Fluorescence lifetime imaging (FLI) is increasingly recognized as a powerful tool for biochemical and cellular investigations, including in vivo applications. Fluorescence lifetime is an intrinsic characteristic of any fluorescent dye which, to a large extent, does not depend on excitation intensity and signal level. In particular, it allows distinguishing dyes with similar emission spectra, offering additional multiplexing capabilities. However, in vivo FLI in the visible range is complicated by the contamination by (i) tissue autofluorescence, which decreases contrast, and by (ii) light scattering and absorption in tissues, which significantly reduce fluorescence intensity and modify the temporal profile of the signal. Here, we demonstrate how these issues can be accounted for and overcome, using a new time-gated single-photon avalanche diode array camera, SwissSPAD2, combined with phasor analysis to provide a simple and fast visual method for lifetime imaging. In particular, we show how phasor dispersion increases with increasing scattering and/or decreasing fluorescence intensity. Next, we show that as long as the fluorescence signal of interest is larger than the phantom autofluorescence, the presence of a distinct lifetime can be clearly identified with appropriate background correction. We use these results to demonstrate the detection of A459 cells expressing the fluorescent protein mCyRFP1 through highly scattering and autofluorescent phantom layers. These results showcase the possibility to perform FLI in challenging conditions, using standard, bright, visible fluorophore or fluorescence proteins.
Bibliographical noteFunding Information:
This work was funded in part by NIH Grant GM 095904 and CRCC Grant CRR-18-523872 (UCLA) and in part by the Swiss National Science Foundation Grant 166289 and The Netherlands Organization for Scientific Research Project 13916. R.A. thanks Prof. Dror Fixler from the Faculty of Engineering at Bar Ilan University, Israel, for financial support of this work.
© 2019 American Chemical Society.
- fluorescence lifetime imaging
- phasor lifetime analysis
- scattering medium
- single-photon detection
- time-gated camera