title: Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently-labeled cells in highly autofluorescent tissue.
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.
authors: Department of Chemical and Biomolecular Engineering, University of South Alabama, 307 University Blvd. N., EGLB 264, Mobile, AL 36688, USA. leavesley@jaguar1.usouthal.edu
authors: Silas J
authors: SJ
authors: Leavesley
authors: Naga
authors: N
authors: Annamdevula
authors: John
authors: J
authors: Boni
authors: Samantha
authors: S
authors: Stocker
authors: Kristin
authors: K
authors: Grant
authors: Boris
authors: B
authors: Troyanovsky
authors: Thomas C
authors: TC
authors: Rich
authors: Diego F
authors: DF
authors: Alvarez
mesh_terms: D000818:Animals; D003952:Diagnostic Imaging / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D049408:Luminescent Agents / Q000737:chemistry; D008168:Lung / Q000166:cytology* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation
chemical_list: D049408:Luminescent Agents; D049452:Green Fluorescent Proteins