DOI: 10.5061/dryad.3j9kd51cs
title: Data from: Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin

description: 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.
description: <i>In vivo</i> bioluminescent images were acquired
 using IVIS Spectrum (FOV 24, f/1, Medium (8)bin, automatic acquisition
 mode for imaging with LH<sub>2</sub>, FOV 24, f/1, Medium
 (8)bin, 120 s acquisition, total imaging time 24 mins for imaging with
 iLH<sub>2</sub>). 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 (<i>Microsoft</i>) and Prism
 (<i>Graphpad</i>) for further analysis
description: Please contact authors if you require any advice or help.