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Article

Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies

1
Normandie University, UNIROUEN, INSERM, PRIMACEN, 76000 Rouen, France
2
INSERM, 76000 Rouen, France
3
Normandie University, UNICAEN, CEA, CNRS, ISTCT/CERVOxy Group, GIP CYCERON, 14000 Caen, France
4
Department of Pathology, CHU de Caen, 14033 Caen, France
5
Department of Neurosciences, Lerner Research Institute, Cleveland, OH 44195, USA
*
Author to whom correspondence should be addressed.
These authors contribute equally to this work.
Academic Editor: Kiryl Piatkevich
Int. J. Mol. Sci. 2021, 22(20), 11092; https://doi.org/10.3390/ijms222011092
Received: 20 August 2021 / Revised: 8 October 2021 / Accepted: 9 October 2021 / Published: 14 October 2021
Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction. View Full-Text
Keywords: live-cell imaging; sample preservation; fluorescence lifetime; confocal; FLIM; STED; FLIM-STED; τ separation live-cell imaging; sample preservation; fluorescence lifetime; confocal; FLIM; STED; FLIM-STED; τ separation
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MDPI and ACS Style

Bénard, M.; Schapman, D.; Chamot, C.; Dubois, F.; Levallet, G.; Komuro, H.; Galas, L. Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies. Int. J. Mol. Sci. 2021, 22, 11092. https://doi.org/10.3390/ijms222011092

AMA Style

Bénard M, Schapman D, Chamot C, Dubois F, Levallet G, Komuro H, Galas L. Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies. International Journal of Molecular Sciences. 2021; 22(20):11092. https://doi.org/10.3390/ijms222011092

Chicago/Turabian Style

Bénard, Magalie, Damien Schapman, Christophe Chamot, Fatéméh Dubois, Guénaëlle Levallet, Hitoshi Komuro, and Ludovic Galas. 2021. "Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies" International Journal of Molecular Sciences 22, no. 20: 11092. https://doi.org/10.3390/ijms222011092

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