Journal
JOURNAL OF BIOPHOTONICS
Volume 5, Issue 1, Pages 67-84Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jbio.201100066
Keywords
Hyperspectral analysis; lung autofluorescence; pulmonary microvascular endothelial cells
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Funding
- NIH [R01HL094455, S10RR027535]
- Alabama Space Grant Consortium
- University of South Alabama
- International Society for the Advancement
- [AHA0835134N]
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Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP-expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single-band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues. (C) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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