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Zeiss LSM 510 User Manual

Laser scanning confocal microscope
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Download this manual See also: Operating Manual
Objectives
5x/0.15NA EC Plan Neofluar
10x/0.3NA EC Plan Neofluar
20x/0.8NA Plan Apo Chromat
40x/1.3NA
oil
DIC EC Plan Neofluar
63x/1.4NA
oil
DIC Plan Apo Chromat
100x/1.4NA
oil
DIC Plan Apo Chromat
Standard coverslip thickness 0.17
(#1.5 coverslip)
Mercury Arc lamp (ocular)
Dapi, Fitc, Rhod, DIC II, DIC III, Brightfield
Laser lines
Argon/2 – 458, 477, 488,
HeNe –
543
HeNe –
633
Diode –
405/30
514
Iron Man
Zeiss LSM 510 Laser Scanning Confocal Microscope
User Guide
v. 1.4 (11/2016)

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Summary of Contents for Zeiss LSM 510

  • Page 1 Iron Man Zeiss LSM 510 Laser Scanning Confocal Microscope User Guide v. 1.4 (11/2016) Objectives 5x/0.15NA EC Plan Neofluar 10x/0.3NA EC Plan Neofluar 20x/0.8NA Plan Apo Chromat 40x/1.3NA DIC EC Plan Neofluar 63x/1.4NA DIC Plan Apo Chromat 100x/1.4NA DIC Plan Apo Chromat Standard coverslip thickness 0.17...

  • Page 2: Table Of Contents

    Contents: Quick Guide Starting up the system The software will open to the ocular tab To look at your slide The Acquisition Tab At the top In the Setup Manager window In the Imaging Setup window In the Channels window In the Online Acquisition window Adjusting PMTs Snap...

  • Page 3: Quick Guide

    Quick Guide: 1) Turn on components in order. Don’t go too fast – PC & monitor won’t connect. 2) Log into Windows with IdentiKey and start Zen software. 3) Put sample on scope coverslip down. If using oil immersion, do not switch back to air objective.

  • Page 4: Starting Up The System

    Starting Up the System: Turn on components in numerical order. 1a) Power to the PC 1b) Power to microscope components (including lasers) 2) Mercury Arc lamp located underneath floating air table 3) PC 4) The microscope – green switch on right side of microscope base ...

  • Page 5: The Software Will Open To The Ocular Tab

    The software will open to the ocular tab: Online mode will allow you to view your sample through the oculars and offline mode allows you to view the sample on the PC screen. This window will allow you to control/view the following microscope positions: 1) Halogen bulb for Brightfield viewing 2) Aperture...

  • Page 6: The Acquisition Tab

    The Acquisition Tab: 1) At the top – select any options you want for your experiment (Z-series, time series) 2) In the Setup Manager window – turn on the appropriate lasers for your experiment. You do not need to turn on lasers you will not be using.

  • Page 7: In The Imaging Setup Window

    3) In the Imaging Setup window – choose and set up the appropriate track(s) for your experiment. You can open a previous configuration (little folder icon button at the bottom of the Imaging Setup window) and edit this as needed, you can reuse settings from a previous .lsm file (open the image and click on Reuse button at the bottom of the Display...

  • Page 8: In The Online Acquisition Window

    5) In the Online Acquisition window – The objective should be the one you’ve selected (don’t recommend changing this here, go back to the ocular tab to change objectives and refocus) frame size indicates how many pixels in each dimension you want your image to be – more pixels = more scanning time and larger file.

  • Page 9: Adjusting Pmts

    6) Adjusting PMTs  These guidelines assume a fixed sample. For live samples, you should try to avoid Auto Expose or Continuous modes as these will expose your sample to large amounts of excitation light prior to image acquisition. Instead use Live mode or optimize in one region of slide and move to another for data acquisition.

  • Page 10: Z Series

    7) Snap – If you just want a single plane, click snap and save your image. The PC does not have a lot of RAM – save and close regularly. 8) Z series – If you selected Z-stack in the top left, you can now control the Multidimensional Acquisition Z-stack window.

  • Page 11: The Information Tab Next To The Image

    SAVE YOUR FILES AS LSM5!!! If you want to Reuse settings and have the Info tab – it must be LSM5. You can open this in FIJI or Zeiss Zen Reader (links on LMCF website). When you’re done - 1) Save and copy your data –...

  • Page 12: Appendix A -Saving To Your Lab's Folder On Mcdb Dept Server

    APPENDIX A – Saving to your lab’s folder on Collie (MCDB Dept Server):  Questions regarding MCDB Server should be directed to Erik Hedl. 1) Map the network drive. Right click on Computer icon on desktop or Computer in Start menu. Click on “Map network drive…”...

  • Page 13: Appendix B - Parts Of The Microscope

    APPENDIX B – Parts of the microscope: (not all these components are present on our microscope) Iron Man User Guide v. 1.4...

  • Page 14: Appendix C - Configuring The Light Path

    APPENDIX C – Configuring the Light Path  Only turn on lasers you actually need To turn on Argon/2 and Diode, first go to Standby and when it says Ready, then turn it on. Argon/2 output [%] should be 50% (do not go higher).
  • Page 15 You need to know the excitation and emission spectra for all the dyes/fluorophores/probes you will be using. This can readily be viewed in various Spectrum viewers on the web (list of which is on the LMCF Website Resources tab at the bottom. Life Technologies’ SpectraViewer is a straighforward and reasonably complete one.
  • Page 16 Now we need to configure the mirrors and filters appropriately. In the light path window, you can click on the different positions to see what available options exist in this microscope. The laser button is self-explanatory. From there, the light goes through a (1) dichroic (which will reflect or transmit certain wavelengths only), then to your (2) sample, back up through the (1) dichroic, through (3, 4) additional dichroics to separate out the different wavelengths, and through (5, 6, 7) emission filters based on your sample.
  • Page 17 So for this example we have three laser wavelengths: 405 (DAPI), 488 (FITC) and 543 (Rhodamine) and would use the 405/488/543/633 dichroic in position (1). This will reflect excitation light down to the (2) sample and block it on the way back. In position (3) we want to split the lower wavelength DAPI and FITC from the Rhodamine by using the NFT543, which will reflect all light below 543nm to the right and allow to pass through all wavelengths greater than 543.
  • Page 18 Using the dichroic in position (3) we sent all light greater than 543 to channel 1. This is further selected for using the (7) emission filter. For rhodamine, we would use a LP615. If you want to image all three channels in the same track, select the track and check on the three channels.

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