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Proceedings Paper

Hyperspectral confocal fluorescence imaging of cells
Author(s): David M. Haaland; Howland D. T. Jones; Michael B. Sinclair; Bryan Carson; Catherine Branda; Jens F. Poschet; Roberto Rebeil; Bing Tian; Ping Liu; Allan R. Brasier
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Paper Abstract

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.

Paper Details

Date Published: 15 October 2007
PDF: 9 pages
Proc. SPIE 6765, Next-Generation Spectroscopic Technologies, 676509 (15 October 2007); doi: 10.1117/12.738152
Show Author Affiliations
David M. Haaland, Sandia National Labs. (United States)
Howland D. T. Jones, Sandia National Labs. (United States)
Michael B. Sinclair, Sandia National Labs. (United States)
Bryan Carson, Sandia National Labs. (United States)
Catherine Branda, Sandia National Labs. (United States)
Jens F. Poschet, Sandia National Labs. (United States)
Roberto Rebeil, Sandia National Labs. (United States)
Bing Tian, The Univ. of Texas Medical Branch at Galveston (United States)
Ping Liu, The Univ. of Texas Medical Branch at Galveston (United States)
Allan R. Brasier, The Univ. of Texas Medical Branch at Galveston (United States)

Published in SPIE Proceedings Vol. 6765:
Next-Generation Spectroscopic Technologies
Christopher D. Brown; Mark A. Druy; John P. Coates, Editor(s)

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