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Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Zresolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET “controls” with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.
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Hyperspectral confocal microscopy in the short-wave infrared range
We demonstrate hyperspectral confocal microscopy in the short-wave infrared (SWIR) range of 1100–1600 nm using a wavelength-scanning laser in tandem with laser scanning confocal microscopy. Confocal microscopy in the SWIR range allows for high-resolution inspection of an integrated circuit (IC) chip, while hyperspectral imaging, together with a chemometric analysis, enables us to identify functional circuit block groups in the acquired image. With the extended capability, the developed instrument can be potentially used for inline inspection and non-invasive failure analysis of IC chips.
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- Award ID(s):
- 1808331
- PAR ID:
- 10433929
- Date Published:
- Journal Name:
- Optics Letters
- Volume:
- 48
- Issue:
- 15
- ISSN:
- 0146-9592
- Page Range / eLocation ID:
- 3993
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1364/OL.498290
