Characterization of fluorescence lifetime of organic fluorophores for molecular imaging in the SWIR window

Abstract

Significance: Fluorescence lifetime imaging in the short-wave infrared (SWIR) is expected to enable high resolution multiplexed molecular imaging in highly scattering tissue. Aim: To characterize the brightness and fluorescence lifetime of commercially available organic SWIR fluorophores and benchmark them against the tail emission of conventional NIR-excited probes. Approach: Characterization was performed through our established Time-domain Mesoscopic Fluorescence Molecular Tomography (TD-MFMT) system integrated around a TCSPC-SPAD array. Brightness and fluorescence lifetime was measured for NIR and SWIR probes above 1000 nm. Simultaneous probe imaging was then performed to assess their potential for multiplexed studies. Results: NIR probes outperformed SWIR probes in brightness while the mean fluorescence lifetimes of the SWIR probes were extremely short. The phantom study demonstrated the feasibility of lifetime multiplexing in the SWIR window with both NIR and SWIR probes. Conclusions: Long tail emission of NIR probes outperformed the SWIR probes in brightness beyond 1000 nm. Fluorescence lifetime was readily detectable in the SWIR window, where the SWIR probes showed shorter lifetimes compared to the NIR probes. We demonstrate the feasibility of lifetime multiplexing in the SWIR window, which paves the way for in vivo multiplexed studies of intact tissues at improved resolution.

Advancements in fluorescence lifetime imaging microscopy Instrumentation: Towards high speed and 3D

“…It holds the potential for achieving micron-scale imaging resolution and penetrating tissue depths of several centimeters, rendering it a highly valuable tool for deep tissue imaging. In particular, recent studies have unveiled that many established NIR-I fluorophores can double as effective contrast agents in NIR-II FLIM imaging, courtesy of their long-tailed spectral emissions [ 69 , 70 ] ( Fig. 5f ).…”

“…SWIR fluorescence enables deep tissue imaging beyond the NIR window [ 69 ]. (f) Lifetime characterization of SWIR fluorophores [ 70 ].…”