An FFT-based method for attenuation correction in fluorescence confocal microscopy
J.B.T.M. Roerdink, M. Bakker. An FFT-based method for attenuation correction in fluorescence confocal microscopy. J. Microscopy 169, 1993, pp. 3-14.
A problem in three-dimensional imaging by a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. In this paper we propose a new method to correct for these effects. Our approach, valid for weak attenuation, consists in multiplying the measured fluorescent intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the Fast Fourier Transform. We give analytical and numerical estimates as to the degree of attenuation under which our method is valid, and apply the method to various test images. We also show a restoration of a real CSLM image. Finally the method is compared with a recent iterative method with regard to numerical accuracy and computational efficiency.
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