Using the Olympus fluorescent microscope to image in vitro experiment slides

This protocol explains how to: use Olympus fluorescent microscopes to image in vitro experiment slides.

 
Protocol written by: Julie Ruble

1. Turn on green burner power button first, and then the microscope and then the camera.
2. Start viewing in fluorescence (change filters using the filter wheel until you reach the green filter). Begin at the corner of the slide. Use the 40X objective lens.
3. When looking for neurons to image, scan through the slide methodically so as not to miss any:
   
4. When you find a neuron, position it with the cell body in the field of vision but off to the side (so you'll be able to get more neurite in the picture and therefore take fewer pictures). NOTE: When you are not looking at or imaging the neuron, close the shutter on the right side of the microscope so as not to bleach the neuron.
5. To take a picture, open SPOT (the program may have a shortcut on the desktop, but you may need to find it in the C folder and create one). Then, to take a fluorescent image:
-Click "manual" on the image settings box. -Adjust the "gain" to ~16 and the exposure time to ~9-10 seconds (can increase exposure time if picture is too dim, or decrease if it's too bright). -Click "More" and make sure it is set to "green." -Open shutter, divert light to camera by pushing in slider on right of microscope, and click the camera icon. -Close the shutter when picture is finished exposing!
6. To take phase images:
   Without moving the stage, turn the filter wheel to phase and turn up the lighting (dimmer switch on right of microscope). Focus on neurites. If you cannot see neurites, you may have to fiddle with light levels, filter on bottom of microscope, focus, etc. Make sure phase ring is set to Ph2 (phase 2).
   
   To take pictures:
   -Click "auto" on image settings box in SPOT.
   -On the "more" dialog box, make sure it's set to "RGB" instead of "green."
   -Direct light to camera and click camera icon.
7. Since neurons are larger than camera view, you will have to take several frames to get the full view. While you take them, "build" them with a drawing in your notebook to make it simpler to photoshop them together later. Frame "A" should always contain the cell body. For instance:
   
8. Saving images:
In this experiment, images are saved by experiment number, slide letter, neuron number, frame of neuron letter, and then a lowercase "p" for phase images or "g" for green fluorescent images. For instance, a picture might be saved as: 4G1Bg.jpg. This indicates that the image is part of experiment 4, slide G, is the first neuron imaged on the slide, is the second frame of the neuron, and is the green fluorescent version.
9. You can stop in the middle of a slide if you need to -- just refresh yourself on your pictures when you return so you don't re-photograph the same neurons (also, you place a piece of tape light on your slide to mark the "row" you were on when you stopped last).
10. When you turn off the microscope, turn off the camera and turn off the burner last. Record your name, time finished, and bulb hours (found on the burner) on the log. Cover microscope. Do not turn microscope and burner back on within 15 minutes of turning it off -- it needs 15 minutes to cool.
11. Put slide back in slide box in fix fridge. Fill out chart on front of slidebox with the number of neuron images you obtained from the slide and your initials.

Examples of images:

40X fluorescent image -- 4G4Ag.jpg


40X phase image -- 4G4Ap.jpg