Three-Dimensional Microscopy: Image Processing For High Resolution Subcellular Imaging

Recent technological advances now make it practical to record three-dimensional data from biological specimens using fluorescence light microscopy. When three-dimensional images are collected using a conventional microscope, each observed section contains in-focus information from the parts of the sample at the focal plane and out-of-focus information from the remainder of the sample. The imaging process can be characterized as a convolution of the sample with the point spread function (PSF) of the microscope. We have experimentally determined the PSF for an epifluorescence microscope using high numerical aperture oil and water immersion lenses. Several methods for the processing of the observed data are discussed, with the best results obtained by constrainediterative deconvolution methods.