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Favreau PF
; Hernandez C
; Heaster T
; Alvarez DF
; Rich TC
; Prabhat P
; Leavesley SJ
J Biomed Opt
2014[Apr]; 19
(4
): 046010
PMID24727909
show ga
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.
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