Darkfield microscopy is a mode in which direct light is prevented from passing through the objective aperture, but a hollow cone of light forms an apex in the plane of the specimen. The image is formed by light scattering from features of the object. Details appear bright white against a dark background. Darkfield can also be used for living bacteria, algae and plankton.

Some materials produce light when excited by short wavelengths of radiation. This effect is called fluorescence or auto-fluorescence. Specimens that do not fluoresce by themselves may be treated with fluorochromes which produce a secondary florescence. By illuminating with a high intensity mercury or xenon source and filtering out all but the desired excitation wave length to contact the specimen, the resulting longer (less energetic) wavelengths of emission from the specimen its self veiwed. Fluorescence microscopy can be used to enhance particular organelles, immunocytochemistry, in-situ hybridization, enzyme cytochemistry and elemental localization.

Figure 7
Fluoresence microscope.

Figure 8
Comparison of a dry and an oil immersion objective.

 

Abbe in order to ease in identification of lens quality devised an equation for numerical aperture. Numerical aperture numbers can assist in comparing angles of dry, water immersion, and oil immersion objectives. Note the similarity to Abbe’s equation for theoretical resolution. This number is found on all objective lenses.

N.A. = n sin u

n = refractive index of medium

u = 1/2 the angle of light rays taken in when focused on the object.

 

When choosing an objective another consideration is depth of field. Depth of field is the distance from the nearest part of the subject in acceptable focus to the farthest part of the subject in acceptable focus. The efficiency (resolution) of a lens is inversely proportional to the depth of field (Table 1).

N.A.	.25	.30	.50	.65	.85	.95
Depth (in microns)	8.0	5.5	2.0	1.0	.25	.10

Table 1
Variation in Depth of Field with Change in N.A.

Two aberrations within lenses detract from Abbe's equation of theoretical resolution. These aberrations are called spherical aberration and chromatic aberration. Spherical Aberration occurs when outer rays entering a lens are diffracted differently from those entering near the center. A solution for reducing spherical aberration is introducing a diaphragm or aperture.

Figure 9
Spherical aberration of a simple lens. A. Under correction. B. Over correction.

The thickness of the cover glass should be chosen according to specifications of a particular objective. Deviation from the required thickness results in over correction or under correction of spherical aberration.

Chromatic Aberration occurs as white light entering a lens is broken into a spectrum from red to violet. Violet rays (more energetic) are refracted more than the red rays (less energetic). Consequently an uncorrected lens will be surrounded by color fringes. The more expensive lenses have a higher degree of correction.

Figure 10
Chromatic aberration of a simple lens. Each spectrum color has a separate focus.