Royal (Dick) School of Veterinary Studies, Easter Bush Campus, University of Edinburgh, Roslin, UK
It is very important to set up your microscope correctly. Incorrect set up results in poor-quality images, eye strain, headaches and motion sickness. This discourages use and compromises the diagnosis.
Correct Microscope Setup
1. Alter the separation between the eyepieces until you see a single, circular image. You can make a note of the distance for quick reference in the future.
2. Most microscopes have wide-field eyepiece lenses suitable for people that wear glasses. If you do, fold the rubber eyecups down and rest your glasses against them. If you don’t, fold the eyecups out and rest your orbit against them. Your eyes will now be the correct distance from the lenses.
3. Focus to get a sharp image with relaxed eyes (i.e., focus on infinity and don’t strain to get the image in focus).
4. Adjust the focus between the eyepieces for your eyesight using the adjustable (independently focusing) eyepiece. The adjustment may be on the eyepiece or on the housing (i.e., on the microscope body itself. There will be a 0 or other mark - when this is aligned both eyepieces are confocal, which is appropriate for perfect or perfectly corrected vision. Most people have slight differences between their eyes.
a. First focus normally on a slide with one eye looking through the fixed (non-adjustable) eyepiece.
b. Next, close your first eye and then correct the focus for your other eye, if necessary, by adjusting the other eyepiece (using its independent focus).
c. You will now have the two binocular images in perfect focus for each eye. This avoids eyestrain and blurring of edges.
5. Koehler illumination focuses the light source on the slide, giving the optical illusion that the light comes from the sample. This avoids transillumination and gives you the best light balance and image quality. It is very easy to set up:
a. Focus on a slide.
b. Close the light source lens diaphragm so that you can see the edges (they look like a hexagon or octagon) in the image field using the x4 or x10 objective lenses.
c. Focus the condenser until edges of the diaphragm are sharp.
d. If necessary, adjust the condenser centring screws until the light source is centred in the field of view.
e. Open the diaphragm so that it is no longer visible and there is even illumination of the field of view.
f. Some older and/or cheaper microscopes do not have a light source lens diaphragm - hold a thin piece of card or paper, a paper-clip, or the tip of a pen or pencil against the light source and focus the condenser until you see a sharp image outline in the image field.
6. You can now adjust the iris diaphragm to give the clearest image for each lens:
a. For parasites and dermatophytes close the iris diaphragm - the image is poorer but the increased contrast makes the parasites or fungi stand out better on unstained samples.
b. For cytology of stained preparations under high power, open the diaphragm to reduce contrast and improve the detail of the nucleus, cytoplasm, granules and microorganisms.
Correct Use of a Microscope
1. Always use a coverslip
a. You should always use a cover-slip (or cover glass).
b. The only exceptions are tape-strip preparations (the acetate backing of the tape acts as its own cover-slip).
c. Microscope lenses are designed to look through a cover-slip and fluid layer, which provides a flat optical surface, puts material in a similar focal field, avoids air-fluid interfaces and reduces contrast.
d. Curved surfaces (including cell layers, hairs, skin debris, and oil on top of the cover-slip) act as mini lenses and cause serious distortion.
e. Cover-slips can also protect the lenses from scratches and the mounting fluid, and provide a defined search area for skin scrapes and hair plucks.
f. It is important to use enough mounting fluid to seal the cover-slip to the slide without gaps or air bubbles, but not so much that it gets onto the microscope lenses or stage.
g. I prefer to use immersion oil as it is more viscous - it adheres the cover slip to the slide better with less leakage. However, if you collect material into liquid paraffin use more liquid paraffin if required before placing the cover-slip. Mixing liquid paraffin and immersion oil results in an opaque emulsion.
2. Scanning slides
a. Initially, visually examine the slide to orientate it, and appreciate the depth of material, degree of staining and possible areas of interest.
b. It is useful to check the quality of staining visually or under low power before placing a cover-slip, as the slide can be re-stained at this stage if necessary.
c. Study the slide at progressively higher magnification.
i. Low-power lenses (x4–x10) are useful to scan large areas; with skin scrapes, hairs plucks etc., start at one corner of the cover slip, proceed down to the opposite edge, across one visual field and back up again to methodically search all the collected material.
ii. Use the high-power lens (x40) to close in on areas of interest.
iii. Use the oil-immersion lens (x100) last of all to avoid getting oil on the other lenses.