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Journal of Biomedical Optics

Excitation-scanning hyperspectral imaging microscope
Author(s): Peter F. Favreau; Clarissa Hernandez; Tiffany Heaster; Diego F. Alvarez; Thomas C. Rich; Prashant Prabhat; Silas J. Leavesley
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Paper 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.

Paper Details

Date Published: 11 April 2014
PDF: 11 pages
J. Biomed. Opt. 19(4) 046010 doi: 10.1117/1.JBO.19.4.046010
Published in: Journal of Biomedical Optics Volume 19, Issue 4
Show Author Affiliations
Peter F. Favreau, Univ. of South Alabama (United States)
Clarissa Hernandez, Univ. of South Alabama (United States)
Tiffany Heaster, Mississippi State Univ. (United States)
Diego F. Alvarez, Univ. of South Alabama (United States)
Thomas C. Rich, Univ. of South Alabama (United States)
Prashant Prabhat, Semrock, Inc. (United States)
Silas J. Leavesley, Univ. of South Alabama (United States)

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