Formyl-met-leu-phe added

Figure 1.5. Human blood cells viewed by bright-field and phase-contrast light microscopy. Arrow indicates a white blood cell. Formyl-met-leu-phe (page 171) causes the white blood cell to spread out and become very thin. It becomes almost invisible by bright-field microscopy but can still be detected by phase-contrast microscopy.

microscopy, the image that reaches the eye consists of the colors of white light less that absorbed by the cell. Most living cells have little color (plant cells are an obvious exception) and are therefore largely transparent to transmitted light. This problem can be overcome by cytochemistry, the use of colored stains to selectively highlight particular structures and organelles. However, many of these compounds are highly toxic and to be effective they often require that the cell or tissue is first subjected to a series of harsh chemical treatments.

A different approach, and one that can be applied to living cells, is the use of phase-contrast microscopy. This relies on the fact that light travels at different speeds through regions of the cell that differ in composition. The phase-contrast microscope converts these differences in refractive index into differences in contrast, and considerably more detail is revealed (Fig. 1.5). Light microscopes come in a number of physical orientations (upright, inverted, etc.) but whatever the orientation of the microscope the optical principles are the same.

IN DEPTH 1.1 Fluorescence Microscopy

Fluorescent molecules emit light when they are illuminated with light of a shorter wavelength. Familiar examples are the hidden signature in bank passbooks, which is written in fluorescent ink that glows blue (wavelength about 450 nm) when illuminated with ultraviolet light (UV) (wavelength about 360 nm), and the whitener in fabric detergents that causes your white shirt to glow blue when illuminated by the ultraviolet light in a club. The fluorescent dye Hoechst 33342 has a similar wavelength dependence: It is excited by UV light and emits blue light. However, it differs from the dyes used in ink or detergent in that it binds tightly to the DNA in the nucleus and only fluoresces when so bound. Diagram a shows the optical path through a microscope set up so as to look at a preparation stained with Hoechst. White light from an arc lamp passes through an excitation filter that allows only UV light to pass. This light then strikes the heart of the fluorescent microscope: a special mirror called a dichroic mirror that reflects light of wavelengths shorter than a designed cutoff but transmits light of longer wavelength. To view Hoechst, we use a dichroic mirror of cutoff wavelength 400 nm, which therefore reflects the UV excitation light up through the objective lens and onto the specimen. Any Hoechst bound to DNA in the preparation will emit blue light. Some of this will be

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