In vitro and in vivo NIR fluorescence lifetime imaging with a time-gated SPAD camera

Abstract

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated intensified charge-coupled device (ICCD) camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

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

“…Following by a single shot of WF acquisition, temporal point spread functions (TPSFs) were captured at each scanning position by the SPAD and fed into a mono-exponential fitting model for the pixel-wise lifetime quantification using the open source software AlliGator. 17, 18 One can follow the workflow of the TD data collection and lifetime quantification laid out in our previous work. 11 WF and SPAD-based intensities measured by the InGaAs camera and the SPAD, the lifetime quantification results, as well as the normalized IRF and representative decays of four fluorophores are shown in Fig.…”

in vivoquantitative FRET small animal imaging: intensity versus lifetime-based FRET

“…5B). Importantly, MFLI FRET has been expanded to measure antibody-target engagement using NIR-labeled trastuzumab, an anti-HER2 clinically relevant antibody in HER2 breast tumor xenograft models (41, 48). Therefore, NIR MFLI FRET imaging is a quantitative and non-invasive tool for the optimization of targeted drug delivery systems based on receptor-ligand or antibody-target engagement in tumors in vivo.…”

“…This method neglects environment-dependent changes of lifetime between mouse organs. Though this assumption is valid herein, it may very well not be for other applications (e.g., pH dependence on lifetime (48)). All other analysis steps and calculation of FRET efficiency were performed similarly as described above for the dsDNA sample.…”

“…Using intensity-and lifetime-based FRET microscopy, we have demonstrated that protein ligands (e. g. transferrin: Tf), do bind extracellular domains of membrane-bound receptors (e. g. transferrin receptor: TfR) (45,46). Moreover, in vivo MFLI FRET measurements have been successfully validated via ex vivo histochemistry, establishing that in vivo FRET signal directly reports on receptor-ligand engagement in intact live animals (40,41,44,47,48).…”

“…transferrin receptor: TfR) (45, 46). Moreover, in vivo MFLI FRET measurements have been successfully validated via ex vivo histochemistry, establishing that in vivo FRET signal directly reports on receptor-ligand engagement in intact live animals (40, 41, 44, 47, 48).…”