We present a novel fluorescence lifetime tomography system applied to a highly scattering autofluorescent phantom containing live cells expressing the fluorophore enhanced green fluorescent protein (EGFP). The fluorescence signal was excited using a fiber-laser-pumped supercontinuum source and detected using wide-field time gating imaging. To facilitate rapid 3D reconstruction of the fluorescence lifetime distribution, the time-resolved data were Fourier-transformed in time to give complex functions that formed a data set for the Fourier domain reconstruction. Initially the presence of an unspecified background autofluorescence signal impeded reconstruction of the lifetime distribution, but we show that this problem can be addressed using a simple iterative technique.

We report a three-dimensional time-resolved tomographic imaging technique for localizing protein–protein interaction and protein conformational changes in turbid media based on Förster resonant energy-transfer read out using fluorescence lifetime. This application of “tomoFRET” employs an inverse scattering algorithm utilizing the diffusion approximation to the radiative-transfer equation applied to a large tomographic data set of time-gated images. The approach is demonstrated by imaging a highly scattering cylindrical phantom within which are two thin wells containing cytosol preparations of HEK293 cells expressing TN-L15, a cytosolic genetically encoded calcium Förster resonant energy-transfer sensor. A $10\mathrm{mM}$ calcium chloride solution was added to one of the wells, inducing a protein conformation change upon binding to TN-L15, resulting in Förster resonant energy transfer and a corresponding decrease in the donor fluorescence lifetime. We successfully reconstruct spatially resolved maps of the resulting fluorescence lifetime distribution as well as of the quantum efficiency, absorption, and scattering coefficients.

Engineering animal models with self-sustained luminescence could enable non-invasive longitudinal monitoring of molecular events in living animals. To create animal models that report physiology with autoluminescence, both luciferin biosynthesis enzymes and the luciferase need to be optimised. Previous work on engineering the autoluminescence pathway from fungi resulted in the development of nnLuz_v3, a version of Neonothopanus nambi luciferase with enhanced thermal stability. Here, we generated an nnLuz_v3 reporter of endogenous Cyp1a1 expression as a measure of aryl hydrocarbon receptor (AHR) activation, assessing the performance of nnLuz_v3 in vivo at physiologically relevant expression levels. As AHR dynamically responds to metabolic, environmental and dietary changes it provides a validated platform to assess novel luminescence approaches. In Cyp1a1-nnLuz mice bioluminescence signal was stable, allowing the generation of well-resolved luminescence images both on standard in vivo imaging equipment and consumer-grade cameras. Using mice and nematode models, we demonstrated limited oral availability of the fungal luciferin, potentially compatible with delivering the substrate via food or the microbiome. Our results are an encouraging first step in the generation of an autoluminescent mammalian model of a molecular event and encourage optimisation of other enzymes of the fungal luciferase pathway.

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1 . To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase ( Fluc ) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo .