image is displayed in the frame at the top left of the [Process] panel.

  • Page 291: Image Or Image

    APPLIED OPERATIONS /Image Processing 6. Click the <Image AND Image> button. A new [Display] panel showing [Image AND <Image AND Image> Image] in the page tab appears, showing the image obtained by the operation. button Fig. 2-85 [Image AND Image] Panel 2-5-3-7 Image OR Image Two different images can be ORed.

  • Page 292: Image Xor Image

    APPLIED OPERATIONS /Image Processing 2-5-3-8 Image XOR Image Two different images can be XORed. The operation method is identical to the AND operation between two different images except for the following point. See section 2-5-3-6, “Image AND Image”. Click the <Image XOR Image> button. A new [Display] panel showing [Image XOR <Image XOR Image>...

  • Page 293: Image Analysis

    APPLIED OPERATIONS /Image Analysis 2-6 Image Analysis Images can be analyzed using the [Analyze] panel. Display the [Analyze] panel at the front. Displays the icon of the image being displayed (image to be subjected to <Begin Analysis> button analysis). Starts analysis. [Measurement Results] box Shows the measurement data of the specified line or region.

  • Page 294: Checking The Intensity Of A Specific Part

    APPLIED OPERATIONS/ Image Analysis 2-6-1 Checking the Intensity of a Specific Part 2-6-1-1 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 [Display] panel of the image to be subjected to the intensity checking at the front.
  • Page 295 APPLIED OPERATIONS /Image Analysis To specify a free line: On the image, drag the mouse pointer along the line <Free Line>button to be checked. The line is displayed on the image together with the handles on it. The intensity profile can be displayed Handle while the handles are displayed.
  • Page 296 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 2-14, “Changing the Chart Display Method”...

  • Page 297: Intensity Values On A Planar Region (Bird's Eye View)

    APPLIED OPERATIONS /Image Analysis 2-6-1-2 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 [Display] panel of the image to be subjected to the intensity checking at the front.
  • Page 298 APPLIED OPERATIONS/ Image Analysis Specify the region to be checked in the image in the [Display] panel. 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 299 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 2-11-6, 2-11-7, 2-11-8 and 2-11-9 in section 2-11, “Entering Comment in Image”.
  • Page 300 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 X/Y, Z or T Statistical channel data including;...
  • Page 301 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 302: Checking The Intensity Distribution Of A Specific Part

    APPLIED OPERATIONS/ Image Analysis 2-6-2 Checking the Intensity Distribution of a Specific Part 2-6-2-1 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 303: 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 Alt key is held depressed, the coordinates can be displayed.
  • Page 304 APPLIED OPERATIONS/ Image Analysis When a desired area is specified by dragging the left button of the mouse on the graph, the specified area can be magnified. When the right button of the mouse is dragged on the graph, the graph can be scrolled.

  • Page 305: Measuring A Part

    APPLIED OPERATIONS /Image Analysis 2-6-3 Measuring a Part 2-6-3-1 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...

  • Page 306: Measuring The Change In Mean Value Of Intensity

    APPLIED OPERATIONS/ Image Analysis 2-6-3-3 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. Display the [Series] sub-panel at the front. Display the [Display] panel of the image that you want to check the intensity at the front.
  • Page 307 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 <Set start position> button the <Set start position> and <set end position> buttons above the images.
  • Page 308 APPLIED OPERATIONS/ Image Analysis Click the <Annotate> button in the toolbar at the bottom of the [Analyze] panel. 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>...
  • Page 309 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 2-93 [Properties] Dialog Box 10. Select the desired color from the [Color Palette] list box. 11.
  • Page 310 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 311 APPLIED OPERATIONS /Image Analysis <Properties> button Displays the [Editing] dialog box for use in detailed setting of the chart or change of the chart display. See section 2-14, “Changing the Chart Display Method” for details. <Copy> button Copies the plotted image in the clipboard.

  • Page 312: Measuring The Change In Integrated Intensity

    APPLIED OPERATIONS/ Image Analysis 2-6-3-4 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 313 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 Alt key is held depressed, the coordinates can be displayed.

