Display Settings:

Format

Send to:

Choose Destination
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
J Biophotonics. 2012 Jan;5(1):67-84. doi: 10.1002/jbio.201100066. Epub 2011 Oct 11.

Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently-labeled cells in highly autofluorescent tissue.

Author information

  • 1Department of Chemical and Biomolecular Engineering, University of South Alabama, 307 University Blvd. N., EGLB 264, Mobile, AL 36688, USA. leavesley@jaguar1.usouthal.edu

Abstract

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.

Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PMID:
21987373
[PubMed - indexed for MEDLINE]
PMCID:
PMC3517021
Free PMC Article

Images from this publication.See all images (8)Free text

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for John Wiley & Sons, Inc. Icon for PubMed Central
    Loading ...
    Write to the Help Desk