Abstract
The availability of the ratiometric Ca2+ indicator dyes, fura-2, and indo-1, and advances in digital imaging and computer technology have made it possible to detect Ca2+ changes in single cells with high temporal and spatial resolution. However, the optical properties of the conventional epifluorescence microscope do not produce a perfect image of the specimen. Instead, the observed image is a spatial low pass filtered version of the object and is contaminated with out of focus information. As a result, the image has reduced contrast and an increased depth of field. This problem is especially important for measurements of localized Ca2+ concentrations. One solution to this problem is to use a scanning confocal microscope which only detects in focus information, but this approach has several disadvantages for low light fluorescence measurements in living cells. An alternative approach is to use digital image processing and a deblurring algorithm to remove the out of focus information by using a knowledge of the point spread function of the microscope. All of these algorithms require a stack of two-dimensional images taken at different focal planes, although the "nearest neighbor deblurring" algorithm only requires one image above and below the image plane. We have used a modification of this scheme to construct a simple inverse filter, which extracts optical sections comparable to those of the nearest neighbors scheme, but without the need for adjacent image sections. We have used this "no neighbors" processing scheme to deblur images of fura-2-loaded mast cells from beige mice and generate high resolution ratiometric Ca2+ images of thin sections through the cell. The shallow depth of field of these images is demonstrated by taking pairs of images at different focal planes, 0.5-microns apart. The secretory granules, which exclude the fura-2, appear in focus in all sections and distinct changes in their size and shape can be seen in adjacent sections. In addition, we show, with the aid of model objects, how the combination of inverse filtering and ratiometric imaging corrects for some of the inherent limitations of using an inverse filter and can be used for quantitative measurements of localized Ca2+ gradients. With this technique, we can observe Ca2+ transients in narrow regions of cytosol between the secretory granules and plasma membrane that can be less than 0.5-microns wide. Moreover, these Ca2+ increases can be seen to coincide with the swelling of the secretory granules that follows exocytotic fusion.