Leica TCS SP8 FSU Manual
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Leica TCS SP8 FSU Manual

Leica TCS SP8 FSU Manual

Las x 3.1
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Living up to Life
TCS SP8
FSU
Manual
Summery
LAS X 3.1
20170703
0

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Summary of Contents for Leica TCS SP8 FSU

  • Page 1 Living up to Life TCS SP8 Manual Summery LAS X 3.1 20170703...

  • Page 3: Table Of Contents

    Contents Ｉ. Start up 2 ＩＩ. Image acquisition 6 III. Viewer 20 IV. MaxProjection 23 V. Quantify menu 24 VI. Data saving 25 VII. Shut down 30 VIII. Microscope operation 32 IX. HyVolution (Cofocal super resolution method) 36...

  • Page 4: Main Switches

    I. Start up Main Switches Indicator lamp of imaging lasers ② ③ ① ④Key switch of imaging laser ④ ①Switch for PC ②Power switch ③Main power supply of for the Scanner imaging lasers and cooling ② ①...
  • Page 5 ① Switch on the PC. ② Switch on the Scanner. ③ Switch on the main power supply of imaging lasers. ④ Switch on the key switch of the imaging lasers (yellow indicator lamp will be on ) ⑤ Switch on the HG lamp. Note) Do not switch on/off frequently and wait about 5min when restart.
  • Page 6 ⑨ The following window comes up. Select modes/switches and click “OK” and wait until the interface opens without operation including microscope. Configuration： machine.xlhw → Imaging through the microscope. SimulatorSP8.xlhw → Starting up without connected hardware(Simulator mode). Turn on PC only. Microscope：...
  • Page 7 ⑪ Change the bit depth Click the Configutation menu and click the Hardware And select an appropriate number of bit depth. ⑫ If using Resonant mode; Define the zoom factor With Resonant mode, the zoom factor set as bigger when the software opened (zoom factor depends on the 8/12kHz.).

