We request funds to set up a facility for 4-D light microscopy. After examining several alternatives, we have selected a microscope with associated light sources, filters, and motor drives, together with a computer, cooled CCD camera and software package, selected and designed by Intelligent Imaging of Denver, Colorado. This proposal describes scientific projects from the labs of five major and three minor users whose research will depend heavily on the capabilities of this instrument to image fluorescent signals from through focus series, obtained with a variety of fluorophores, and to deconvolve the out-of- focus transfer function of the microscope from these data, producing more reliable 3-D models of cellular fluorescence. The projects described also include work with live cells. Here we will use differential interference contrast, and/or the fluorescence of tagged cellular components, to follow event in vivo. The efficient optical train requested, together with the sensitive CCD camera, will be used in conjunction with a motorized stage with X,Y,Z control to allow 4-D imaging of multiple cells seriatim, greatly increasing the rate of data acquisition. Specific applications will use vital fluorescent dyes, different alleles of the green fluorescent protein (GFP), and/or purified proteins labeled with different fluorophores and injected back into living cells. A capability for ratio imaging is included to allow both normalization of specific protein fluorescence with non-specific cellular labeling and to permit measurement of ion concentrations in different cellular compartments. The proposal describes facilities and personnel for technical support of the instrument and our plans for instrument management and maintenance over a period extending far beyond the duration of the grant itself.
