An epifluorescence microscope will be modified with novel hardware and software to simultaneously determine the fluorescence excitation spectrum of every pixel (or feature) in a field of view. Such technology will later be extended to the more difficult problem of obtaining fluorescence emission spectra. This Fluorescence Imaging Micro-Spectrophotometer (FIMS) will be based on KAIROS' High Resolution Imaging Microscope (HIRIM) which currently measures the ground state absorption spectrum of every feature in a scene. Hardware modifications to the microscope will include replacement of the standard (fixed wavelength) epifluorescence 'cubes' with a tunable system capable of scanning the entire visible spectrum. Software will be written to simultaneously acquire, process, and display hundreds of fluorescence spectra. Using a novel method for radiometrically calibrating the system response, this massively parallel fluorimeter will obtain excitation and emission spectra of a quality similar to that of a conventional fluorimeter. Using spectral deconvolution techniques, FIMS technology will enable cell biologists to quantitate the amount of fluorochromes present within individual features, even in cases where significant spectral overlap occurs. FIMS technology is essential in deconvoluting the overlapping spectra of recently engineered derivatives of the Green Fluorescent Protein (GFP), already in use for a variety of cellular assays.
FIMS constitutes the first example of the application of massively parallel imaging spectroscopy to fluorescence microscopy. This technology is essential in deconvoluting fluorophores with overlapping excitation or emission spectra so that meaningful quantitative information from each dye can be determined at subcellular resolution. We specifically address FRET and multispectral analysis of Green Fluorescent Protein (GFP). An estimated 8,000 biotech and biomedical researchers are now working with GFP.
