FIGURE 12.21 Schematic layout of a near-field scanning optical microscope. The NSOM probe is a tapered optical fiber. Laser light is passed into the fiber and is used to excite fluorophores as the probe scans the sample surface. The probe-sample distance is maintained constant at <10 nm during scanning by shear-force-based distance detection in combination with an electronic feedback system controlling the piezoelectric scan stage. Fluorescence is collected by a conventional inverted microscope. Dual-channel optical detection allows wavelength and/or polarization discrimination. Reproduced from de Lange et al. (2001) with permission of the authors and The Company of Biologists Limited.
by a beam that must be perpendicular to the detection beam. Were this same approach used for microscopy, inner-filter effects would create a horizontal gradient in the intensity of the excitation beam, much like that shown for the cuvette in Fig. 4.14. While it is possible to correct for inner-filter effects in fluorimeters, fluorescence microscopy works best when the sample is illuminated uniformly from above. A major advance in the development of fluorescence microscopy was the invention in the 1960s of dichroic mirrors that are reflective over only relatively narrow wavelength ranges. Such "partial" mirrors behave as a chromatic beamsplitter that simultaneously uses the objective lens as an illumination condenser and observation objective (Fig. 12.22).
Image brightness is determined by (a) the illumination intensity, (b) the fluorophore's quantum yield, and (c) the microscope's light-gathering power. Fluorescence signal strength therefore increases with illumination intensity and quantum yield. The image brightness will increase until all of the fluorophores become fully saturated. Modern epi-fluorescence microscopes are fitted with objectives that serve both as condenser and objective, and the illuminating light is first passed through pure water to remove any infra red light that would otherwise heat and possibly damage the specimen. The illuminating light beam passes through the excitation filter and is reflected from the dichromatic mirror surface within the band-pass filter cube, whereupon it passes through the objective to form a cone of illumination that excites the specimen. Secondary fluorescence emitted by fluorophores attached to the specimen is gathered by means of the same objective lens and is then passed back through the dichromatic mirror and barrier filter and projected into the eyepiece or imaging system. A high-numerical aperture (NA) objective (where the NA is a dimensionless parameter that depends on the half-angle 0 of the maximum cone of light that can enter or exit the lens with respect to a defined point) increases the signal intensity in a manner that is proportional to the square of the numerical aperture. Because the light gathering power of the objective is likewise proportional to the numerical aperture squared,
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