Citation Index 1
Fluorescence Imaging Micro-Spectrophotometer (FIMS)
Douglas C. Youvan, William J. Coleman, Chris M. Silva, Julien Petersen,
Edward J. Bylina, and Mary M. Yang
Bldg. 62*
3350 Scott Blvd.
Santa Clara, CA 95054 USA
Contributed June 5, 1997
Abstract
A prototype spectrophotometer is described which combines the spatial resolution of an epifluorescence microscope (<1 mm) with the spectral resolution of a conventional fluorimeter (~2 nm). This Fluorescence Imaging MicroSpectrophotometer (FIMS) acquires fluorescence excitation or emission stacks within minutes and then rapidly compresses the data into a pseudocolored image, wherein each color represents a different spectral category. FIMS technology replaces the fixed-wavelength excitation and emission filters of a conventional epifluorescence microscope with fully tunable wavelength selection, thus allowing the fluorescence spectrum of every pixel in a scene to be simultaneously determined. Hyperspectral pseudocoloring software has been written which reduces these image stacks into a single image, while maintaining the radiometric calibration of each pixel’s spectrum and providing overall spectral statistics on each pseudocolored category. Categories are determined by rapid processing of all the pixels within a scene according to a variety of sorting algorithms which group together pixels exhibiting similar spectra. FIMS data acquisition and processing are demonstrated on spectral stacks containing data from: (1) six different fluorescent microspheres emitting between 520 and 720 nm, and (2) bacteria expressing various forms of the Green Fluorescent Protein (i.e., wild-type and variants with red-shifted excitation and red-shifted emission) excited between 355 and 505 nm.
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© Biotechnology et alia, 1997
*Current address: 10225 Barnes Canyon Rd., A110 San Diego, CA 92121 USA