Abstract

Live-cell fluorescence microscopy provides powerful insights into dynamic cellular processes but poses the challenge of maintaining cell viability during extended imaging sessions. This webinar presents an optimized protocol that carefully balances spatial resolution, temporal depth, and cell preservation using red and near-infrared viable probes combined with advanced imaging strategies to minimize phototoxicity.

The session will guide viewers through the full workflow, including probe selection, optimized concentrations, and incubation conditions for effective multi-labeling. The protocol employs advanced confocal laser scanning microscopy with hybrid detectors and a tunable white laser, integrated with fast fluorescence lifetime imaging (FLIM) and stimulated-emission-depletion (STED) nanoscopy to enable long-term imaging with high temporal and spatial resolution.

Additionally, lifetime-based approaches such as dye unmixing, environmental sensitivity analysis, and image denoising are utilized to enhance signal clarity and support flexible multiplexing. These combined techniques allow researchers to capture high-quality, long-duration live-cell and subcellular dynamics with minimal light-induced stress.

This webinar will walk participants through each critical step—from labeling to image acquisition and processing—demonstrating how FLIM and STED can be effectively applied for reproducible and minimally invasive live-cell imaging.

Highlights

- Strategies to minimize phototoxicity using red and near-infrared viable dyes.

- Optimized probe concentration and incubation for multi-labeling.

- Long-duration time-lapse imaging using advanced confocal microscopy with white laser and hybrid detectors.

- Integration of fast FLIM for lifetime-based multiplexing and dye unmixing.

- Combining STED with FLIM to improve signal-to-noise ratio and spatial resolution.