Zeiss 800 upright microscope SK5

Michael Cammer & Yan Deng are the contact people for the microscope and to use it you need to be part of the group that purchased it or have permission from Dr. Steve Burden.


Signup to use microscope http://signups.med.nyu.edu/facilities/equipment/signups/zeiss-800-confocal

 

All files must be saved on Drive D:
All files should be copied to Medical Center maintained server. Read these instructions or look for these shortcuts which are in multiple locations on the computer

If you are asked to install an update, just say no!

About the microscope

Laser lines Useful for...
405 nm Dapi, CFP, Pacific Blue
488 nm FITC, Alexa488, GFP, YFP
561 nm RFP, mCherry, rhodamine, Cy3, Alexa568, Alexa594, propidium iodide
640 nm Cy5, Alexa647, To-Pro

There are two ways to choose the spectral detection. One is by filter and the other is by a user adjustable mask. If you are using multiple channels, imaging will be much faster if the mask does not move between channels.

Emission filters & splitter Useful for...
  Dapi, CFP, Pacific Blue
  FITC, Alexa488, GFP, YFP; (May collect spillover from blue)
  RFP, mCherry, rhodamine, Cy3, Alexa568, Alexa594, propidium iodide; (May collect spillover from green)
  Cy5, Alexa647, To-Pro; (May collect spillover from red)

 

Lenses:

Use oil that says RI 1.518 on the bottle.

For best results, use #1.5 coverslips as explained here.

 

Startup:

  1. Sign in the paper logbook.
  2. Box to left of air table, "Systems" switch.
  3. "Components" switch.
  4. DO NOT TOUCH THE KEY.
  5. Power strip strapped to right side of air table, turn on.
  6. Computer on.
  7. Log in to LSM User account. 
  8. Make sure the touchpad on the right side of the microscope is fully ready before next step.
  9. Run Zen software (icon is above center of screen).
    DO NOT TOUCH THE MICROSCOPE WHILE THE SOFTWARE IS LOADING.
  10. There are two tabs in the upper left of the software labeled “Locate” and “Acquisition”.

    The Locate tab is for looking through the microscope oculars.
    The Acquisition tab is for using the confocal microscope.
    The Processing tab is for working on images after they have been collected and saved.
    Analysis can generate numbers from the images, but you may find ImageJ or Volocity better.
  11. The Locate tab has buttons to turn the light on/off to look by eye.

    Do not use the touch pad on the microscope.
    Do not change any of the settings in the window titled "Microscope Control".
  12. Best way to set up imaging method:
    Acquisition tab
    Open image from earlier session with same settings.
    Click Reuse button.

To look by eye, the slider on the right side of the microscope must be pushed in, all the way to the left.
To use the confocal, the slider must be pulled out all the way to the right.
Put slide on the stage.

Put it in the recessed are of the stage closest to you and push it back under the metal tabs.
This means that samples should be at the center of the slide, not at the far edge.
Use joystick to move stage.  F1 button in top right corner toggles from fine to coarse and back.
Find sample. 
Then set up tracks in Acquisition tab.

The behavior of the Live scan button is different than previous versions of the Zeiss confocals. You cannot image multiple tracks simultaneously, details here.

 

Always leave the Digital Offset at 0 and the Digital Gain at 1.
DO NOT use these to reduce background or brighten the image. Especially if you want to do intensity quantification later, using these will complicate or nullify the ability to do so.
Pinhole always at 1. Greater than 1, go use widefield microscope with camera instead.
In the images below, note that you can click off the Show All box at the top of the Channels window so that the sliders for Digital Gain and Offset will disappear so you won't be tempted to touch them.

 

Save all images on drive D: only.  Files stored on drive C: may be deleted without warning.
All files should be copied to Medical Center maintained server. Read these instructions or look for these shortcuts which are in multiple locations on the computer

Click here for screen snap of how to export TIFs from the Processing tab, especially useful after stitching.

 

 

Shutdown:

  1. Quit software.
  2. Shut down computer.
  3. Switches in reverse order.
  4. Sign out of paper logbook.
  5. Before you leave the room, stop, look down and around, and if you see stuff on the floor, pick it up and throw it out.

 

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comments, questions, suggestions for this web page: Michael.Cammer@med.nyu.edu or mcammer@gmail.com