  • Page 314: Building An Image From A Different Viewpoint

    APPLIED OPERATIONS /Building an Image from a Different Viewpoint 2-7 Building an Image from a Different Viewpoint 2-7-1 Building Extended Focus Image from XYZ Image 2-7-1-1 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 315 APPLIED OPERATIONS /Building an Image from a Different Viewpoint (Image No. 0) (Image No. 1) (Image No.2) (Image No. 3) Fig. 2-97 Four Images Used in Building Extended-Focus Image Fig. 2-98 Panel Showing an Extended-Focus Image 2-205 Page...
  • Page 316 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 317: Turning Built Image Into Single Image

    APPLIED OPERATIONS /Building an Image from a Different Viewpoint 2-7-1-2 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 318 APPLIED OPERATIONS /Building an Image from a Different Viewpoint Fig. 2-100 Panel Showing Extended-Focus Image 2-208 Page...

  • Page 319: Turning Built Image Into Time Series Image

    APPLIED OPERATIONS /Building an Image from a Different Viewpoint 2-7-1-3 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 320 APPLIED OPERATIONS /Building an Image from a Different Viewpoint 1. Display the [Display] panel of the XYZ (multiple sections) image. 2. Click the <Make Multi Plane View> option button in the [Rendered View] panel. <Make Multi Plane View> The [3D-] panel is created to start the line image building. button Line image display in XZ-direction Display the image of the line...

  • Page 321: Viewing 3D Image

    APPLIED OPERATIONS /Viewing 3D Image 2-8 Viewing 3D Image Use the [Visualize] panel to view an image three-dimensionally. First display the [Visualize] panel. [Display] panel Shows the image. The file name of the [Begin Visualize] button image is shown in the page tab of the Starts building the images for 3D panel.

  • Page 322: Successive Display Of Images

    APPLIED OPERATIONS /Viewing 3D Image 2-8-1 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 [Display] panel as shown below. 1.

  • Page 323: 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 [Display] panel.

  • Page 324: Animation

    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. 2-8-2 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...
  • Page 325 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 326: Building Stereo 3D Images

    APPLIED OPERATIONS /Viewing 3D Image 2-8-3 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 327 APPLIED OPERATIONS /Viewing 3D Image Fig. 2-106 Panel Showing Stereo 3D Images 2-217 Page...

  • Page 328: Building A 3D Image To Be Viewed Through Color (Red/Green) Eyeglasses2-218

    APPLIED OPERATIONS /Viewing 3D Image 2-8-4 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 329 APPLIED OPERATIONS /Viewing 3D Image Fig. 2-107 Panel Showing 3D Image to be Viewed Through Color Eyeglasses 2-219 Page...

  • Page 330: Viewing Images Following The Progress Of Time

    APPLIED OPERATIONS /Viewing Images Following the Progress of Time 2-9 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. 2-9-1 Displaying Images Together The change over time can be viewed at a glance by displaying multiple image slices together.
  • Page 331 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 332 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 [Display] panel.

  • Page 333: Transferring Data To Another Application

    APPLIED OPERATIONS /Transferring Data to Another Application 2-10 Transferring Data to Another Application 2-10-1 Transferring Analysis Data to Another Application Analysis data can be transferred to Excel. 1. Display the [Analyze] panel and executes analysis. After it, display the [Enhanced Profile Plot], [Intensity Map], [Enhanced Histogram Plot], {Average Intensity Trace] or [Integrated Intensity Trace].
  • Page 334 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. 2-109 [Save As] Dialog Box 3.
  • Page 335 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. 2-111 Dialog Box When the File is Opened by Excel 2 / 3 8.

  • Page 336: 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]. 2-10-2 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 337: Transferring Image Data To Another Application (Microvoxel, Paint, Etc.)2-227

    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 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 338: Entering Comment In Image

    APPLIED OPERATIONS /Entering Comment in Image 2-11 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 at the bottom left of the screen. Click the <Annotate>...
  • Page 339 APPLIED OPERATIONS /Entering Comment in Image From the drop-down list in the dialog box, select one of the following labels. Z Position <z> Time Instant <t> Channel # <channel> Display Zoom <display zoom> Is bounded <bounds> Experiment Name <name> <display zoom> <z> <t> <animation> <channel> <stereo> <bounds> Desired characters can also be entered.

  • Page 340: Displaying The Image Intensity

    APPLIED OPERATIONS /Entering Comment in Image Select the character font and size using the [Font], [Font Style] and [Size] list boxes. Click the <OK> button to close the [Font] dialog box. Click the <Close> button to close the [Active Overlays] dialog box. Place and click the mouse pointer on the image position you want to enter characters.

