![]() However, the actual dye concentration can be low to minimize the disturbance of biological systems: some instruments can track single fluorescent molecules. Biological samples are often treated with fluorescent dyes to make selected objects visible. Ĭonfocal microscopy provides the capacity for direct, noninvasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution compared to wide-field microscopy. To offset this drop in signal after the pinhole, the light intensity is detected by a sensitive detector, usually a photomultiplier tube (PMT) or avalanche photodiode, transforming the light signal into an electrical one. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity – so long exposures are often required. As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. ![]() ![]() In contrast, a confocal microscope uses point illumination (see Point Spread Function) and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal – the name 'confocal' stems from this configuration. ![]() All parts of the sample can be excited at the same time and the resulting fluorescence is detected by the microscope's photodetector or camera including a large unfocused background part. In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded evenly in light from a light source. The principle of confocal imaging was patented in 1957 by Marvin Minsky and aims to overcome some limitations of traditional wide-field fluorescence microscopes. ![]()
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