Combined FLIM, Confocal Microscopy, and STED Nanoscopy for Live-Cell Imaging.

Area of Science:

• Cell biology
• Microscopy techniques
• Biophotonics

Background:

• Live cell imaging requires balancing visualization with minimizing phototoxicity.
• Developing robust protocols is crucial for maintaining cell health during long-term experiments.
• Advanced microscopy offers enhanced resolution but necessitates careful parameter optimization.

Purpose of the Study:

• To provide a comprehensive guide for optimizing live cell fluorescence imaging.
• To detail strategies for minimizing light-induced cellular stress and maximizing cell survival.
• To enable high-resolution visualization of cellular dynamics using advanced microscopy.

Main Methods:

• Utilized time-lapse fluorescence microscopy with a pleural mesothelial cell line (H28).
• Employed red and near-infrared probes with low cytotoxicity and passive membrane penetration.
• Optimized advanced confocal laser scanning microscopy parameters, including white laser, STED nanoscopy, hybrid detectors, and fast FLIM module.
• Applied lifetime analysis techniques including lifetime imaging, dye unmixing, and denoising.

Main Results:

• Developed a labeling protocol balancing probe concentration and incubation time for multi-labeling.
• Achieved long-term time-lapse acquisitions with good spatial and temporal resolutions.
• Demonstrated successful multi-labeling of spectrally similar dyes using FLIM-based unmixing.
• Enhanced signal-to-noise ratio in confocal-STED images combined with FLIM.

Conclusions:

• The developed strategies effectively preserve cell viability during live cell fluorescence imaging.
• Advanced microscopy, combined with optimized probes and protocols, allows for detailed study of cellular dynamics.
• FLIM-based analysis provides flexibility for multiplexing and improving image quality in complex live cell experiments.