  • Page 341: Displaying The X-Coordinate/Y-Coordinate Of The Image

    APPLIED OPERATIONS /Entering Comment in Image 2-11-3 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 [Display] panel of the image that you want to display the X-coordinate position or the Y-coordinate position at the front.

  • Page 342: Drawing A Figure In Image

    APPLIED OPERATIONS /Entering Comment in Image 2-11-4 Drawing a Figure in Image This facility is used to draw figures in the image. FLUOVIEW provides seven figure drawing modes. Display the [Display] panel 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 343: Drawing A Scale In Image

    APPLIED OPERATIONS /Entering Comment in Image 2-11-5 Drawing a Scale in Image A scale can be drawn between two points in an image. Display the [Display] panel 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 344 APPLIED OPERATIONS /Entering Comment in Image The size of characters in the scale can also be changed. The size of characters in the scale is determined by the size of characters written using the <Text> button. Perform the following operation before starting <Text>...

  • Page 345: Drawing An Arrow In Image

    APPLIED OPERATIONS /Entering Comment in Image 2-11-6 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 [Display] panel of the image you want to draw an arrow. Click the <Arrow>...

  • Page 346: Drawing Color Bars In Image

    APPLIED OPERATIONS /Entering Comment in Image 2-11-7 Drawing Color Bars in Image This facility is used to draw color bars in an image. Display the [Display] panel 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 <Color Bar>...

  • Page 347: Deleting Comment

    APPLIED OPERATIONS /Entering Comment in Image 2-11-8 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 348 APPLIED OPERATIONS /Entering Comment in Image A comment can also be moved by selecting it, placing the mouse pointer on it so that the mouse pointer turns into a cross (+), then dragging the mouse. In this case, the final positioning of the comment can be determined by placing the mouse pointer outside the areas enclosed by the handles and clicking the left button of the mouse.

  • Page 349: Changing The Comment Size

    APPLIED OPERATIONS /Entering Comment in Image 2-11-10 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. 2-118 appears. Select [Size] from the menu.

  • Page 350: Changing The Comment Color

    APPLIED OPERATIONS /Entering Comment in Image 2-11-11 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. 2-118 appears. Select [Properties] from the menu.
  • Page 351 APPLIED OPERATIONS /Entering Comment in Image Fig 2-120 [Properties] Dialog Box Set the font type and size using the drop-down lists and select the effect. 2-241 Page...

  • Page 352: Image Output At Printer

    APPLIED OPERATIONS /Image Output at Printer 2-12 Image Output at Printer Display the [Display] panel 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 353 APPLIED OPERATIONS /Image Output at Printer One Point! The [Print] dialog box can also be displayed by mouse operation on the image. Display the image to be output at the printer at the front of the [Display] panel, and click the right button of the mouse on the image. A pop-up menu as shown below appears.

  • Page 354: Merger/Extraction Of Image Channels

    APPLIED OPERATIONS /Merger/Extraction of Image Channels 2-13 Merger/Extraction of Image Channels 2-13-1 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 355: 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 [Display] panel. 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 356 APPLIED OPERATIONS /Merger/Extraction of Image Channels 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. 2-122 [Experiments in Memory] Dialog Box Click the <ADV>...
  • Page 357 APPLIED OPERATIONS /Merger/Extraction of Image Channels 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 of the [Experiment Editor] group box. The icon of the first image is displayed in the frame at the top left of the [Experiment Editor] group box.
  • Page 358 APPLIED OPERATIONS /Merger/Extraction of Image Channels Clicking a channel line in this area switches the channel between the selected and deselected status. NOTE Up to 6 channels can be selected together. To select another channel after having selected 6 channels, deselect the unnecessary channels before selecting required channels.

  • Page 359: Extracting Channels From Image

    APPLIED OPERATIONS /Merger/Extraction of Image Channels 2-13-3 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 360 APPLIED OPERATIONS /Merger/Extraction of Image Channels Click the <ADV> button in the [Experiments in Memory] dialog box. The [Experiment Editor] group box appears as shown below. <ADV> button Fig. 2-126 [Experiment Editor] Group Box Displayed by <ADV> Button The frame at the top left of the [Experiment Editor] group box shows the icon of the image file displayed at the front of the [Display] panel.
  • Page 361 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 of the [Experiment Editor] group box. The icon of the image is displayed in the frame at the top left of the [Experiment Editor] group box.
  • Page 362 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 [Experiment Editor] group box. A new [Display] panel showing [Extract] in the page tab appears and the image of the extracted channel(s) is displayed in the panel.

