Abstract
In this paper, we demonstrate the ability of structured illumination microscopy to enhance the ability of fluorescence lifetime imaging to resolve fluorescence lifetimes in relatively thick samples that possess distinct but spectrally overlapping fluorescent layers. Structured illumination fluorescent lifetime imaging microscopy (SI-FLIM) is shown to be able to accurately reconstruct lifetime values in homogenous fluorophore samples (POPOP, NADH, and FAD) as well as accurately measure fluorescent lifetime in two layer models that are layered with NADH/FAD over POPOP, where NADH/FAD and POPOP have spectral overlap. Finally, the ability of SI-FLIM was demonstrated in a hamster cheek pouch ex vivo to show that more accurate lifetimes could be measured for each layer of interest in the oral mucosa (epithelium and submucosa).
Highlights
Fluorescence lifetime imaging microscopy (FLIM) is a widely studied imaging modality that is most commonly used for its ability to distinguish multiple fluorophores of similar spectral characteristics, allowing for the chemical analysis of microscopy samples via both their fluorescence spectral and temporal properties
Several techniques exist for measuring lifetimes such as time correlated single photon counting (TCSPC), direct decay via high bandwidth photomultiplier tube (PMT), and wide-field imaging via intensified CCD (ICCD) [2]
The Structured illumination fluorescent lifetime imaging microscopy (SI-FLIM) system was validated in multiple steps with both the FLIM and Structured illumination microscopy (SIM) components being validated independently
Summary
Fluorescence lifetime imaging microscopy (FLIM) is a widely studied imaging modality that is most commonly used for its ability to distinguish multiple fluorophores of similar spectral characteristics, allowing for the chemical analysis of microscopy samples via both their fluorescence spectral and temporal properties. It is attractive because it allows for high resolution spatial analysis of chemical distributions with little to no dependence on the intensity or concentration of the fluorophores under scrutiny [1]. In contrast to other FLIM methods that are typically based on laser scanning techniques, wide-field imaging decouples field of view from imaging speed, allowing for faster acquisition that is less susceptible to motion artifacts, a necessity for in vivo FLIM
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