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Abstract Nonlinear optical imaging modalities, such as stimulated Raman scattering (SRS) microscopy, use pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we used bulk RNA sequencing, quantitative measurement of cell proliferation, and fluorescent measurement of the generation of reactive oxygen species to assess phototoxic effects of near-IR pulsed laser radiation, at different time scales, for laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant change in mouse Neuro2A cells after laser exposure. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.
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Tear Down the Fluorescent Curtain: A New Fluorescence Suppression Method for Raman Microspectroscopic Analyses
Abstract The near exponential proliferation of published Raman microspectroscopic applications over the last decade bears witness to the strengths and versatility of this technology. However, laser-induced fluorescence often severely impedes its application to biological samples. Here we report a new approach for near complete elimination of laser-induced background fluorescence in highly pigmented biological specimens (e.g., microalgae) enabling interrogation by Raman microspectroscopy. Our simple chemiphotobleaching method combines mild hydrogen peroxide oxidation with broad spectrum visible light irradiation of the entire specimen. This treatment permits observing intracellular distributions of macromolecular pools, isotopic tracers, and even viral propagation within cells previously not amenable to Raman microspectroscopic examination. Our approach demonstrates the potential for confocal Raman microspectroscopy becoming an indispensable tool to obtain spatially-resolved data on the chemical composition of highly fluorescent biological samples from individual cells to environmental samples.
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- Award ID(s):
- 1833053
- PAR ID:
- 10153843
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 9
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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