Notes on using the Nikon Eclipse E800 microscope for DIC and fluorescence imaging

There is a scope in the Buck’s morphology core in the basement that can be used for DIC and fluorescence microscopy with yeast.

Things to bring to the scope:

Yeast with GFP reporters, slides, cover slips, pipettor and tips, notebook, agar for fixing if needed

Steps to operating the scope to look at GFP containing yeast:

1) Power supplies are to the left of the scopes, hit switch labeled “1” then press “2” until you hear a click to ignite the light source, then turn on three (when you’re done switch off 3 then 1)

2) Put some yeast on a slide and cover with a slip, since you will likely be using the 60X or 100X objective add a drop of oil

3) Mount the slide, lower the stand all the way down and set to desired objective

4) The switch on the bottom of the scope should be set to DIA for bright field

5) Log in to the computer using your Buck credentials and you can create a folder on the Diablo or Big Rock servers so you can access images from your desk in lab

6) Open the ACT-1 program on the desktop, at the right side of the home screen click on the “Save” tab and change the directory to your folder, for each sample you can change the text field to “YOURGENE_” and keep the “Body” as $### and it will save each consecutive image as YOURGENE_1, YOURGENE_2, etc automatically

7) For the best images, you will need to make some adjustments collectively called the Kohler process: bring cells into focus, adjust the diaphragm (bottom right base of scope) so that it’s as small as possible in the field of view (but still likely fuzzy), then use the small dial under the left side of the stage to focus the octagon formed by the diaphragm, then adjust the diaphragm so that it is no longer visible on the computer image. This process focuses the column of light and creates a double image that gives more pronouncement to the cells and their structures

8) To see GFP fluorescence, turn the switch just below the eyepieces to FITC, rotate the polarizer out of the way since it’s not necessary, and turn the bright-field “DIA” to “OFF”

9) Capture images

10) Sign off the computer, turn off the power supplies. There is a glass waste box in the room behind the scopes

Danielle runs the core alongside Kathy, a technician, and they are very helpful when you need help operating the scope or computer. They can usually be found the office right next to the scope room

A link to the manual is here.