KEYWORDS: Near infrared, Fluorescence, Tumors, Fluorescence lifetime imaging, In vivo imaging, Multiplexing, 3D modeling, Fluorescence tomography, Fluorescence imaging, Fluorescent proteins
The development of fluorescent proteins emitting in the near infrared (NIR) range (i.e., 650 nm-950 nm) has improved our capabilities for lifetime multiplexing and fluorescence imaging in vivo. Wavelengths in the NIR window experience reduced scattering and increased penetration depth through living tissue. Additionally, autofluorescence of cells and tissues is less prevalent in the NIR range, further improving signal to noise ratio. We performed fluorescence lifetime imaging (FLI) on breast cancer (AU565) and ovarian cancer (SKOV3) cell lines expressing the NIR fluorescent proteins (FPs), miRFP680 and emiRFP670. Confocal microscopy with time-correlated single-photon counting (TCSPC) reveals unique fluorescence decays for these NIR FPs, allowing for lifetime-based multiplexing on a single channel. Despite similar emission spectra, we were able to unmix fluorescence signals from a co-culture of SKOV3 expressing emiRFP670 and AU565 expressing miRFP680 based upon their unique fluorescence decays. We then generated 3D liquid overlay tumor spheroids using SKOV3 expressing emiRFP670 or miRFP680 for lifetime imaging via mesoscopic fluorescence molecular tomography (MFMT). 2D lifetime values and images acquired from MFMT corroborated our findings. Future investigation includes 3D light sheet mesoscopic imaging of tumor spheroids, as well as imaging of in vivo tumor xenografts expressing NIR-FPs. The long wavelengths and unique fluorescence lifetimes of emiRFP670 and miRFP680 make them ideal for multiplexed imaging, as well as for defining tumor volumes in vivo, while also leveraging the benefits of NIR imaging.
SignificanceFluorescence lifetime imaging in the shortwave infrared (SWIR) is expected to enable high-resolution multiplexed molecular imaging in highly scattering tissue.AimTo characterize the brightness and fluorescence lifetime of commercially available organic SWIR fluorophores and benchmark them against the tail emission of conventional NIR-excited probes.ApproachCharacterization was performed through our established time-domain mesoscopic fluorescence molecular tomography system integrated around a time-correlated single-photon counting-single-photon avalanche diode array. Brightness and fluorescence lifetime were measured for NIR and SWIR probes >1000 nm. Simultaneous probe imaging was then performed to assess their potential for multiplexed studies.ResultsThe 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.ConclusionsLong-tail emission of NIR probes outperformed the SWIR probes in brightness >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.
The novel Time-Domain Mesoscopic Fluorescence Molecular Tomography (TD-MFMT) imaging modality is aimed to resolve intra-tumoral heterogeneity and monitoring target engagement at the mesoscopic regime; however, its spatial resolution is limited by the scan strategy employed for imaging. Imaging in the Short-Wave Infrared (SWIR) can improve resolution due to reduced scattering effect. We therefore implement data-fusion of low-resolution fluorescence lifetime images with high-resolution SWIR intensity images via deep learning (DL). This approach was validated through phantom study where results show resolution enhancement of fluorescence lifetime imaging in scattering media, demonstrating potential for expansion to preclinical studies.
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