  • Page 363: Changing The Chart Display Method

    APPLIED OPERATIONS /Changing the Chart Display Method 2-14 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 364 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 365 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 366 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 367 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 368 APPLIED OPERATIONS /Changing the Chart Display Method [Legend] sub-panel Sets the items to be Used to set the chart legend display. displayed in the legend. Enables or disables the legend Usually set [Automatic]. display. Sets the legend display style. Sets the legend background This setting is effective when color.
  • Page 369 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 370 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 371: Series] Panel

    APPLIED OPERATIONS /Changing the Chart Display Method 2-14-2 [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 372 APPLIED OPERATIONS /Changing the Chart Display Method [Points] sub-panel Used to set the inflection points in the chart. Enables or disables the inflection point display. Sets the width of inflection points. Enables or disables the 3D Sets the height of inflection inflection point display.
  • Page 373 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 374: Pop-Up Menus

    APPLIED OPERATIONS /Pop-up Menus 2-15 Pop-up Menus Selection of function panels and display panels 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 function panel When the right button of mouse is clicked on the page tab of a function panel, a pop-up menu appears to allow selection of the function panel to be displayed at the front.
  • Page 375 APPLIED OPERATIONS /Pop-up Menus Pop-up menu of display panel When the right button of mouse is clicked on the page tab of a [Display] panel, a pop-up menu appears to allow selection of the [Display] panel to be displayed at the front. Click the mouse right button here.
  • Page 376 APPLIED OPERATIONS /Pop-up Menus 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. Fig. 2-129 Image Comment with Pop-up Menu Copies the comment.
  • Page 377 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 378 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 379: Appendix A List Of Tools

    Appendix A List of Tools /List of Tools Appendix A List of Tools Appendix A-1 List of Tools Appendix A-1-1 Toolbar The horizontal array of buttons at the bottom left of the panel form the toolbar. The most frequently-used FLUOVIEW functions are gathered here. <Help>...

  • Page 380: Appendix A-1-2 Tools At The Top Of Display Panel

    Appendix A List of Tools /List of Tools Appendix A-1-2 Tools at the Top of Display Panel These refer to the horizontal array of buttons at the top of the display panel. The functions frequently used with images are gathered here. <Set end position>...

  • Page 381: Appendix B List Of Hot Keys

    Appendix B List of Hot Keys Appendix B 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). Cttrl Switches the status of the Ch2 check box (Ch2 scanned/not scanned).
  • Page 382 Appendix B 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 383 Appendix B List of Hot Keys Target Operation Piezo: Returned to the 0.0 position. Home Moves the stage till the Stop Z position. Moves the stage till the Start Z position. Transmitted illumination lamp ON/OFF keys Target Operation Ctrl + Switches the transmitted illumination ON/OFF.
  • Page 384 Appendix B List of Hot Keys [Acquire] sub-panels Target Operation Alt + Selects the [Scan] sub-panel. Alt + Selects the [Z Stage] sub-panel. Alt + Selects the [Time Series] sub-panel. Selects the [Dyes] sub-panel. Alt + Selects the [Config] sub-panel. Alt + [Process] sub-panels Target Operation...

  • Page 385: Appendix C Glossary

    Appendix C Glossary Appendix C Glossary Command button A figure in the shape of button in the window. Clicking a command button with the mouse allows AOTF the function indicated on the button to be executed. AOTF represents Acoustic Optical Tunable Filter. AOTF is the sound optical element having optical Confocal anisotropy.
  • Page 386 Appendix C Glossary ¡ Pixel. Icon Double-click An icon is a small figure with characters below it. Action of pressing and releasing the mouse button The icon indicates the status in which the window is quickly twice, without moving the moue. closed (or minimized).
  • Page 387 Appendix C Glossary Mouse A device which was named because it looks like a Resolution mouse. It is used to give instructions in the window. Number of dots composing the image on a screen or printer. When the number of dots is increased, i.e. Mouse pointer when the resolution is increased, the gradation can When the mouse is moved on the desk, arrow...
  • Page 388 Appendix C Glossary Text box A box in the window that accepts the input of character strings. Clicking a text box displays blinking |. This “|” indicates the position where the input character is inserted. Title bar The horizontal bar at the top of the window, that shows the title of the window or dialog box.

