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Updated: May 24, 2025

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals
Published on: August 22, 2019
High-Speed Hyperspectral Imaging for Near Infrared Fluorescence and Environmental Monitoring
Jan Stegemann1,2, Franziska Gröniger2, Krisztian Neutsch1
1Department of Chemistry and Biochemistry, Ruhr University Bochum, 44801, Bochum, Germany.
Abstract:
Hyperspectral imaging captures both spectral and spatial information from a sample but is intrinsically slow. The near infrared (NIR, > 800 nm) is advantageous for imaging applications because it falls into the tissue transparency window and also contains vibrational overtone and combination modes useful for molecular fingerprinting. Here, fast hyperspectral NIR imaging is demonstrated using a spectral phasor transformation (HyperNIR). A liquid crystal variable retarder (LCVR) is used for tunable, wavelength-dependent sine- and cosine-filtering that transforms optical signals into a 2D spectral (phasor) space. Spectral information is thus obtained with just three images. The LCVR can be adjusted to cover a spectral range from 900 to 1600 nm in windows tunable from 50 to 700 nm, which enables distinguishing NIR fluorophores with emission peaks less than 5 nm apart. Furthermore, label-free hyperspectral NIR reflectance imaging is demonstrated to identify plastic polymers and monitor in vivo plant health. The approach uses the full camera resolution and reaches hyperspectral frame rates of 0.2 s-1, limited only by the switching rate of the LCVR. HyperNIR facilitates straightforward hyperspectral imaging for applications in biomedicine and environmental monitoring.
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