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A major benefit of fluorescence microscopy is the now plentiful selection of fluorescent markers. These labels can be chosen to serve complementary functions, such as tracking labeled subcellular molecules near demarcated organelles. However, with the standard 3 or 4 emission channels, multiple label detection is restricted to segregated regions of the electromagnetic spectrum, as in RGB coloring. Hyperspectral imaging allows the user to discern many fluorescence labels by their unique spectral properties, provided there is significant differentiation of their emission spectra. The cost of this technique is often an increase in gain or exposure time to accommodate the signal reduction from separating the signal into many discrete excitation or emission channels. Recent advances in hyperspectral imaging have allowed the acquisition of more signal in a shorter time period by scanning the excitation spectra of fluorophores. Here, we explore the selection of optimal channels for both significant signal separation and sufficient signal detection using excitation-scanning hyperspectral imaging. Excitation spectra were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Tunable filters had bandwidths between 13 and 17 nm. Scans utilized excitation wavelengths between 340 nm and 550 nm. Hyperspectral image stacks were generated and analyzed using ENVI and custom MATLAB scripts. Among channel consideration criteria were: number of channels, spectral range of scan, spacing of center wavelengths, and acquisition time.
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On-chip hyperspectral detectors for fluorescence lifetime imaging
Fluorescence Lifetime Imaging (FLIM) is a powerful technique that measures the decay time of fluorophores present in tissue samples alluding to their constituent molecules. FLIM has gained popularity in biomedical imaging for applications such as detecting cancerous tumors, surgical guidance, etc. However, conventional FLIM systems are limited by a reduced number of spectral bands and long acquisition time. Moreover, the large footprint, complexity, and cost of the instrumentation make it difficult for clinical applications. In this paper, we demonstrate a reconstruction-based hyperspectral detector that can resolve decay time and intensities in broad spectral ranges while providing high sensitivity, high gain, and fast response time. The hyperspectral detector is comprised of an array of efficient, ultrafast avalanche photodetectors integrated with nanophotonic structures. We utilize different nanostructures in the detectors to modulate light-matter interactions in spectral channels. This allows us to computationally reconstruct the spectral profile of the incoming fluorescence spectrum without the need for additional filters or dispersive optics. Also, the nanophotonic structures enhance efficiency (by a factor of 2 to 10 over different wavelengths) while providing fast response time. An innovative detector design has been employed to reduce the breakdown of the avalanche photodetectors to -7.8V while maintaining high gain (~50) across the spectral range. Therefore, enabling low light detection with a high signal-to-noise ratio for FLIM applications. Added spectral channels would provide valuable information about tissue materials, morphology, and disease diagnosis. Such innovative hyperspectral sensors can now be integrated on-chip capable of miniaturizing the FLIM system and making it a commercially viable tool for clinical use. This technology has the potential to revolutionize the current FLIM system with improved detection capabilities opening doors for new horizons.
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- Award ID(s):
- 2329884
- PAR ID:
- 10513196
- Editor(s):
- Goda, Keisuke; Tsia, Kevin K
- Publisher / Repository:
- SPIE
- Date Published:
- Journal Name:
- Proc. SPIE 12853, High-Speed Biomedical Imaging and Spectroscopy IX
- ISBN:
- 9781510669659
- Page Range / eLocation ID:
- 10
- Subject(s) / Keyword(s):
- Hyperspectral Imaging Reconstruction-based spectroscopy Photon-trapping nanostructures Fluorescence lifetime imaging microscopy Avalanche Photodiodes On-chip hyperspectral detectors
- Format(s):
- Medium: X
- Location:
- San Francisco, United States
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1117/12.3002476
