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Proceedings Paper

Compact spectrograph for hyperspectral imaging with overlapping fluorescent reporters
Author(s): Nathan A. Hart; Holly C. Gibbs; Alvin T. Yeh
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Paper Abstract

A compact, prism-based spectrograph was designed for 2-photon light sheet microscopy based on broadband, ultrashort optical pulses that corrects for chromatic aberrations and distortion. Gaussian or Bessel beams of broadband optical pulses may be used to create narrow cylindrical, nonlinear excitation volumes from which fluorophores emit characteristic spectra. In practice, a slit aperture is often used to optically section the excitation volume before being imaged onto the camera chip. Optical dispersion of the fluorescence in the transverse direction of the imaging slit allows for hyperspectral image acquisition. Hyperspectral imaging systems may be used to simultaneously image and segment multiple fluorescent reporters in biological tissue. However, current systems have properties which are undesirable for low-light microscopy including chromatic aberrations, distortion, low optical transmission, and large footprint which consumes precious laboratory real estate. Here, we present a novel spectrograph that has sufficient optical transmission, achromaticity, and distortion correction for microscopy of fluorescent reporters spanning the visible spectrum (400 – 650 nm). Across the spatial dimension of the excitation volume, the spectrograph has uniform separation of the spectral bands while maintaining a compact size and profile. A second order deconvolution algorithm is used to spectrally deconvolve overlapping fluorophores.

Paper Details

Date Published: 20 February 2020
PDF: 10 pages
Proc. SPIE 11245, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVII, 112450L (20 February 2020); doi: 10.1117/12.2546894
Show Author Affiliations
Nathan A. Hart, Texas A&M Univ. (United States)
Holly C. Gibbs, Texas A&M Univ. (United States)
Alvin T. Yeh, Texas A&M Univ. (United States)

Published in SPIE Proceedings Vol. 11245:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVII
Thomas G. Brown; Tony Wilson; Laura Waller, Editor(s)

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