Iii

▲ EXPERIMENTAL FIGURE 5-42 Optical microscopes are commonly configured for both bright-field (transmitted) and epifluorescence microscopy. (a) In a typical light microscope, the specimen is usually mounted on a transparent glass slide and positioned on the movable specimen stage. The two imaging methods require separate illumination systems but use the same light gathering and detection systems. (b) In bright-field light microscopy, light from a tungsten lamp is focused on the

A) or gathering more light (increasing either N or a). Note that the magnification is not part of this equation.

Owing to limitations on the values of a, A , and N, the limit of resolution of a light microscope using visible light is about 0.2 ^m (200 nm). No matter how many times the image is magnified, the microscope can never resolve objects that are less than «0.2 ^m apart or reveal details smaller than «0.2 ^m in size. Despite this limit on resolution, the light microscope can be used to track the location of a small bead of known size to a precision of only a few nanometers. If we know the precise size and shape of an object—say, a 5-nm sphere of gold—and if we use a video camera to record the microscopic image as a digital image, then a computer can calculate the position of the center of the object to within a few nanometers. This technique has been used to measure nanometer-size steps as molecules and vesicles move along cytoskeletal filaments (see Figures 19-17, 19-18, and 20-18).

specimen by a condenser lens below the stage; the light travels the pathway shown. (c) In epifluorescence microscopy, ultraviolet light from a mercury lamp positioned above the stage is focused on the specimen by the objective lens. Filters in the light path select a particular wavelength of ultraviolet light for illumination and are matched to capture the wavelength of the emitted light by the specimen.

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