Notes

1. Specimens that are to be further processed for in situ hybridization with RNA probes or that are labeled with a volatile dye, such as Dil, can be mounted in fixative in the same way.

2. A general exception is x1 objectives, which are highly unlikely to be parfocal with any other objective lens.

3. Each eyepiece has a diopter adjustment ring, which can be aligned with engraved markings on the eyepiece tube. Observers who would normally wear glasses should adjust the diopter with respect to each eye. With the ring aligned to the zero position, the specimen should be brought into focus at high power. To achieve parfocality, the same field should be viewed at low magnification and the specimen focused again using the diopter adjustment ring on the eyepiece. This should be repeated for each of the lower-power objective lenses and separately for each eyepiece.

4. NA is calculated as the product of the refractive index of the medium between the objective lens and the specimen multiplied by the sine of the half-angle of the cone of light entering the objective. This cone becomes narrower (and therefore the NA smaller) as the working distance increases (4).

5. Spatial resolution is proportional to the wavelength of transmitted light divided by the NA.

6. For an oil immersion lens, using a mounting medium with a refractive index lower than glass or oil will result in a corresponding loss in numerical aperture (effectively that achievable with the less dense medium). Any area of low refractive index in the object space, e.g., a water or air bubble, will have this effect.

7. Stopping the aperture diaphragm right down can produce "false resolution," a diffraction image that gives the impression of a fine ultrastructure within the specimen.

8. If dehydrating through an alcohol series, reduce the overall exposure to ap-prox 2 min.

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