  • Page 389: Appendix D U-Mcb Setting

    Appendix D U-MCB Setting Appendix D U-MCB Setting Please set U-MCB before using this system. NOTE Switch the display for setting of the multi control box to MAIN to operate it through the FLUOVIEW software after the setting has been completed.

  • Page 390: Appendix D-1 Mirror Cube Data Input(Cube

    Appendix D U-MCB Setting /Mirror Cube Data Input(CUBE) Appendix D-1 Mirror Cube Data Input(CUBE) Please register the fluorescence cubes for fluorescence observations. NOTE Please do not do full registration. Leave at least one position “NONE” for use in LSM observation. When the CUBE button on the INIT.

  • Page 391: Appendix D-2 Objective Data Input(Ob

    Appendix D U-MCB Setting /Objective Data Input(OB.) Appendix D-2 Objective Data Input(OB.) To display the OB. LENS DATA SET screen (Fig.D-7), press OB. (1) on the INIT. input screen (Fig.D-6). Fig. D-6 When the objective number (2) to be entered is pressed, the screen shown in FIg.D-8 will appear.
  • Page 392 Appendix D U-MCB Setting /Objective Data Input(OB.) The following table shows the objectives Objective PLAPO 40X PLAPO 60XO PLAPO 100XO PLAPO 40XWLSM PLAPO 60XWLSM PLAPO 60XOLSM UPLAPO 10X UPLAPO 20X UPLAPO 20XO UPLAPO 40X UPLAPO 40XOI UPLAPO 60X UPLAPO 60XW UPLAPO 60XWPSF UPLAPO 100XOI UPLFL10X...

  • Page 393: Appendix D-3 Transmitted Light Dic Prism Setting (Ucd

    Appendix D U-MCB Setting /Transmitted light DIC prism Setting (UCD) Appendix D-3 Transmitted light DIC prism Setting (UCD) Please register the DIC prism is suitable for a objective. NOTE In the case that you do not have the suitable prism for a objective lens, please register the prism temporarily.
  • Page 394 Appendix D U-MCB Setting /Transmitted light DIC prism Setting (UCD) The following table shows which DIC prism is suitable for a objective lens. Objective Transmitted light DIC prism PLAPO 60X0 ODPAO60 UPLAPO 10X DP10 UPLAPO 20X DPA20 UPLAPO 20X0 DPA20 UPLAPO 40X DPA40 UPLAPO 40XOI...

  • Page 395: Appendix D-4 Setting Pc Communications

    Appendix D U-MCB Setting /Setting PC Communications Appendix D-4 Setting PC Communications Set the RS232C communication DIP switch as follows. SW1: SW2: SW3: SW4: SW5: SW6: SW7: SW8: With the above settings, the communication rate is set to 9600 bps, data length to 7 bits, parity check to None, stop bit to 2 bits and delimiter code to “CR+LF”.

  • Page 397: Appendix E User Registration

    Appendix E User Registration Appendix E User Registration FLUOVIEW manages the system setting data on a per-user basis. Use the following procedure to register yourself as a user. Log in the system with Administrator. Register yourself as a new user. From the [Start] menu, select [Programs] - [Administrative Tools(Common)] - [User Manager].
  • Page 398 Appendix E User Registration Enter the user name in the [Username] text box. It is not permitted to enter an existing user name or the same user name as the group name. The user name can be a character string of up to 20 characters and can contain any uppercase or lowercase characters except for the following characters.
  • Page 399 Appendix F Formatting of Magnetic Optical Disk Appendix F Formatting of Magnetic Optical Disk Use the Disk Administrator tool of Windows for formatting a magnetic optical (MO) disk. Note that this tool can be used only by a person who have the authority of Administrator. Click the <start>...
  • Page 400 Appendix F Formatting of Magnetic Optical Disk From the [Partition] menu of the [Disk Administrator] window, select the [Create] command. The dialog box as shown below appears. Set the value of the [Create partition of size] edit box in the [Create Primary Partition] dialog box to “Maximum size”, and click the <OK>...
  • Page 401 Appendix F Formatting of Magnetic Optical Disk 10. From the [Tool] menu of the [Disk Administrator] window, select the [Format] command. The dialog box as shown below appears. Fig. Appendix F-2 [Format] Dialog Box 11. Select [Removable Media (Unknown Size)] from the [Capacity] drop-down list in the [Format] dialog box. Then select [FAT] from the [File System] drop-down list and select [FAT] and select [Default Allocation Unit Size] from the [Allocation Unit Size] drop-down list.

