Precise fluorophore lifetime mapping in live-cell, multi-photon excitation microscopy.
Fluorophore excited state lifetime is a useful indicator of micro-environment in cellular optical molecular imaging. For quantitative sensing, precise lifetime determination is important, yet is often difficult to accomplish when using the experimental conditions favored by live cells. Here we report the first application of temporal optimization and spatial denoising methods to two-photon time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to improve lifetime precision in live-cell images. The results demonstrated a greater than five-fold improvement in lifetime precision. This approach minimizes the adverse effects of excitation light on live cells and should benefit FLIM applications to high content analysis and bioimage informatics.
Chang
Ching-Wei
CW
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109-2099, USA.
Mycek
Mary-Ann
MA
D000465:Algorithms; D000818:Animals; D002452:Cell Count; D002460:Cell Line; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D017785:Photons; D034421:Sus scrofa; D013997:Time Factors
D005456:Fluorescent Dyes