In Vivo Tomographic Imaging of Deep-Seated Cancer Using Fluorescence Lifetime Contrast

Abstract

Preclinical cancer research would benefit from non-invasive imaging methods that allow tracking and visualization of early stage metastasis in vivo. While fluorescent proteins revolutionized intravital microscopy, two major challenges which still remain are tissue autofluorescence and hemoglobin absorption, which act to limit intravital optical techniques to large or subcutaneous tumors. Here we employ time-domain technology for the effective separation of tissue autofluorescence from extrinsic fluorophores, based on their distinct fluorescence lifetimes. Additionally, we employ cancer cells labelled with near infra-red fluorescent proteins (iRFP) to allow deep-tissue imaging. Our results demonstrate that time-domain imaging allows the detection of metastasis in deep-seated organs of living mice with a more than 20-fold increase in sensitivity compared to conventional continuous wave techniques. Furthermore, the distinct fluorescence lifetimes of each iRFP enables lifetime multiplexing of three different tumors, each expressing unique iRFP labels in the same animal. Fluorescence tomographic reconstructions reveal 3D distributions of iRFP720-expressing cancer cells in lungs and brain of live mice, allowing ready longitudinal monitoring of cancer cell fate with greater sensitivity than otherwise currently possible.

Near-infrared fluorescent proteins engineered from bacterial phytochromes

“…The rejection of tissue autofluorescence substantially increases the detection sensitivity. This approach allowed visualization of ∼5×10 4 iRFP720-labelled cancer cells in mouse lungs [46]. Tomographic implementation of fluorescence lifetime detection, such as asymptotic fluorescence lifetime tomography – XCT imaging, uses anatomical information obtained in XCT for better reconstruction.…”

“…Tomographic implementation of fluorescence lifetime detection, such as asymptotic fluorescence lifetime tomography – XCT imaging, uses anatomical information obtained in XCT for better reconstruction. Using fluorescence lifetime tomography, the 3D distribution of iRFP720-labelled cells in deep-seated organs, such as lungs and brain, was demonstrated (Figure 3i) [46]. …”

Novel uses of fluorescent proteins

“…In spite of low molecular brightness, IFPs far outperform GFP-like proteins in whole body imaging in mice, where near-infrared light penetrates tissues much deeper compared to shorter-wavelength light [26,27 ]. The use of a reversibly photoswitchable variants (PAiRFP1 and PAiRFP2) enables strong enhancement of signal-to-noise ratio by controlled optical modulation of the signal in in vivo imaging, where high autofluorescence represents a significant problem [28].…”

Optimized longitudinal monitoring of stem cell grafts in mouse brain using a novel bioluminescent/near infrared fluorescent fusion reporter

“…only for monitoring tumor progression using FLI. 30 In addition, iRFP720 has shown potential for photoacoustic imaging of tumor progression. 31 However, in both applications, millions of cells need to be transplanted.…”