  • Page 403: Appendix G Converting Analysis Data Into A Chart Using Excel

    Appendix G Converting Analysis Data into a Chart Using EXCEL Appendix G Converting Analysis Data into a Chart Using EXCEL Start up Excel. From the [File] menu of Excel, select the [Open] command to open the analysis data file saved after analysis using FLUOVIEW. When the dialog box as shown below appears, click the [Delimited] option button in the [Original Data Type] group box, then select [Windows [ANSI]] from the [File Origin:] drop-down list.
  • Page 404 Appendix G Converting Analysis Data into a Chart Using EXCEL Click the <Next> button. When the dialog box as shown below appears, click the [General] option button in the [Columns Data Format] group box, then click the <Finish> button. Fig. Appendix G-3 Dialog Box Displayed When File is Opened with Excel (3/3) Drag the data to be converted into a chart.
  • Page 405 Appendix G Converting Analysis Data into a Chart Using EXCEL Click the <ChartWizard> button. <ChartWizard> button On the sheet, drag the area where the chart is to be inserted. The dialog box as shown below appears. Fig. Appendix G-4 Dialog Box of Chart Wizard (1/5) Click the <Next>...
  • Page 406 Appendix G Converting Analysis Data into a Chart Using EXCEL 11. Select the desired chart format and click the <Next> button. The dialog box as shown below appears. Fig. Appendix G-7 Dialog Box of Chart Wizard (4/5) 12. Click the [Columns] option button under [Data Series in:]. 13.
  • Page 407 Appendix G Converting Analysis Data into a Chart Using EXCEL 17. Click the <Finish> button. A chart will be drawn on the sheet. Fig. Appendix G-9 Sheet with Inserted Chart When the mouse is clicked on the chart, black handles will appear around the chart. The following operations are available in this condition.
  • Page 408 Appendix G Converting Analysis Data into a Chart Using EXCEL When the mouse is double-clicked on the chart, a frame composed of oblique lines appear around the chart. (The frame may become a window when the chart is large.) The format settings are available in this condition. 1.
  • Page 409 Appendix H USER REGISTRATION OF FV500 FLUOVIEW FV500 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 FV500 software.

  • Page 410: Appendix H-2 Logging Into The Fv500

    Appendix H USER REGISTRATION OF FV500 /Logging into the FV500 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 411: Appendix H-3 Deleting A User

    Appendix H USER REGISTRATION OF FV500 /Deleting a User Enter the user name in the [User Name:] text box and click the <OK> button. The FLUOVIEW software starts. If no user name has been registered, the [FLUOVIEW User Login] dialog box is not displayed, but the system is automatically started for the Administrator.
  • Page 412 Appendix H USER REGISTRATION OF FV500 /Deleting a User Display the [Users] panel at the front. <Add a user and Log in> button Used to add a new user. <Reset user to Factory Defaults> button <Delete a user> button Resets the system setups of the users selected in Deletes the user selected in the list box.
  • Page 413 Appendix H USER REGISTRATION OF FV500 /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 415: Appendix I Change Of Default Folder For [File I/O] Paneli-1

    Appendix I Change of Default Folder for [File I/O] Panel FLUOVIEW FV500 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 416 Appendix I 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. Page...
  • Page 417 Appendix I Change of Default Folder for [File I/O] Panel Select the [Environment] sub-panel. User name logging in Windows NT. Select [FLUOVIEWIMAGES] under [Variable] in the [User Variables for Administrator:] list box. “Administrator” of [User Variables for Administrator:] is variable depending on the user name logging in Windows NT.
  • Page 418 Appendix I Change of Default Folder for [File I/O] Panel In the [Value:] text box, enter the path name of the default folder to be changed by delimiting the drive and folder names using backslashes ( ). (The figure shows an example in which folder IMAGE in drive C is entered.) [Value:] text box Enter the path name of the default folder.
  • Page 419 Appendix I Change of Default Folder for [File I/O] Panel Click the <OK> button to close the [System Properties] 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 421: Appendix J List Of Functions In The [Active Overlays] Dialog Box

