Histogram Warping based Correction of the Light Attenuation with Depth of Data Sets Captured by a Confocal Microscope

M. Čapek, L. Kubínová, K. Hána, and P. Smrčka (Czech Republic)


Histogram warping, light attenuation, brightness and contrast correction, confocal microscopy


A confocal laser scanning microscope (CLSM) gives possibility to capture images from a biological specimen in different depths and obtain a stack of precisely registered fluorescent images, i.e. a 3D image. However, fluorescent image intensities from a CLSM suffer from light loss distortions. Particularly, images captured from deep layers of the specimen are often darker than images from the topmost layers. This effect causes problems in subsequent analysis of biological objects. Therefore, methods for compensation of the light attenuation with depth must be used. It is possible to solve this problem by a feedback-based tuning of the CLSM hardware during scanning, but in this paper we pay attention to post processing methods and developed such one. It is a two step algorithm. In the first step the algorithm determines a standard histogram of the stack of images and in the second step histograms of individual images are warped according to this standard histogram. The algorithm results in more stable brightness and contrast of images through the stack.

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