When to use bright field microscopy

Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms. It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections. Here is a not-so-complete list of specimens that might be observed using bright-field microscopy, and appropriate magnifications (preferred final magnifications are emphasized).

• Prepared slides, stained - bacteria (1000x), thick tissue sections (100x, 400x), thin sections with condensed chromosomes or specially stained organelles (1000x), large protists or metazoans (100x).
• Smears, stained - blood (400x, 1000x), negative stained bacteria (400x, 1000x).
• Living preparations (wet mounts, unstained) - pond water (40x, 100x, 400x), living protists or metazoans (40x, 100x, 400x occasionally), algae and other microscopic plant material (40x, 100x, 400x). Smaller specimens will be difficult to observe without distortion, especially if they have no pigmentation.

DARK FIELD

Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field illumination uses a carefully aligned light source to minimize the quantity of directly-transmitted (unscattered) light entering the image plane, collecting only the light scattered by the sample. Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation. However, the technique suffers from low light intensity in final image of many biological samples, and continues to be affected by low apparent resolution.

Rheinberg illumination is a special variant of dark field illumination in which transparent, colored filters are inserted just before the condenser so that light rays at high aperture are differently colored than those at low aperture (i.e. the background to the specimen may be blue while the object appears self-luminous red). Other color combinations are possible but their effectiveness is quite variable

A diatom under Rheinberg illumination

Principle

To view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye (figure 2). Instead of coming up through the specimen, the light is reflected by particles on the slide. Everything is visible regardless of color, usually bright white against a dark background. Pigmented objects are often seen in "false colors," that is, the reflected light is of a color different than the color of the object. Better resolution can be obtained using dark field as opposed to bright field viewing.

You don't need sophisticated equipment to get a dark field effect, although the effect is most dramatic when the occulting disk is built into the condenser itself. You do need a higher intensity light, since you are seeing only reflected light. At low magnification (up to 100x) any decent optical instrument can be set up so that light is reflected toward the viewer rather than passing through the object directly toward the viewer.

Date: 2015-12-11; view: 2442