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

Smith, Jason T.; Rudkouskaya, Alena; Gao, Shan; Gupta, Juhi M.; Ulku, Arin; Bruschini, Claudio; Charbon, Edoardo; Weiss, Shimon; Barroso, Margarida; Intes, Xavier; Michalet, Xavier

doi:10.1364/optica.454790

Cited by 34 publications

(40 citation statements)

References 54 publications

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“…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.…”

Section: Methods and Resultsmentioning

confidence: 99%

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

Chavez

Gao

Intes

2022

Preprint

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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.

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Section: Methods and Resultsmentioning

confidence: 99%

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

Chavez

Gao

Intes

2022

Preprint

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 90%

“…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).…”

Section: Introductionmentioning

confidence: 97%

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

Smith

Sinsuebphon

Rudkouskaya

et al. 2023

Preprint

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Förster Resonance Energy Transfer (FRET) microscopy is used in numerous biophysical and biomedical applications to monitor inter- and intramolecular interactions and conformational changes in the 2-10 nm range. FRET is currently being extended toin vivooptical imaging, its main application being in quantifying drug-target engagement or drug release in animal models of cancer using organic dye or nanoparticle-labeled probes. Herein, we compared FRET quantification using intensity-based FRET (sensitized emission FRET analysis using 3-cube IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a time-resolved ICCD system in small animal opticalin vivoimaging. The analytical expressions and experimental protocols required to quantify FRET efficiency 𝐸 and the fraction of donor molecules actually involved in FRET, 𝑓𝐷, are described in detail for both methodologies. Dynamicin vivoFRET quantification of transferrin receptor-transferrin binding was acquired in live intact nude mice upon intravenous injection of near infrared-labeled transferrin FRET pair and benchmarked againstin vitroFRET using hybridized oligonucleotides. Even though bothin vivoimaging techniques provided similar FRET quantification trends of receptor-ligand engagement, we demonstrate that MFLI FRET has significant advantages. Whereas the sensitized emission FRET approach using IVIS imager required 9 measurements (8 of which are used for calibration) acquired from three mice, MFLI FRET needed only one measurement collected from a single mouse. Hence, MFLI represents the method of choice for FRET measurements in intact, live mice to assess targeted drug delivery in longitudinal preclinical studies, as well as for many otherin vivobiomedical applications.

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Fluorescence Lifetime Imaging for Quantification of Targeted Drug Delivery in Varying Tumor Microenvironments

Verma,

Pandey,

Sherry

et al. 2024

Advanced Science

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Trastuzumab (TZM) is a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2) and is clinically used for the treatment of HER2‐positive breast tumors. However, the tumor microenvironment can limit the access of TZM to the HER2 targets across the whole tumor and thereby compromising TZM's therapeutic efficacy. An imaging methodology that can non‐invasively quantify the binding of TZM‐HER2, which is required for therapeutic action, and distribution within tumors with varying tumor microenvironments is much needed. Near‐infrared (NIR) fluorescence lifetime (FLI) Forster Resonance Energy Transfer (FRET) is performed to measure TZM‐HER2 binding, using in vitro microscopy and in vivo widefield macroscopy, in HER2 overexpressing breast and ovarian cancer cells and tumor xenografts, respectively. Immunohistochemistry is used to validate in vivo imaging results. NIR FLI FRET in vitro microscopy data show variations in intracellular distribution of bound TZM in HER2‐positive breast AU565 and AU565 tumor‐passaged XTM cell lines in comparison to SKOV‐3 ovarian cancer cells. Macroscopy FLI (MFLI) FRET in vivo imaging data show that SKOV‐3 tumors display reduced TZM binding compared to AU565 and XTM tumors, as validated by ex vivo immunohistochemistry. Moreover, AU565/XTM and SKOV‐3 tumor xenografts display different amounts and distributions of TME components, such as collagen and vascularity. Therefore, these results suggest that SKOV‐3 tumors are refractory to TZM delivery due to their disrupted vasculature and increased collagen content. The study demonstrates that FLI is a powerful analytical tool to monitor the delivery of antibodydrugs both in cell cultures and in vivo live systems. Especially, MFLI FRET is a unique imaging modality that can directly quantify target engagement with the potential to elucidate the role of the TME in drug delivery efficacy in intact live tumor xenografts.

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

Cited by 34 publications

References 54 publications

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

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

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

Fluorescence Lifetime Imaging for Quantification of Targeted Drug Delivery in Varying Tumor Microenvironments

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