  • Page 8: Ｉｉ. Image Acquisition

    II. Image Acquisition Configuration： Acquire： Process： Quantify： Set up menu for Main menu for Image processing Quantify menu for microscope, lasers imaging menu measuring and and systems analyses Open Acquire Menu Wavelength settings: Acquire menu Selecting lasers, detectors, emission wavelength, objective lenses.
  • Page 9 Wavelength settings Settings single color Laser wavelength selection and power setting Objective setting Panel box, notch filter, Dye assistant settings Detection setting detectors (Prism-slit) （PMT or HyD） Transmission detector (Bright field)
  • Page 10 Select objective and observe specimen, find the position and the focus. ① Load the fluorescence setting For Single staining observation Select the light path setting from the pull-down menu of “Leica Settings” of “Load/Save single settings”. *For the multi staining, go to ③.
  • Page 11 ③－３ Several “Seq.” buttons are displayed depend on the number of fluorophores. Activate each “Seq”button and define for the each staining (laser power, gain). Refer ④ and after how to define these parameters. ＊Sequential mode Each excitation changes in line by line. between lines Suitable for live imaging since it is almost simultaneous scanning, time lag is only a...
  • Page 12 Loading the settings from the acquired/saved data By using the Apply button on the Experiments tab, the settings of the acquired/saved data can be loading (reproducible).Activate the raw image data on the Projects tab and click Apply. All the parameters of wavelength(Excitation, emission), detectors will be reproduced. Apply button Activate certain data on the Project tab and click Apply button, wavelength settings will restore according to that data.
  • Page 13 ④Image acquisition Click the Live button at the lower left on the monitor. Scan starts and align the parameters as follows. Notes: Live button expose lasers your specimens. So it should be stopped often in order to avoid the bleaching. ■...
  • Page 14 Defining the focus, and the detector gains Adjust focus and the detector gains and offsets by using each dial of the control panel (option) as below. For the multistaining, the “Smart Gain” and the “Smart Offset”dials can adjust each staining by activating channel image respectively on the monitor.
  • Page 15 Quick Look Up Table (Q LUT) button For easy tuning of signal intensity, click Q LUT button and the look up table (image display color) changes to “Grow Over/Under”. Image display color changes like follows by clicking this button. Original （pseudo color like green, red, etc.） →Grow Over/Under （for checking）...
  • Page 16 ⑤ Other parameters（format, scan speed, zoom, etc） If necessary, Format (pixel numbers of the acquired image), Speed(scan speed), Zoom factor(optical zoom) can be also changed. The slower the scan speed, the smoother the image quality. Please note, the bleaching effects becomes bigger with the higher the zoom factor. Format：Pixel numbers of the image Speed：scan speed Zoom：minimum is 0.75.
  • Page 17 ⑧ Z stack acquisition Define start position with “Begin” button and end position with “End” button while scanning by “Live”button. Focus can be moved by Z position dial of the Control panel. Z position Adjust z position/focus ⑧－１ Define Begin/End position Delete defined...
  • Page 18 ⑨ Acquisition of the Zstack Data acquisition starts by clicking following button. XYZ (or for several continuous image acquisition： click “Start” button. Image data will be kept in the Project tab as a name of “Series001”temporarily. Note: keep away from the microscope and the anti-vibration table while scanning in order to avoid the vibration noise to the images.
  • Page 19 （ Hybrid Detector *option） HyD has higher sensitivity, higher S/N, and extremely low noises. Photoncounting mode can be selected. Caution: Please separate mobile items (cellar phone and other items which send electric wave) from HyD about 1m. If they are close to locate each other, HyD would detect noises from them. If HyD detects extremely high intensity, its shutter closes automatically.
  • Page 20 Counting mode (Photon Counting mode) This is a mode to count and accumulate photons in each pixel. In this case, the intensity means the number of photons. Detector noises are almost zero and the S/N and the quantitativity improves quite a lot. Theoretically it is not possible to use Gain with Counting mode, the image shows less bright than Standard mode, to get brighter image with Counting mode, please refer following settings.
  • Page 21 ⑩ timelapse ⑩－１．Select mode Select “xyｔ” or “xyzｔ” from the Acquisition Mode. ｘｙｔ ： XY timelapse imaging without Z stack ｘｙｚｔ ： XYZ timelapse imaging with Z stack. ⑩－２．Following panel will open according to the selected mode. Define the “Duration”(whole acquisition time) and “Time interval” Whole acquisition Time interval time...

  • Page 22: Viewer

    III.Viewer Viewer：Window which is shown the acquired images. There are function buttons around the images. The colors of the image can be changed by clicking this LUT bar. Tiled/full display can be changed by double clicking the image. Tiled display Full display...
  • Page 23 Icons of the viewer Select tool Delete selected/activated item Insert scale. Activate the scale by using button and right mouse click, select “Properties” then define the length, angle, etc. Display scale. On/Off this button and the image changes 100% display or shrink or zoom depend on the image resolution and the display resolution.
  • Page 24 Overlay Display or not display “overlay image” of the multiple channels by activating/not activating this button. Maximum projection Create Maximum projection image from stack data temporarily. maximum intensity in the z stack for each pixel will be shown as a single 2D image.

  • Page 25: Maxprojection

    IV. MaxProjection Create Maximum Projection image data from the Z stack image. Open the “Process” menu and select the “Projection” in the Process Tools. Activate the Z stack data on the Experiments tab and click “Apply” button. Maximum Projection image data will create/add in the Experiment tab.

  • Page 26: Quantify Menu

    V. Quantify menu Functions to measure lengths, intensities, areas, histograms, ratio, etc. There are three mode as follows. Lengths and intensities and other statistical data are shown by drawing line on the images. This function is supposed to use for Line single image.