    Appendix J List of Functions in the [Active Overlays] Dialog Box /Coordinate Position Data Appendix J 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 422: Appendix J-1-2 Y-Coordinate

    Appendix J List of Functions in the [Active Overlays] Dialog Box /Coordinate Position Data Appendix J-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 423: Appendix J-1-3 Other

    Appendix J List of Functions in the [Active Overlays] Dialog Box /Coordinate Position Data Appendix J-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 424: Appendix J-2 Intensity Data

    Appendix J List of Functions in the [Active Overlays] Dialog Box /Intensity Data Appendix J-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 425: Appendix J-3 Other

    Appendix J List of Functions in the [Active Overlays] Dialog Box Appendix J-3 Other The following image data can be displayed in addition to the X- and Y-coordinate positions and intensity value. Appendix J-3-1 Channel Number The channel number can be displayed. When images are overlapped, the displayed channel numbers are connected by the “+”...
  • Page 427 On This Volume This volume describes the user maintenance procedures of the FLUOVIEW FV500 system. Please read this volume so that you can use the system for an extended period of time.
  • Page 429 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 ..........1-6 1-3-1 Overall Setting of FLUOVIEW ..............1-6 1-3-2 Setting the [BX Control Panel]..............1-14 1-4 Adding the dyeing method ............1-20 2 Maintenance of Major System Units 2-1 Laser Scanning Microscope ............2-1 2-2 UV-Ar Laser..................2-2...

  • Page 431: 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 432 Software Setup/ Re-setup or Updating of the Software NOTE Current settings are performed after <Start Update> button selection. Be sure to save the current settings before proceeding to the removal of the software. If the software is removed without saving the current settings, the settings in the software program obtained by re-setup will be reset to the initial settings.

  • Page 433: 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. Select the [Start] button to display the [Start] menu, then select commands [Settings] - [Control Panel] - [Add/Remove Programs].
  • Page 434 Software Setup/ New Setup of the Software When the [Run Installation Program] dialog box appears, ensure that the “<CD- ROM drive name> SETUP.EXE” is displayed correctly and select the <Finish> button. When the [Choose Destination Location] dialog box appears, confirm the setup destination drive name and directory and select the <Next>...
  • Page 435 Software Setup/ New Setup of the Software The [Start Copying Files] dialog box appears. Confirm the installation destination directory and select the <Next> button. This will start copying of files. 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 436: Setting The System Configuration

    [FLUOVIEW Setup] icon Enter the user name in the [User Name : ] text box and click the <OK> button to log into FLUOVIEW FV500. Refer to Appendix H-2 “Logging into the FV500” for details. The dialog as shown below appears for use in saving the system configuration.
  • Page 437 Software Setup/ Setting the System Configuration 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 438 Software Setup/ Setting the System Configuration 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.) <To delete an unnecessary objective from the list>...
  • Page 439 Software Setup/ Setting the System Configuration Display the [Fluorescence] panel in the front position. In this panel, select the items to be included in the list of dyes used by the FLUOVIEW application. (Each user should set the dyes that the user wants to use with FLUOVIEW.) List of dyes that can List of all dyes...
  • Page 440 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 441 Software Setup/ Setting the System Configuration Display the [Hardware] panel in the front position. In this panel, select Enabled/Disabled to enable/disable the automatic black-level adjustment, 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 442 Software Setup/ Setting the System Configuration 10. Display the [Lasers] panel in the front of position. Set the lasers to be used with the FLUOVIEW application and the initial ND filter values here. [Laser Install] check box Select and check the lasers to be used with the FLUOVIEW application.
  • Page 443 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. (Selecting <Quit w/o Saving>...

