ABSTRACT
Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In this biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control. Analogously, we tested the use of melanophore cells from Xenopus laevis, containing compartments (melanosomes) filled with strongly absorbing melanin, as whole-cell sensors for optoacoustic imaging. Our results show that pigment relocalization in these cells, which is dependent on binding of a ligand of interest to a specific G protein-coupled receptor (GPCR), can be monitored in vitro and in vivo using photoacoustic mesoscopy. In addition to changes in the photoacoustic signal amplitudes, we could furthermore detect the melanosome aggregation process by a change in the frequency content of the photoacoustic signals. Using bioinspired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoacoustic imaging of GPCR-mediated signaling molecules.
Funding Sources We are grateful for financial support from the European Research Council under grant agreements ERC-St: 311552 (A.L., G.G.W.), the DFG Reinhart Koselleck project “High resolution near-field thermoacoustic sensing and imaging” NT 3/9-1 (D.S., M.O., V.N.) and the Federal Ministry of Education and Research, Photonic Science Germany, Tech2See-13N12624 (D.S., M.O., V.N.).
ABBREVIATIONS BF, brightfield; cAMP, cyclic adenosine 3’-5’-monophosphate; CNO, clozapine N-oxide; CPS, cumulative power spectrum; DREADD, designer receptor exclusively activated by designer drugs; FACS, fluorescence-activated cell sorting; FFT, fast Fourier transform; FOV, field of view; GFP, green fluorescent protein; GPCR, G-protein-coupled receptors; MCH, melanin-concentrating hormone; MIP, maximum intensity projection; MORSOM, multi-orientation raster-scan optoacoustic mesoscopy; MSH, melanin-stimulating hormone; NA, numerical aperture; PaPiReS, photoacoustic pigment relocalization sensor; PCR, polymerase chain reaction; PBS, phosphate buffer saline; ROI, region of interest; RSOM, raster-scan optoacoustic mesoscopy;
Footnotes
Author Contributions A.L. conducted all biological experiments and performed the brightfield imaging and the ROI-based analysis, D.S. performed the optoacoustic imaging experiments and analysis, supported by M.O. and supervised by V.N. who suggested the frequency analysis. A.S. contributed to culturing melanophores and zebrafish and provided technical support in the experiments. G.G.W. conceived the study. D.S., A.L., and G.G.W. discussed the results and wrote the manuscript.
