Research Papers: Imaging

Excitation-scanning hyperspectral imaging microscope

[+] Author Affiliations
Peter F. Favreau

University of South Alabama, Department of Chemical and Biomolecular Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

University of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

Clarissa Hernandez

University of South Alabama, Department of Chemical and Biomolecular Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

Tiffany Heaster

Mississippi State University, Department of Agricultural and Biological Engineering, Bos 9632, 130 Creelman St., Starkville, Mississippi 39762

Diego F. Alvarez

University of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

University of South Alabama, Department of Pharmacology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

University of South Alabama, Department of Internal Medicine, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

Thomas C. Rich

University of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

University of South Alabama, Department of Pharmacology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

University of South Alabama, College of Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

Prashant Prabhat

Semrock Inc., A Unit of IDEX, Corporation, 3625 Buffalo Road, Suite 6, Rochester, New York 14624

Silas J. Leavesley

University of South Alabama, Department of Chemical and Biomolecular Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

University of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

University of South Alabama, Department of Pharmacology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688

J. Biomed. Opt. 19(4), 046010 (Apr 11, 2014). doi:10.1117/1.JBO.19.4.046010
History: Received January 10, 2014; Revised March 19, 2014; Accepted March 20, 2014
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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 (300ms/wavelength band with excitation scanning versus 3s/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.

Figures in this Article
© 2014 Society of Photo-Optical Instrumentation Engineers

Citation

Peter F. Favreau ; Clarissa Hernandez ; Tiffany Heaster ; Diego F. Alvarez ; Thomas C. Rich, et al.
"Excitation-scanning hyperspectral imaging microscope", J. Biomed. Opt. 19(4), 046010 (Apr 11, 2014). ; http://dx.doi.org/10.1117/1.JBO.19.4.046010


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