  • Page 444: Setting The [Bx Control Panel]

    Software Setup/ Setting the System Configuration 1-3-2 Setting the [BX Control Panel] The <Microscope Setting> button as shown below appears when selecting “BX 51,52” or “BX51,52WI” and checking the [Automatic] check box or selecting “BX61,62” or “BX61,62WI” in the [Microscope] group box in the [Microscope] panel in the [FLUOVIEW Setup] dialog box.
  • Page 445 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 446 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 447 Software Setup/ Setting the System Configuration Select “Edit”. The [Edit] dialog box as shown below appears. [Recommended Confocal Pinhole] text box Enter the number of the confocal pinhole with keyboard. [Name Objective] drop-down list Select the name of the objective in the list or enter the name with keyboard.
  • Page 448 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 449 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 450: 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 FV500. When using the system for one user, the [FLUOVIEW User Login] dialog box does not appear.
  • Page 451 Software Setup/ Adding the dyeing method Display the [Fluorescence] 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 452 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 453: Maintenance Of Major System Units

    Maintenance of Major System Units /Laser Scanning Microscope 2 Maintenanc e of Major System Units This section describes the maintenance of the Microscope. 2-1 Laser Scanning Mi croscope According to IEC60825 “Safety of Laser Product” and IEC60825(EN60825), this product is a CLASS 3B laser product. Therefore, all of activities such as attachment or removal of the microscope modules listed below are defined as the service activities.

  • Page 454: 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 455: Checking, Refilling And Replacing Water In Chiller

    Maintenance of Major System Units/ UV-Ar Laser 2-2-2 Checking, Refilling an d 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 456 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 Page...
  • Page 457 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 459 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 461 CONTENTS 1 TROUBLESHOOTING GUIDE Fluorescence image cannot be observed ........1-1 Transmitted image cannot be observed........1-2 Image is disturbed................1-2 Image is striped................1-2 Image looks poor................1-2 Image is irregularly blurred or the brightness is uneven...1-3 Image is flared................1-3 Circular flare is observed at the center of image......1-3 Image is blurred or out of focus............1-3 10.

  • Page 463: Troubleshooting Guide

    TROUBLESHOOTING GUIDE 1 TROUBLESHOOTING GUIDE Under certain conditions, performance of this system may be adversely affected by factors other than defects. If a problem occurs, please review the following list and take remedial action as needed. If you cannot solve the problem after checking the entire list, please contact your local Olympus representative for assistance.

  • Page 464: Fluorescence Image Cannot Be Observed

    TROUBLESHOOTING GUIDE Phenomenon Cause Treatment Manual Ref. Pages 1. Fluorescence image The fluorescent dye and Select a laser suitable for the 1-2-7, OPERATION cannot be observed excitation wavelength does fluorescent dye method. INSTRUCTIONS not match. Change the ND filters by reading 1-2-7, “Selecting the ND Filters”...

  • Page 465: Image Is Irregularly Blurred Or The Brightness Is Uneven

    TROUBLESHOOTING GUIDE Phenomenon Cause Treatment Manual Ref. Pages 5. Image looks poor. The fluorescent dye does not Select the barrier filter 1-2-7, OPERATION match the barrier filter. matching the fluorescent INSTRUCTIONS dye. Change the ND filters by reading 1-2-7, “Selecting the ND Filter”...

  • Page 466: The [Acquire] Panel Cannot Be Displayed

    TROUBLESHOOTING GUIDE Phenomenon Cause Treatment Manual Ref. Pages 12. The [Acquire] panel The power unit is not Check the connection 1-2-1, OPERATION cannot be displayed. recognized. between the computer and INSTRUCTIONS power unit. Turn the power unit ON. If the combination with the Check the connections of the 1-2-1, OPERATION AX70A is used, the...

  • Page 467: The Mouse Pointer In The Screen Cannot Be Moved By Moving The Mouse

    TROUBLESHOOTING GUIDE Phenomenon Cause Treatment Manual Ref. Pages 18. The mouse pointer in The software is 1-2-1 & 1-2-11, Press Ctrl + Alt + the screen cannot be malfunctioning. OPERATION Delete on the keyboard moved by moving the INSTRUCTIONS and exit from FLUOVIEW by mouse.
  • Page 470 OLYMPUS OPTICAL CO., LTD. 2-43-2,Hatagaya, Shibuya-ku, Tokyo, Japan OLYMPUS OPTICAL CO.(EUROPA) GMBH. Postfach 10 49 08, 20034, Hamburg, Germany OLYMPUS AMERICA INC. 2 Corporate Center Drive, Melville, NY 11747-3157, U.S.A. OLYMPUS SINGAPORE PTE LTD. 491B River Valley Road, #12-01/04 Valley Point Office Tower, Singapore 248373 OLYMPUS OPTICAL CO.

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