Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH₂) analogue infraluciferin (iLH₂), near-infrared dual BLI can be achievedin vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5’-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH₂ reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH₂ is superior to LH₂ for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model.

In vivo bioluminescent images were acquired using IVIS Spectrum (FOV 24, f/1, Medium (8)bin, automatic acquisition mode for imaging with LH₂, FOV 24, f/1, Medium (8)bin, 120 s acquisition, total imaging time 24 mins for imaging with iLH₂). These parameters are calculated to keep the binning, exposure time and f/stop within an optimal range for quantification. Up to 5 animals could be imaged at once and the stage was heated to 37°C. Open filter images were acquired prior to and post spectral imaging to confirm the stability of photon emission during spectral acquisition. Spectral imaging acquired images through 14 and 12 of the 20nm bandpass filters on the IVIS Spectrum depending on substrate used (530-830nm for LH2 and 590-830nm for iLH2), starting from the lowest to the highest filter. It was not necessary to acquire images through all filters as the bioluminescent emissions of FLuc mutants did not cover the full spectral range from 490- 850nm. Living image software was used for ROI analysis of spectral images and spectral unmixing analysis. Radiance values for bioluminescence are shown using pseudo-colour scales detailed in each image. Image analysis involved placing an ROI over the tumour signal for every animal in each model. If a series if spectral images were acquired, the same ROI was placed over tumour signal in every image for each mouse. For spectral unmixing analysis, guided spectral unmixing was first used on pure expressing FLuc_green and FLuc_red populations from spectral characterisation experiments to create a library spectra for each mutant with each substrate. The relevant library spectra was then used to perform spectral unmixing on mixed FLuc_green and FLuc_red populations. Data exported to Excel (Microsoft) and Prism (Graphpad) for further analysis

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