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Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based
Magalie Bénard1, Damien Schapman1, Christophe Chamot2
1Normandie University, UNIROUEN, INSERM, PRIMACEN, 76000 Rouen, France.
International Journal of Molecular Sciences
|October 23, 2021
Summary
This study optimizes live-cell imaging by reducing light exposure using red/near-infrared dyes and fluorescence lifetime (τ) analysis. This approach enhances sample preservation and enables flexible, fast imaging of multi-labeled cells.
Area of Science:
- Microscopy and imaging techniques
- Cell biology
- Biophotonics
Background:
- Live-cell imaging is crucial for understanding cellular processes but is limited by light exposure and sample preservation.
- Advanced light microscopy requires optimization to balance image quality with minimal phototoxicity.
Purpose of the Study:
- To optimize advanced light microscopy for live-cell imaging by reducing light exposure.
- To exploit fluorescence lifetime (τ) of red/near-infrared dyes for improved imaging strategies.
- To provide a guide for selecting appropriate lifetime-based imaging approaches.
Main Methods:
- Characterization of red/near-infrared laser lines and high numerical aperture objectives for signal transmission and low irradiance.
- Utilized hybrid detectors (HyD-S, HyD-X, HyD-R) for red/near-infrared photon counting and τ separation.
- Compared lifetime-based imaging methods: confocal microscopy with coarse τ separation, fluorescence lifetime microscopy (FLIM) with phasor plot analysis, and stimulated emission depletion (STED) nanoscopy with lifetime weighting.
Main Results:
- Red/near-infrared laser lines with a high NA objective provided high fluorescence transmission, low irradiance, and super-resolution.
- Hybrid detectors demonstrated suitability for red/near-infrared photon counting and τ separation.
- Phasor plot analysis in FLIM and photon-counting mode of HyDs enabled flexible and fast imaging of multi-labeled living cells.
Conclusions:
- The choice of imaging approach depends on fluorochrome properties and compatibility with techniques like STED.
- Combining red/near-infrared dyes with lifetime-based strategies offers new avenues for live-cell imaging.
- This method enhances sample preservation by reducing acquisition time and light exposure.
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