Nikon inverted microscope Room MSB 595B

To sign up for microscope in advance
http://signups.med.nyu.edu/facilities/equipment/signups/nikon-fluorescence-microscope

Microscopes uses Nikon Elements software. [User's Guide v. 4.0 PDF]

This microscope is good for routine snapshots of Blue, Green, Red, Far-Red fluorescence.

Warnings:
1.) The Channels are not in perfect XY registration due to old filter blocks.
2.) Chromatic aberration, especially at low magnifications, may require refocusing between channels, especially blue.

Do not use for brightfield histology in color.

 

Instructions for turning on:

  1. Make sure you have instrument reserved in online calendar.
  2. Turn on lamp for fluorescence.
  3. Turn on power strip for microscope.
  4. Turn on or wake up computer.
  5. If Nikon Elements software is running, quit it and run again.
  6. When you change lens, make sure you select the lens in the Nikon software so data in your image files will be correct.
  7. Control filter wheel for fluorescent channels with buttons in Nikon Software. (Above image is 10X lens with filter for red fluorescence.)
  8. Light turns on/of with knob on front of lamp box.
  9. Light goes to eyes or camera ("SIDE") with knob on front right of the microscope base.
  10. Set up channels you want to take pictures of and whether you want to use time lapse in the "ND Acquisition" window.
    Make sure "Save to file" is checked and the paths and file name is correct.
    Uncheck the "Time" tab if only taking single pictures.
    "Run now" button in lower right corner of window to take pictures.
  11. Files are saved in ND format. Always save files in this format to preserve instrument information and to keep color channels in original bit depth and unique for quantification. These files open in ImageJ with information you need.
    TIF and JPG throw away information!!!

Shutdown:

  1. Quit Nikon Elements software
  2. Turn off lamp for fluorescence.
  3. Turn off power strip for microscope.
  4. Drop focus to lowest position
  5. Transfer files to server
  6. Clean up. During covid pandemic this includes wiping the bench, focus knobs, mouse, etc with Kimwipes saturated with 70% EtOH.

 

  

 

 

 

 

Nikon DS-Qi1 camera installed on 1 April 2015.

Recommend using at full 1280 x 1024 with binning 1 X 1. New spatial scale information here.

Other changes:

When you turn on the computer, it will log into a common account.

When you run NIS Elements, the first thing you should do (if you have used the system before) is load your favorite settings. There are two parts to this.
1. Load Optical Configurations. These are the camera and other microscope settings.
Calibration > Optical Configurations... > Import
2. Change the layout of the screen.

You may customize the look of the program screen. In the lower left there are tabs.
When you have the screen arranges the way you want, right click on the active tab and Save As with your name. You can also find more options inthe Layout Manager.

Saving/load your own settings.
When you have all your exposure times, channels, etc set, you may save them all.
Calibration > Optical Configurations... > Export

Recommend not using 100X except for 1 ms exposures due to shaking.

Save Files Efficiently
Using the ND Acquisition method for snapping pictures, especially multiple channel ones, will save you a lot of time!


DO NOT STORE FILES ON THE DESKTOP. Files on the Desktop may be deleted without warning.
You may put files in a Users Files directory or My Documents.

DO NOT SURF THE WEB FROM THE COMPUTER.
When you run Explorer there will be tabs for this web page so you can see this info or the Sign Up webpage.
Do Not Ever look at an off campus webpage.

 

Aligning Light for Brightfield

If imaging by brightfield, phase contrast, or DIC/Nomarski, always set condenser for Kohler illumination.
1. Focus on sample
2. set condenser for BF
3. Close Field Diaphagm
4. Move condenser up and down using knob on right until center octagon is in focus
5. Center by turning two knobs on condenser.
6. Open field diaphragm to edge of visible field, not all the way.

 

Standard lenses on this microscope:

4x10x

For best images use #1.5 coverslips. The lenses are designed specifically for #1.5 coverslips. Except for the 40x/0.60 lens which needs to be adjusted for each sample, see below.
We recommend you do not use the 100X lens.

 

There are two 40X lenses. Which 40X lens should you use?

The 40X lens with the numbers printed in green is designed for tissue culture plastic.  It will work with coverslips, but has a ring on it that needs to be adjusted.  It is an air objective.  DO NOT put oil on it!

The 40X N.A. 1.3 lens with oil is designed for higher resolution.

Click here to see a comparison of these two lenses.

To get the best resolution, you need to adjust the collar.

As pictured, the adjustment is a little bit too far open for a #1.5 coverslip, but close enough. If you were imaging through a plastic tissue culture dish, the setting would likely be between 1 and 2. (The units are mm.)

You cannot easily see the collar underneath the stage. Therefore:
1. Focus on sample best possible.
2. Reach under stage to turn ring on objective a little.
3. Refocus, turn ring, and repeat until image has maximum crispness.

Also, this may also be done when in the fluorescence mode. It is important to make this adjustment or else your pictures will not be in proper focus.

 

 

 

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