33.6: Confocal Fluorescence Microscopy
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore, very useful for examining thick specimens such as biofilms, which can be examined alive and unfixed.
The confocal microscopes use two pinholes—illumination, and emission pinhole, to modulate the laser beam to obtain clear, crisp images. The laser passes through the illumination pinhole and gets reflected by the dichroic mirror to scan the sample surface. An emission pinhole confocal with the illumination plane; focuses the emitted light reaching the detector. It eliminates light from non-focused z-planes reaching the detector to obtain high contrast two-dimensional images called the optical sections. A computer software program then merges optical sections from different focal planes to reconstruct a three-dimensional image.
Two types of confocal microscope are widely used based on their method of scanning the samples; laser scanning (LSCM) and spinning disc laser (SDLM) microscopy. In LSCM, a point laser scans each focal plane across the sample and collects the emitted fluorescence through a pinhole in detectors. These two-dimensional images, called the optical sections, can be stacked to reconstruct the three-dimensional image. In contrast, SDLM consists of two linked spinning disks with hundreds of pinholes. It allows rapid scanning of the sample surface at different planes and faster image capturing.
Limitations of confocal microscopy
In confocal microscopy, the limited wavelengths of light in the lasers are a disadvantage. The traditional fluorescence microscopes offer a wide range of illumination wavelengths, using mercury or xenon arc lamps as their illumination sources. The high intensity of the laser in early confocal microscopes was damaging for the cells, which has been overcome to a great extent in the multiphoton microscope systems.
