Compensation of the Brightness and Contrast Attenuation with Depth in Confocal Microscopy

Intensities of fluorescent images captured by a confocal laser scanning microscope (CLSM) from deep layers of a specimen are often darker than images from the top most layers due to absorption and scattering of both excitation and fluorescent light. These effects cause problems in subsequent analysis of biological objects. Methods [e.g. 1, 2] used for solution of this problem are generally based on the post-processing of data. We have chosen, first, an approach, which lies in software optimization of the microscope hardware during scanning and, second, a postprocessing method based on a simple optimization of image intensities in the image stack.