Zeiss LSM 510 Quick Start Manual
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Zeiss LSM 510 Quick Start Manual

Zeiss LSM 510 Quick Start Manual

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Quick Start
Zeiss LSM 510

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Summary of Contents for Zeiss LSM 510

  • Page 1 Quick Start Zeiss LSM 510...

  • Page 2: Table Of Contents

    Table of Contents START HARDWARE........................3 START SOFTWARE........................4 START LASERS ..........................5 FIND THE SPECIMEN ..................... 6 (AXIOPLAN 2) FIND THE SPECIMEN ....................7 (AXIOVERT 200M) CONFOCAL FILTER SET CONFIGURATION.................. 8 ACQUIRE PRELIMINARY CONFOCAL IMAGE................9 OPTIMIZING THE SETTINGS......................10 7.1.

  • Page 3: Start Hardware

    1. Start Hardware • Turn on the mercury short arc lamp light switch. Note: Whenever the mercury lamp is turned on, it should be left on for at least 30 minutes. Once the lamp has been turned off, it should not be turned on again for 30 minutes.

  • Page 4: Start Software

    If you are using the UV laser, start this no before starting the LSM software. • Double click the LSM 510 desktop icon. • The Zeiss LSM 510 switchboard window will appear • Make sure Scan New Images is pressed and then click the Start expert mode button.

  • Page 5: Start Lasers

    3. Start Lasers Acquire Click the button on the toolbar. The lower toolbar will now change to the Acquire sub-toolbar and show the acquisition controls. Laser Click the button. Turn on the desired lasers position: HeNe’s to ON, Argon to Standby. When the Status reads Ready, click the On buttons.

  • Page 6: Find The Specimen

    4. Find the specimen (Axioplan 2) light path select, focus knob stage are manual controls. The rest of the microscope is controlled by the software. To set up the Axiovert microscope to locate your light path selector specimen first move the VIS.

  • Page 7: Find The Specimen

    Find the specimen (Axiovert 200M) • Change the microscope-light path to direct the emitted fluorescence is sent to the eyepieces by clicking on the button in the LSM software toolbar. • Move the correct objective into place using the Objective buttons on the microscope button.

  • Page 8: Confocal Filter Set Configuration

    5. Confocal filter set configuration Click the Config button (in the Acquire sub-toolbar). To activate multi-tracking simply chose the MultiTrack button in the Configuration Control window. Load the configuration that matches your fluorophores (referred to as ‘tracks’) by clicking Config button.

  • Page 9: Acquire Preliminary Confocal Image

    6. Acquire preliminary confocal image Scan Click the button in the Acquire sub-toolbar to open then Scan control window. Click the Find button and the computer will open a new image window and calculate the approximate levels to generate a starting image. You will see an image looking something like this (left).

  • Page 10: Optimizing The Settings

    7. Optimizing the settings Due to the sequential nature of ‘Multi-track’ acquisition optimizing the settings in this mode is difficult. It is easier to set the imaging parameters for each Track individually. You can do this by switching off each track with the checkbox along side it.
  • Page 11 Your image window will now look like this.

  • Page 12: Scan Control: Channels Window

    7.2. Scan control: Channels window Click on the Channels button of the Scan control window. The active channel will appear here. The settings below need to be adjusted for each channel separately. 1. Set Pinhole of each channel: The pinhole diameter determines the thickness of the optical section – i.e. the axial resolution.
  • Page 13 You need to set the detector range to match the dimmest and brightest signal from the specimen. Setting the detector incorrectly results in the loss of information from the specimen. 3. Set Ampl. Offset – setting the ‘min’ signal This should be set next, but may need re-setting later. While acquiring with the Fast scan button, adjust the Amp.
  • Page 14 A few red speckles; a few blue speckles After you have set both channels, stop scanning by pressing the Stop button in the Scan Control window. Your image will look like this (above). Turn off the optimised channel in the Configuration Control window.

  • Page 15: Acquiring Your Final Image

    7.3. Acquiring your final image Once you are satisfied that each channels detector is set optimally to the range of the image, you can create your final image. Ensure that all Tracks are checked when acquiring the final image. Go to the Scan control window and click the Mode button. Change the Frame size by clicking on the Optimal button (see Appendix 1 for an...
  • Page 16 Click on the Single button in the scan control window to collect your final image (bottom). Info Click on the image window button. This will bring up a bar on the left of the image window with the image information on it.

  • Page 17: Saving Your Final Image

    8. Saving your final image When you are satisfied with your image you need to save it to your database. Images are saved to Databases. A database can be single or multiple images or stacks. Press Save as in the image window and the Save Image and Parameter window will open.

  • Page 18: Acquiring A Z-Series

    9. Acquiring a Z-series Having set the system to acquire a satisfactory image, you can acquire a z-series. It may be worthwhile changing the frame size to 512×512 to minimize file size and to speed acquisition. Click on the Z-Stack button in the Scan control window.
  • Page 19 In the Scan control window, click the slice button to bring up the Optical Slice dialog Optimal Interval Click the optical section button. Check the for each channel is the same and close. If you optical sections are different for each wavelength go to Mode/Channels and adjust the Pinhole so that each channel has the same optical section –...

  • Page 20: Advanced Options

    10. Advanced Options 10.1. Single-Track – simultaneous acquisition Multi-track can solve the problem of cross-talk. Typically this occurs with bleed through of green fluorescence in to the red channel. Multi Track avoids this by acquiring the fluorescent channels sequentially, not simultaneously as with Single Track acquisition.

  • Page 21: Z-Attenuation Compensation

    10.2. Z-attenuation compensation As images are collected deeper in to the sample there can be significant loss of signal. This can be caused but refractive index mismatch, light scattering and absorption. This signal loss can be compensated for collecting the attenuated slices with higher gains and/or with higher laser intensity.

  • Page 22: Transmitted Light Image

    10.3. Transmitted light image Whilst the laser is scanning the field of view a certain amount of the excitation laser light passes through the specimen. This light can be detected and its intensity in each part of the field of view corresponds to the transmitted light optical properties of the specimen.

  • Page 23: Frap

    10.4. FRAP Edit Bleach toolbar button to open up Bleach Control dialog. 1. Click on 2. Set Bleach parameters in Bleach Control dialog Check Bleach after Number of Scans Set “Scan Number” to for pre-bleach imaging Set “Iterations” value – this may require empirical determination.
  • Page 24 Experimental progress shown here. Progress bar will pause during the bleach process. Bleached area Save experiment 8. Create ROI reference image Turn ROI white by clicking the ROI colour button and selecting white. Export this image for reference via the menu command File/Export.

  • Page 25: Shutting Down The System

    Cover the microscope avoiding the hot lamp housing. 11.3. Exit the software Exit the Zeiss LSM . A message will come up reminding you not to power down the system until the laser is cool. Click OK. If you have left the lasers on for the next user, you will also be asked whether you want the lasers switched off.

  • Page 26: Opening Your Images Offline

    12. Opening your images offline 12.1. Zeiss image browser http://www.zeiss.de/lsm Follow link in lower right hand corner: “Free LSM Image Browser” Requires a registration form to be completed and also the download and installation of an extra DAO file if you have Microsoft Office 2000.

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