SHARPENING THE FOCUS AND SEEING CELLS IN THREE DIMENSIONS
The images obtained by conventional fluorescence microscopy are blurred as a result of out-of-focus fluorescence. These images can be improved by a computational approach called image deconvolution, in which a computer analyzes images obtained from different depths of focus and generates a sharper image than would have been expected from a single focal point. Alternatively, confocal microscopy allows images of increased contrast and detail to be obtained by analyzing fluorescence from only a single point in the specimen. A small point of light, usually supplied by a laser, is focused on the specimen at a particular depth. The emitted fluorescent light is then collected using a detector, such as a video camera. Before the emitted light reaches the detector, however, it must pass through a pinhole aperture (called a confocal aperture) placed at precisely the point where light emitted from the chosen depth of the specimen comes to a focus (Figure the plane of focus is able to reach the detector. Scanning across the specimen generates a two-dimensional image of the plane of focus, a much sharper image than that obtained with standard fluorescence microscopy (Figure 1.34). Moreover, a series of images obtained at different depths can be used to reconstruct a three-dimensional image of the sample.
Figure 1.33 Confocal microscopy A pinpoint of light is focused on the specimen at a particular depth, and emitted fluorescent light is collected by a detector. Before reaching the detector, the fluorescent light emitted by the specimen must pass through a confocal aperture placed at the point where light emitted from the chosen depth of the specimen comes into focus. As a result, only in-focus light is detected.
Figure 1.34 Confocal micrograph of human cells Microtubules are yellow, actin filaments are blue, and nuclei are red.
Multiphoton microscopy is an alternative to confocal microscopy that can be applied to living cells. The specimen is illuminated with a wavelength of light such that excitation of the fluorescent dye requires the simultaneous absorption of two or more photons (Figure 1.35). The probability of two photons simultaneously exciting the fluorescent dye is only significant at the point in the specimen upon which the input laser beam is focused, so fluorescence is only emitted from the plane of focus of the input light. This highly localized excitation automatically provides three-dimensional resolution, without the need for passing the emitted light through a pinhole aperture, as in confocal microscopy. Moreover, the localization of excitation minimizes damage to the specimen, allowing three-dimensional imaging of living cells.
Figure 1.35 Two-photon excitation microscopy Simultaneous absorption of two photons is required to excite the fluorescent dye. This only occurs at the point in the specimen upon which the input light is focused, so fluorescent light is only emitted from the chosen depth.