Abstract
Bioluminescence imaging became a widely used technique for noninvasive study of biological processes in small animals. Bioluminescent probes with emission in near-infrared (NIR) spectral region confer the advantage of having deep tissue penetration capacity. However, there are a very limited number of currently available luciferases that exhibit NIR bioluminescence. Here, we engineered two novel chimeric probes based on RLuc8 luciferase fused with iRFP670 and iRFP720 NIR fluorescent proteins. Due to an intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, the chimeric luciferases exhibit NIR bioluminescence with maxima at 670 nm and 720 nm, respectively. The 50 nm spectral shift between emissions of the two iRFP chimeras enables combined multicolor bioluminescence imaging (BLI) and the respective multicolor fluorescence imaging (FLI) of the iRFPs. We show that for subcutaneously implanted cells, NIR bioluminescence provided a 10-fold increase in sensitivity compared to NIR FLI. In deep tissues, NIR BLI enabled detection of as low as 104 cells. Both BLI and FLI allowed monitoring of tumor growth and metastasis from early to late stages. Multimodal imaging, which combines concurrent BLI and FLI, provides continuous spatiotemporal analysis of metastatic cells in animals, including their localization and quantification.
Highlights
In vivo bioluminescence imaging (BLI) is a powerful and simple technique for studies of living animals and cells[1,2,3]
Using iRFP670 and iRFP720 we engineered a pair of spectrally distinct NIR chimeric luciferases that we further tested in multicolor BLI and fluorescence imaging (FLI) in cells and in mice
In the chimeric protein consisting of a luciferase and an iRFP protein the intramolecular bioluminescence resonance energy transfer (BRET) occurs via Soret band (Fig. 1a), resulting in NIR emission at iRFP spectral maximum
Summary
In vivo bioluminescence imaging (BLI) is a powerful and simple technique for studies of living animals and cells[1,2,3]. It is widely used for interrogating various ongoing biological processes such as tracking of luciferase-labeled cells, monitoring gene expression, assessing protein stability and function, and sensing small bioactive molecules. RLuc[8] is a small (35.7 kDa), stable, bright and ATP-independent luciferase with a variety of available substrates, which makes it an advantageous donor of bioluminescence for in vivo imaging. We proceeded to demonstrate that high sensitivity of bioluminescence combined with NIR fluorescence enables continuous and sustained analysis of cellular processes on different scales, from isolated cells to whole organs, using the same NIR chimeric probes
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