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Proceedings Paper

Hyperspectral unmixing for removing autofluorescence from paraffin-embedded formalin-fixed tissue sections
Author(s): P. Constantinou; B. C. Wilson; S. Damaskinos
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Paper Abstract

The use of digital fluorescence confocal microscopy in biological sciences has grown in recent decades due to the versatility of fluorescence imaging. The ability to selectively label specific morphological features, genetic mutations and/or chemical micro-environmental changes with discreet fluorescent labels allows a better understanding of the complex systems that regulate cellular processes. Specimens can range in size from single cells to tissue sections and tissue arrays, which can occupy the entire surface of a microscope slide (25mm x 70mm). Using a confocal scanning laser MACROscope, a wide-area confocal imaging system (Biomedical Photometrics Inc.), it is possible to image these large specimens at high resolution, without the need to tile many small microscope fields. A hyperspectral imaging (HSI) mode has been added to the MACROscope system to assess the use of HSI in the removal/separation of tissue autofluorescence from digital images of fluorescently-labeled paraffin-embedded, formalin-fixed tissue sections. In pathology and immunohistochemistry applications this autofluorescence can hinder, or even prevent, detection of the applied fluorescent label(s). In the present study, fluorescence emission from the specimen was sampled at ~7 nm bandwidths across 32 channels, amounting to viewing ~220 nm of the visible spectrum as a hyperspectral data cube. The data cube was then processed to remove the contributions from autofluorescence, leaving only the signal from the fluorophore(s) of interest. Comparisons are drawn from HSI obtained with a commercial hyperspectral confocal microscope (Zeiss LSM 510 META) employing image tiling. The initial results demonstrate the ability to spectrally unmix the tissue autofluorescence in large tissue sections.

Paper Details

Date Published: 13 October 2005
PDF: 10 pages
Proc. SPIE 5969, Photonic Applications in Biosensing and Imaging, 59692E (13 October 2005); doi: 10.1117/12.628178
Show Author Affiliations
P. Constantinou, Univ. of Toronto (Canada)
B. C. Wilson, Univ. of Toronto (Canada)
S. Damaskinos, Biomedical Photometrics Inc. (Canada)


Published in SPIE Proceedings Vol. 5969:
Photonic Applications in Biosensing and Imaging
Brian C. Wilson; Richard I. Hornsey; Warren C. W. Chan; Ulrich J. Krull; Robert A. Weersink; Kui Yu, Editor(s)

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