A Fluorescence Image Deconvolution-Reconstruction Microscope will be used for a variety of different applications with living cells. Use will be restricted to observations of living cells that are present singly, or in thin specimens. This microscope generates a stack of fluorescent images at different focal planes, and then employs a set of sophisticated algorithms to determine point-spread functions for sources of fluorescence in the specimen. Out-of focus noise is subtracted from the signals in the image slices, and the slices are restacked to generate a three-dimensional reconstruction of the object at high resolution. Since small, bright objects placed against a dark background are detected as spots, it is possible to image fluorescence sources that are smaller than the theoretical limit of resolution for the microscope.
During the last twenty years, developments in the design of novel indicator fluorescent probes have enabled biologists to attack formerly intractable problems in cell biology and cell physiology. The cellular processes that have been amenable to this kind of analysis include photoreception, neuronal transmission, animal development, hormonal signaling, triggered gene expression, mitotic regulation, and chemotaxis. The developments in fluorescent dye design have moved in parallel with improvements in our ability to visualize and measure low light intensity signals from small numbers of molecules in living cells, with newly-designed optical microscopes and large pixel array CCD camera detectors. It is reasonable to expect that a significant expansion of analysis of processes in living cells will result from combined developments of reporter molecules and digital imaging technologies. It seems clear that the convergence of developments in photochemistry, biochemistry, cell biology, physiology and microscopy are all about to intersect within the living cell, and when accurate assessments of changing abundance and activity of a variety of molecules can be made in vivo and through time.
This microscope will be placed in an imaging facility that provides faculty, postdocs, graduate and undergraduate students with access to several high performance microscopes equipped with the capacity to view small quantities of fluorescent reporter molecules in living cells.
