ArticleInternational journal of molecular sciences2021
Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies.
Article in International journal of molecular sciences, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- Enhancing STED microscopy via fluorescence lifetime unmixing and filtering in two-species SPLIT-STED.Methods in microscopy · 2026Article
- Combining Fluorescence and Magnetic Resonance Imaging in Drug Discovery-A Review.Pharmaceuticals (Basel, Switzerland) · 2025Review
- Investigation of quantitative structural changes in astrocyte cells after laser-induced shockwave.Biophotonics discovery · 2025Article
- Article
- Article
- Combining sophisticated fast FLIM, confocal microscopy, and STED nanoscopy for live-cell imaging of tunneling nanotubes.Life science alliance · 2024Article
- Identifying STEDable BFMolecules (Basel, Switzerland) · 2023Article
- Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes.ACS chemical biology · 2022Article
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Authors and funding
7 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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