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Confocal fluorescence imaging of biological systems is an important method by which researchers can investigate
molecular processes occurring in live cells. We have developed a new 3D hyperspectral confocal fluorescence
microscope that can further enhance the usefulness of fluorescence microscopy in studying biological systems. The new
microscope can increase the information content obtained from the image since, at each voxel, the microscope records
512 wavelengths from the emission spectrum (490 to 800 nm) while providing optical sectioning of samples with
diffraction-limited spatial resolution. When coupled with multivariate curve resolution (MCR) analyses, the microscope
can resolve multiple spatially and spectrally overlapped emission components, thereby greatly increasing the number of
fluorescent labels, relative to most commercial microscopes, that can be monitored simultaneously. The MCR algorithm
allows the "discovery" of all emitting sources and estimation of their relative concentrations without cross talk, including
those emission sources that might not have been expected in the imaged cells. In this work, we have used the new
microscope to obtain time-resolved hyperspectral images of cellular processes. We have quantitatively monitored the
translocation of the GFP-labeled RelA protein (without interference from autofluorescence) into and out of the nucleus
of live HeLa cells in response to continuous stimulation by the cytokine, TNFα. These studies have been extended to
imaging live mouse macrophage cells with YFP-labeled RelA and GFP-labeled IRF3 protein. Hyperspectral imaging
coupled with MCR analysis makes possible, for the first time, quantitative analysis of GFP, YFP, and autofluorescence
without concern for cross-talk between emission sources. The significant power and quantitative capabilities of the new
hyperspectral imaging system are further demonstrated with the imaging of a simple fluorescence dye (SYTO 13)
traditionally used to stain the nucleus of live cells. We will demonstrate the microscope system's ability to actually
discover and quantify the presence of two separate SYTO 13 fluorescent species shifted in wavelength by only a few
nm. These two emission components exhibit very different spatial distributions in macrophage cells (i.e., nucleus vs.
cytoplasm + nucleus). Two highly overlapped autofluorescence components in addition to the two SYTO 13
components were also observed, and the spatial distributions of the two autofluorescence components were
quantitatively mapped throughout the cells in three dimensions.
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