FIGURE 4-18 Light paths for two types of confocal microscopy. Both types of microscopy are assembled around a conventional fluorescence microscope (yellow shading). (a) Light path in a point-scanning confocal microscope. A single-wavelength point of light from an appropriate laser is reflected off a dichroic mirror and bounces off two scanning mirrors and from there passes through the objective lens to illuminate a spot in the specimen. The scanning mirrors rock back and forth in such a way that the light scans the specimen in a raster fashion (see green lines in the specimen). The fluorescence emitted by the specimen passes back through the objective lens and is bounced off the scanning mirrors onto the dichroic mirror. This allows the light to pass through toward the pinhole. This pinhole excludes light from out-of-focus focal planes, so the light reaching the photomultiplier tube comes almost exclusively from the illuminated spot in the focal plane. A computer then takes these signals and reconstructs the image. (b) Light path in a spinning disk confocal microscope. Here, instead of using two scanning mirrors, the beam from the laser is spread to illuminate pinholes on the coupled spinning disks, the first consisting of microlenses to focus the light on pinholes in the second disk. The excitation light passes through the objective lens to provide point illumination of a number of spots in the specimen. The fluorescence emitted passes back through the objective lens and through the holes in the spinning disk, and is then bounced off a dichroic mirror into a sensitive digital camera. The pinholes in the disk are arranged so that as it spins, it rapidly illuminates all parts of the specimen several times. As the disk spins as fast as 3000 rpm, very dynamic events in live cells can be recorded.