Excitation-scanning hyperspectral imaging microscope

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....

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Bibliographic Details
Published in:Journal of biomedical optics 2014-04, Vol.19 (4), p.046010-046010
Main Authors: Favreau, Peter F, Hernandez, Clarissa, Heaster, Tiffany, Alvarez, Diego F, Rich, Thomas C, Prabhat, Prashant, Leavesley, Silas J
Format: Article
Language:English
Subjects:
Animals
Emission
Excitation
Filtering
Filtration
Green Fluorescent Proteins
Hyperspectral imaging
Image Processing, Computer-Assisted - methods
Lung - chemistry
Male
Microscopes
Microscopy, Fluorescence - methods
Optical Imaging - methods
Rats
Research Papers: Imaging
Scanning
Signal-To-Noise Ratio
Wavelengths
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Summary: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.
ISSN:1083-3668
1560-2281
DOI:10.1117/1.JBO.19.4.046010