Abstract
Bioluminescent probes are widely used to monitor biomedically relevant processes and cellular targets in living animals. However, the absorption and scattering of visible light by tissue drastically limit the depth and resolution of the detection of luminescence. Here we show that bioluminescent sources can be detected with magnetic resonance imaging by leveraging the light-mediated activation of vascular cells expressing a photosensitive bacterial enzyme that causes the conversion of bioluminescent emission into local changes in haemodynamic contrast. In the brains of rats with photosensitized vasculature, we used magnetic resonance imaging to volumetrically map bioluminescent xenografts and cell populations virally transduced to express luciferase. Detecting bioluminescence-induced haemodynamic signals from photosensitized vasculature will extend the applications of bioluminescent probes.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. The MRI atlas used for this study was downloaded from NITRC via https://www.nitrc.org/projects/whs-sd-atlas (ref. 55). The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We acknowledge grants to A.J. from the National Institutes of Health (UG3 MH126868, U01 EB031641, R21 EY032369 and R01 NS121073) and the G. Harold and Leila Y. Mathers Foundation. We also gratefully acknowledge research funding from L. McGovern, G. Hendrie and B. Fikes. R.O. was funded by a fellowship from the Deutsche Forschungsgemeinschaft. M.S. was funded by a Marie Skłodowska-Curie fellowship from the European Union. Y.K. was supported by a Y. Eva Tan Fellowship, and Y.J. was supported by a J. Douglas Tan Fellowship, both from the McGovern Institute for Brain Research. A. Becker and K. Backman are thanked for technical support.
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Contributions
R.O. and A.J. designed the BLUsH principle. R.O. performed most of the in vitro experiments. N.L. and R.O. performed the in vivo MRI experiments with contributions from V.D.P.V., M.S., M.D. and W.T.Z. R.O., V.D.P.V., Y.K. and B.S. performed histology analyses. Y.K. and Y.J. performed the BLI experiments. S.D. performed the in vitro CTZ titration. N.L., R.O. and A.J. analysed the imaging data. R.O., N.L. and A.J. wrote the paper.
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Extended data
Extended Data Fig. 1 Vascular expression of bPAC.
(a) Expression of bPAC (red, left) in two fields of view (top and bottom), compared with the tomato lectin vascular stain (green, right) in two fields of view from a different animal, showing similarity of labeled features as in Fig. 2b. Scale bar = 200 µm. (b) Images showing bPAC expression in individual cells spaced along individual vessels, analogous to Fig. 2c. Scale bars = 100 µm (top) and 200 µm (bottom).
Extended Data Fig. 2 Brain-wide vascular expression of bPAC in transgenic mouse.
(a) Immunohistochemical visualization of bPAC expression in a coronal slice from a representative SM22α-Cre transgenic mouse infected with a Cre-activated bPAC vector, showing strong and homogeneous labeling of brain-wide vasculature in the slice. Scale bar = 1 mm. (b) Close-up of the boxed region in a. Scale bar = 1 mm. (c) Close-up of the boxed region in b. Scale bar = 100 µm.
Extended Data Fig. 3 Analysis of nitrotyrosine accumulation.
Nitrotyrosine (NitroY) staining (blue, left) was performed to examine whether elevations of this marker colocalize with bPAC expression (red, middle) in virally transduced rat brain regions. Merged image is shown at right. Scale bars = 1 mm (top) and 500 µm (bottom).
Extended Data Fig. 4 Microglial staining in bPAC-expressing tissue.
Comparison of bPAC expression profile (top) with distribution of the microglial marker Iba1 (middle) and overlay (bottom), showing absence of clear microglial activation in bPAC-rich areas in a representative BLUsH-treated brain hemisphere. Panels at right represent enlargements of the dashed areas in the panels at left. Areas 1 and 2 were chosen to be regions with and without substantial bPAC expression, respectively. Scale bars = 1 mm (left) or 500 µm (right).
Extended Data Fig. 5 Raw time series data during light stimulation.
Individual image frames from an experiment as in Fig. 2d, showing raw MRI scans (top) and signal changes from baseline (bottom) at time points (t) relative to the beginning of the experiment, denoted in minutes below each frame. Grey scales denote arbitrary intensity units (AU, top) or percent signal change (%SC, bottom). The onset of signal changes coincident with bilateral LED illumination (blue bar) is indicated by the yellow arrowheads.
Extended Data Fig. 6 BLI of luciferase-expressing xenografts.
(a) BLI signal (color) and reflectance images (grayscale) of individual rats implanted with bioluminescent xenografts prepared and implanted as for the experiments of Fig. 3. The implant in the left animal expressed GLucM23, as in Fig. 3, while the implant in the rat on the right expressed Akaluc, a red-shifted luciferase. (b) FWHM of luminescence signals measured by BLI (closed purple circles = GLucM23, open purple circles = Akaluc), compared with values measured by MRI-based BLUsH (red circles). Horizontal lines denote means for BLI and BLUsH data points. The difference between BLI and BLUsH size measurements is significant with t-test p = 0.01, n = 4, 5.
Extended Data Fig. 7 BLUsH responses to repeated CTZ administration.
(a) Snapshots of the responses to three doses of CTZ infused over a luciferase-expressing cell pellet in a single animal, following the experimental paradigm of Fig. 3. CTZ was injected in 2.5-min epochs (blue shading at top), separated by 10 minute intertrial intervals. Timing with respect to infusion start is denoted at top. (b) Time courses of MRI signal change observed in ROIs defined around the cell pellet. Labeled traces denote CTZ injection trials 1, 2, and 3, and the infusion periods are denoted by blue shading. (c) Maps of mean response amplitude (5 min following CTZ start) and recovery amplitude (8 min after CTZ infusion end) averaged over the three CTZ infusion trials in an ROI around the luminescent xenograft.
Extended Data Fig. 8 Raw time series data following intravenous CTZ delivery.
Individual image frames from an experiment as in Fig. 4a, showing raw MRI scans (top) and signal changes from baseline (bottom) at time points (t) relative to the signal peak time (red bar), denoted in minutes below each frame. Grey scales denote arbitrary intensity units (AU, top) or percent signal change (%SC, bottom). The location and timing of the peak BLUsH response in this experiment is indicated by the yellow arrowhead.
Extended Data Fig. 9 BLUsH and control imaging of virally driven luciferase expression.
(a) Imaging data from five rats that underwent BLUsH imaging of AAV-driven GLucM23 expression following intravenous CTZ delivery in the experiments of Fig. 4. Data are shown over six coronal slices with bregma coordinates noted in yellow at top. (b) Corresponding MRI data obtained from three control virus-treated animals that also express GLucM23 following AAV infection.
Extended Data Fig. 10 Extended histology data from BLUsH and control experiments.
(a) Data corresponding to Fig. 4d, showing BLUsH MRI maps (left), GLucM23 staining (blue), bPAC staining (red), and merged images for three individual rats examined by postmortem histology. (b) Analogous MRI and histology data from three animals that received control viruses in place of BLUsH components. The control treatment results in vascular tdTomato expression (red) in place of bPAC. AAV-directed GLucM23 delivery is identical between BLUsH and control animals and results in robust expression (blue) without corresponding MRI signal changes (left).
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Ohlendorf, R., Li, N., Phi Van, V.D. et al. Imaging bioluminescence by detecting localized haemodynamic contrast from photosensitized vasculature. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01210-w
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DOI: https://doi.org/10.1038/s41551-024-01210-w
