Page 133 - Fundamentals of Light Microscopy and Electronic Imaging

116 PHASE CONTRAST MICROSCOPY AND DARK-FIELD MICROSCOPY

Exercise: Dark-Field Microscopy

1. Adjust the microscope for dark-field mode using a low-NA objective
(10 or 20 ) and a high-NA phase contrast or dark-field annulus. These
annuli produce steeply pitched cones of light that are not accepted by low-
NA, 10 or 20 objectives. Only the scattered light is accepted, which is
what you want.
2. With the microscope adjusted for Koehler illumination, focus on a few
isolated buccal epithelial cells (obtained by scraping of the underside sur-
face of the tongue) and compare with the image obtained by phase con-
trast microscopy. Notice that object edges are enhanced by both of these
modes of microscopy. In dark-field, each speck of dirt is imaged as a
beautiful Airy disk surrounded by higher-order diffraction rings. This is
an excellent chance to see the Airy disk, the diffraction pattern of a point
source of light.
3. Using an oil immersion dark-field condenser (NA 1.4) and a 60 or
100 oil immersion objective, focus on the cilia and flagella of unicellu-
lar protozoa and algae. Cultures of protozoa can be obtained commer-
cially, but a drop of pond water will do as well. Note: If the objective lens
contains an aperture diaphragm, try stopping it down to improve contrast
and visibility, as minute structures are difficult to detect.

The following specimens are suggested for dark-field examination:

• Buccal epithelial cells
• Culture of protozoa
• Axons and glial cells in sections of rat brain labeled with antibodies adsorbed
on gold colloid particles
• Blood cells in a 60 L drop of phosphate-buffered saline
• Taxol-stabilized microtubules in 15 mM imidazole, 1 mM Mg-GTP, 5 M
taxol. The microtubule stock is 5–10 mg/mL. Dilute to 0.1 mg/mL for
observation. Difficult specimen!
• Flagella of Escherichia coli bacteria. Difficult specimen!