Troubleshooting

Related section numbers are given in parentheses.

1. Cannot see an image, or image is vignetted.

a. Check that light path to specimen is clear. Is the specimen evenly illuminated? i. Is a condenser lens of the correct maximum NA in place? For lower magnifications, it might be necessary to remove "swung-in" lenses—objectives of x1 magnification are normally used without a substage condenser.

ii. Is the dark-field condenser prism out of the light path?

iii. Are neutral density or color filters fully out of the light path?

iv. Is field diaphragm sufficiently open?

v. Is aperture diaphragm sufficiently open?

vi. Is DIC analyzer filter swung out (particularly for fluorescence illumination)?

b. Check that light path to eyepieces is clear, and in particular, that the beam is being directed to the eyepieces and not to the camera.

c. For fluorescent specimens, check that the appropriate filter combination is in place, and that the fluorescent lamp is correctly aligned and focused.

2. Cannot bring the specimen into focus at high power.

a. Is the thickness of the specimen too great for NA of the objective lens (Subheading 3.2.3.)? Specifically, is the objective touching the slide?

b. Is a dry objective being used incorrectly with immersion oil or vice versa (Subheading 3.2.4.)?

c. Is the immersion medium matched to the refractive index of the mountant (Subheading 3.2.4.)?

d. Inverted compound microscopes only—is slide placed correctly with cover-glass facing downward?

3. Insufficient contrast:

a. Focus condenser to achieve "Köhler illumination" (Subheading 3.4.1.).

b. Adjust aperture diaphragm of substage condenser to correct NA (Subheading 3.4.2.).

4. DIC not producing a "pseudorelief" image (Subheading 3.5.2.)

a. Is the analyzer filter in place?

b. Are DIC prisms in the substage condenser matched to the NA of the objective (in more sophisticated microscope systems)?

c. Are aperture and field diaphragms correctly adjusted (see Note 3)?

d. Is polarizer rotated to achieve optimal illumination?

5. Photomicrograph exposure is seemingly infinite:

a. Is all light (particularly for fluorescence) being directed to the camera (i.e., no image should be seen through the eyepieces)?

b. Is film speed set correctly?

c. Are camera batteries exhausted (where a separate 35-mm camera body is attached via a tube)?

6. Color photomicrographs have variable color balance and brightness.

a. Was camera voltage supply set to PHOTO, 3200 K or a constant voltage?

b. Was an artificial (tungsten) light source film used?

c. If using "daylight" film (see Subheading 3.9.), was a compensating blue filter (Kodak Wratten 80A) in place?

d. Were exposure times longer than 2 s? If so:

i. Was the correct reciprocity index programmed into camera exposure mechanism with respect to film type?

ii. Were the correct color compensation filters used?

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