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Compensating for depth-dependent light attenuation at 3-D imaging with a confocal microscope

An account has been given previously of a 3-D image processing method to compensate for depth dependent light attenuation in images from a confocal microscope, working in the epifluorescence mode. A basic assumption is that there are regions of the specimen that are homogeneous in the sense that the attenuation is constant within the region. It is shown that a stack of 2-D histograms of adjacent images usually shows distinguishable features through which homogeneous regions of this kind can be traced. It is also shown how the attenuation factor of the region is obtained. Its inverse is the correction factor applicable to the region. The region may be extracted from the stack to be dealt with separately. The method has been further developed by introducing a dynamic display procedure. It makes both the search and the extraction more efficient. Further, techniques have been implemented to perform the compensation automatically by changing the PM tube voltage during the recording using computer control. In the present paper an account is given on these improvements. A review is also given of some fundamentals of the method and of an application of the method.