  • Page 27: Data Saving

    VI. Data saving All image data in the single file will be saved as a .lif file. Open Projects tab Example） Here, there are three image data in the single file “Project001”. They will be saved as a single .lif file. Each single file on the LAS X.
  • Page 28 ①Right mouse click the data and select”Save As”. Image data in the same file will be saved as a single file as explained above. All image data in the same file will be saved as a single .lif file. To rename each data ( Series001,Image001, etc）, right mouse click and select “Rename”...
  • Page 29 ③ Overwrite saving (refresh saving ) Adding/editing data after saving .lif file, data should be overwritten by executing “Save Project” ④ Acquiring new data in the new file To create new file, click “New” button, new data will be acquired in the activated new file. New button Created new file Activate this file and acquire new data, the data...
  • Page 30 ⑤ Exporting file as “.Tiff”, “.Jpg” format Right mouse click the data and select ”Export” and select ”As Tiff...” or ”As JPEG..”. Then following dialog will open. Select the path and click save. Destination Folder by clicking Browse button to select the path Overlay Channels For multicolor staining, select “Overlay”...
  • Page 31 LAS X Download site 1. Access to following site. http://www.leica-microsystems.com/products/microscope-software/software-for-life-science-resea rch/las-x/ 2. There is a link at the most end of the page, click to download. ① For ① ② ②...

  • Page 32: Shut Down

    VII.Shut down 1. Switch off the lasers on the software. Open the Configuration tab and click “Laser Config”. Ar laser: Decrease the laser power down to 0% and switch off. WLL laser: Switch off while keeping laser power 70% 2. Lens cleaning Using lens paper/ cotton-tipped stick and lens cleaner, clean up liquid immersion lens.
  • Page 33 5.Switch off the Microscope. 6.Switch off main switches ① If the Windows is already finished, switch off the PC. ② Switch off the scanner ③ (If Ar laser is equipped) after the cooling fan was stopped (about 5min after deactivate Ar laser on the software), switch off the key switch.

  • Page 34: Microscope Operation

    Focus the image by turning the focus dials on the left and right sides of the stand. Alternatively, rear rotatory knob on the Leica Smart Move control element can also be used. XY stage can be controlled by Leica Smart Move control element.
  • Page 35 Immersion forming on the objective lens. ※ Immersion (IMM) Objectives For immersion objectives use the appropriate immersion medium. OIL: Use optical immersion oil from Leica only W: Water immersion Gly: glycerin IMM: Universal objective for water, glycerin, Oil immersion. (Require setting of the appropriate correction circle to use each IMM) Notre）If the immersion liquid is attached to dry lens, clean the dry lens certainly.
  • Page 36 ．Microscope observation method ( by your eyes.) ３ ３－１． Transmitted method（Including DIC） ① On the Touch Screen use the tab to ① configure the contrast method. ② Select contrast method from Transmitted method list. ② BF： Brightfield Transmitted Light DIC： Differential Interference Contrast ③...
  • Page 37 ３－２．Operating the fluorescence ① On the Touch Screen use the tab to configure the contrast method. ② ① ②Select FLUO ③ Select desired cube using corresponding key from FLUO-Filtercubes. ※Set of Filter cubes are depending on the system. Please refer the Fig1.1about the spec ④...

  • Page 38: Hyvolution (Cofocal Super Resolution Method)

    Leica Microsystems K.K. Tokyo 1-29-9 Takadanobaba,Tokyo, 169-0075 Japan Phone: +81-3-6758-5630 Fax: +81-3-5155-4333 Osaka 10F 5-4-9 Toyosaki, Kitaku, Osaka City, Osaka 531-0072 Japan Phone: +81-6-6374-9771 Fax: +81-6-6374-9772 Nagoya 2F 2-15-20 Nishiki, Nakaku, Nagoya City, Aichi, 460-0003 Japan Phone: +81-52-222-3939 Fax: +81-52-222-3784...

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