Does digital deconvolution improve two-photon microscopy in deep tissue imaging?

In the past scientists have had difficulties visualizing cellular processes in thick tissue specimens. With conventional imaging techniques, fluorescence signals from above and below the focal plane create background noise that significantly degrades the resultant images. Today we have two-photon excitation microscopy, which surpasses other imaging systems in its ability to see further and more clearly into thick tissue. The two-photon system has an infrared pulsed laser light illumination that hits only at the focal plane, thus reducing autofluorescence and photobleaching. Although this technique is superior to others in fluorescence imaging, the two-photon images can still have a considerable amount of background glow deep down in the tissue. Due to this problem, we propose that digital deconvolution be used to improve these signals by eliminating the background noise in the two-photon images. The purpose of deconvolution is to undo the degradation of the image that was created by convolution. Deconvolution uses the entire fluorescent signal and digitally reverses it through the use of a point spread function. We have developed a model system for measuring the depth penetration of our two-photon system integrated with the Biorad MRC 600. With this system the deconvolved signal at different depths shows a better signal-to-noise (S/N) of the two-photon images than those without deconvolution. The biological applications of this process will be discussed in this paper.