Mr. Ryan Kanai Profile

Ryan Kanai

at Albany Medical College

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Proceedings Article | 13 March 2024 Presentation + Paper

Multiplexed fluorescence lifetime imaging of near-infrared fluorescent proteins in 3D tumor spheroids

Catherine Sherry, Luis Chavez, Ryan Kanai, Shan Gao, John Lamar, Xavier Intes, Margarida Barroso

Proceedings Volume 12862, 1286208 (2024) https://doi.org/10.1117/12.3003280

KEYWORDS: Near infrared, Fluorescence, Tumors, Fluorescence lifetime imaging, In vivo imaging, Multiplexing, 3D modeling, Fluorescence tomography, Fluorescence imaging, Fluorescent proteins

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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.

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