Microscopy

   I use dissecting, compound light, and confocal microscopy in my research. I've even done a bit of scanning electron microscopy and magnetic resonance microscopy too. Here are some points of training in microscopes for students that occasionally help me.

   Mr. Charlie Christensen, a friend and Zeiss master technician says that there are entire books on how to maintain microscopes, but has passed along his favorite basic rules to ALWAYS follow around microscopes, based on many years of experience. You may not know how to get the job done, but at least you won't get fired if you:

  1. NEVER touch any microscope objective lenses with bare fingers. The oil on your fingers will kill those little $1000+ optics
  2. If something bad does happen ALWAYS report it before somebody finds the problem the hard way. That problem is almost always becomes more expensive if left alone.
  3. Wipe up liquids on the microscope right away. Drips from pond water or preserved specimen slides or even worse, permount puddles can really mess up a scope if left alone.
  4. NEVER force any drive knobs... never force anything on a microscope. If the rack and pinions aren't "creamy" smooth or if something's tight, chances are the scope needs some maintenance, and by forcing it, you've just made it worse.
  5. Microscopes like plastic covers. Lubricated scope mechanisms and precision surfaces don't do well when they're dusty.

    Everyone knows how to focus a microscope, all you do is use the macrofocus knob, and then, hopefully before putting the objective through the slide, use the fine focus knob to bring it into clear focus. My Ph.D. advisor, Dr. Kier, a fine microscopist, taught me that the other thing you should always do, to get the absolute best resolution and contrast in your microscope is to focus the light! Makes sense, but how? Follow along as I bring my beloved Wild M12 into Kohler illumination (and take some snapshots down the eyepiece while I'm doing it.)

1. Turn on the microscope and adjust the macro and micro focus knobs to get the slide plane in focus. Make sure the light intensity is about half way, then close the field diaphragm (the diaphragm associated with the light bulb) until you can see the edges in the field of view. The illumination is probably out of focus, so you'll see something like the above.

2.Now you have to bring the field diaphragm into sharp focus. You do this by adjusting the height of the condensor (the small stage that moves up and down below the slide stage). Try and get it between the red and blue fringe.

3. Next pull out an ocular lens and, using the condenser aperture diaphragm, adjust the light level. The little circle of light you see on the back of the objective lens, when you look down the ocular tube should be at about 2/3 maximum diameter. (Actually, now that I think about this, you may want to do this step at the end so that the light level setting will take into account what's coming next... although I don't think it matters that much.)

4. Go back to the field diaphragm and open it up so that the dark perimeter is just slightly smaller than the field of view.

5. You'll notice that the center of the field of view and the center of the field diaphragm don't exactly match up, so center the light source base, by pushing it lightly until the edges of the field diaphragm are centered. Then open it up just a bit more until it is just hidden by the edge of the field of view.
   And there you have it! Dr. Kier told me that it's best to refocus the illumination everytime you change objectives or put on a new slide (coverslips all have slightly different thicknesses). I noticed that sometimes he even does it when he focuses on a different side of a big slide, especially when he's taking microphotographs.
Thanks Bill!

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