image acquisition with the Zeiss LSM410

typical image acquisition session using the
Zeiss LSM410 confocal laser scan microscope

1. Starting the system

1.1     Switch on the HBO transformer.
1.2     Switch on the laser line switch and turn on the laser fan.
1.3     Switch the laser ignition key to start and after 40 seconds release to on.
1.4     Switch the LSM control unit key to start and then release to on and wait
          for the boot sequence. 2. General operation 2.1     Put the specimen on the stage (coverslip downward), with the epi-fluorescence filter
          (EFS) in the left, visual position and select Control Panel:Conv:T.
2.2     Choose an objective and set Lens = to that objective.
2.3     Select a field of interest in bright field (or epi-fluorescence by setting Control
          Panel:Conv:F)
2.4     Slide the epi-fluorescence filter (EFS) block to the right to the LSM position.
2.5     Switch to the Control Panel:LSM:R/FL1 and set the wavelength and lens being used.
2.6     Choose the laser line selection filter (LSF) for the excitation wavelength.
2.7     Choose the first beam splitter (DBS 1) for the longer emission wavelength
          (typically to FT 488/568 nm).
2.8     Choose the second beam splitter (DBS 2) for the shorter emission wavelength
          (typically to FT 630 nm) when working with dual labeled specimens.
2.9     Choose the emission filter (EMF 1) for PMT 1 (typically to 590 nm).
2.10   Choose the emission filter (EMF 2) for PMT 2 (typically to 515-540 nm).
2.11   Choose Control Panel:Setup:1, selecting LSM:R/FL1 and
          Display Control:Image 1 (R).
2.12   Choose Control Panel:Setup:2, selecting LSM:R/FL2 and and
          Display Control:Image 2 (G) for a dual labeled specimen.
2.13   Choose Control Panel:Setup:3, selecting LSM:T and and
          Display Control:Image 3 (B) for a transmitted light overlay.
2.14   Choose Control Panel:Extras:Confoc and Control Panel:Extras:Averag
          and press Control Panel:Scan:Start.
          You will now see an image on the right hand monitor. 3. Optimizing resolution 3.1     Launch the pinhole box and set Pinhole:Airy Units to about 1.0 for optimal resolution
          (this will decrease image brightness).
3.2     Press the AUTOBC button to adjust the screen image intensity.
3.3     Check Resinfo and increase the Control Panel:Zoom if necessary so that the pixel
          size is about 40% of the lateral resolution.
3.4     Check Resinfo again and repeat step 1 if necessary.
3.5     Select Display Control:Image:1 and press the AUTOBC button, adjusting brightness
          as needed to see the longer wavelength emission.
3.6     Select Display Control:Image:2 and press the AUTOBC button, adjusting brightness
          as needed to see the shorter wavelength emission.
3.7     Select Display Control:Image:3 and press the AUTOBC button, adjusting brightness
          as needed to see the transmitted light overlay.
3.8     Select Display Control:Image:RGB to see all three images overlayed. 4. Building and viewing a Z Stack 4.1     Focus to the bottom of the specimen and press the ZERO button.
4.2     Focus up through the specimen to top and note the Z height above the ZERO position.
4.3     Set T= 8 seconds.
4.4     Set the Z Sectioning z interval in mm and the number of sections desired and then
          press OK to collect the Z scan series of images.
4.5     Choose 3D Projection and set the Overlay to Maximum and press Calculate.
4.6     Choose Z Animate; Duration = 10; Startto view a rotation of the stack.
4.7     Choose Z Extended depth of focusz interval in mm and the number of sections
          desired to make a single increased depth of focus image.
4.8     Choose Z Gallery to display a series of images.
4.9     Choose File Store Image or File Store Sequence if desired. 5. Shutting down the LSM 410 5.1     Exit from the LSM software (Alt + F4) and close Windows (Alt + F4).
          Ignore the "Unstable Winsock" message.
5.2     Turn the LSM control unit key off.
5.3     Switch the HBO transformer off.
5.4     Switch the laser ignition key off.
5.5     Wait 10 minutes for the laser to cool and then switch the laser fan off.
5.6     Turn the laser line switch off.

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