Signal strength and noise in confocal microscopy: Factors influencing selection of an optimum detector aperture | CU Experts

AbstractIn confocal microscopy, several factors influence the selection of an optimum size and geometry of detector aperture. These include (1) strength of signal from the specimen, (2) noise level in the system, (3) optical configuration of the microscope (e.g., reflection or fluorescence), (4) time available for signal accumulation, (5) specimen thickness, and (6) amount of reduction in axial and transverse resolution (from the theoretical maximum) that can be tolerated. It is shown both theoretically and experimentally that the size of the detector aperture critically influences the type and amount of system noise that is detected along with the specimen signal. It is also demonstrated that increasing the size of the detector pinhole does not appreciably increase the signal strength from any single thin plane, but only increases the sampling depth, enhancing brightness at the cost of a reduction in axial resolution. As a result, it is shown that there is no advantage, from the standpoint of signal strength, to using a slit aperture rather than a circular detector pinhole. Finally, it is concluded that all confocal microscopes should be designed to allow the user the capability of selecting ah optimum compromise detector aperture setting based on the particular specimen properties, type of microscopy (e.g., fluorescence or reflection) and resolution required.

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