title: Excitation-scanning hyperspectral imaging microscope.
abstract: Hyperspectral imaging is a versatile tool that has recently been applied to a variety of biomedical applications, notably live-cell and whole-tissue signaling. Traditional hyperspectral imaging approaches filter the fluorescence emission over a broad wavelength range while exciting at a single band. However, these emission-scanning approaches have shown reduced sensitivity due to light attenuation from spectral filtering. Consequently, emission scanning has limited applicability for time-sensitive studies and photosensitive applications. In this work, we have developed an excitation-scanning hyperspectral imaging microscope that overcomes these limitations by providing high transmission with short acquisition times. This is achieved by filtering the fluorescence excitation rather than the emission. We tested the efficacy of the excitation-scanning microscope in a side-by-side comparison with emission scanning for detection of green fluorescent protein (GFP)-expressing endothelial cells in highly autofluorescent lung tissue. Excitation scanning provided higher signal-to-noise characteristics, as well as shorter acquisition times (300  ms/wavelength band with excitation scanning versus 3  s/wavelength band with emission scanning). Excitation scanning also provided higher delineation of nuclear and cell borders, and increased identification of GFP regions in highly autofluorescent tissue. These results demonstrate excitation scanning has utility in a wide range of time-dependent and photosensitive applications.
authors.affiliation: University of South Alabama, Department of Chemical and Biomolecular Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688bUniversity of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Favreau
authors.affiliation: University of South Alabama, Department of Chemical and Biomolecular Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688.
authors.forename: Clarissa
authors.initials: C
authors.lastname: Hernandez
authors.affiliation: Mississippi State University, Department of Agricultural and Biological Engineering, Bos 9632, 130 Creelman St., Starkville, Mississippi 39762.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Heaster
authors.affiliation: University of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688dUniversity of South Alabama, Department of Pharmacology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688eUniversity of South Alabama, Department of Interna.
authors.forename: Diego F
authors.initials: DF
authors.lastname: Alvarez
authors.affiliation: University of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688dUniversity of South Alabama, Department of Pharmacology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688fUniversity of South Alabama, College of Engineerin.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Rich
authors.affiliation: Semrock Inc., A Unit of IDEX, Corporation, 3625 Buffalo Road, Suite 6, Rochester, New York 14624.
authors.forename: Prashant
authors.initials: P
authors.lastname: Prabhat
authors.affiliation: University of South Alabama, Department of Chemical and Biomolecular Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688bUniversity of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688dUniversity of South Alaba.
authors.forename: Silas J
authors.initials: SJ
authors.lastname: Leavesley
mesh_terms: D000818:Animals; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008168:Lung / Q000737:chemistry; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D051381:Rats; D059629:Signal-To-Noise Ratio
chemical_list: D049452:Green Fluorescent Proteins
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