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Photoacoustic Pigment Relocalization Sensor bioRxiv preprint doi: https://doi.org/10.1101/455022; this version posted October 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Photoacoustic pigment relocalization sensor Antonella Lauri1,2,4, ‡,†, Dominik Soliman1,3, ‡, Murad Omar1, Anja Stelzl1,2,4, Vasilis Ntziachristos1,3 and Gil G. Westmeyer1,2,4* 1Institute of Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg, Germany 2Institute of Developmental Genetics (IDG), Helmholtz Zentrum München, Neuherberg, Germany 3Chair for Biological Imaging and 4Department of Nuclear Medicine, Technical University of Munich (TUM), Munich, Germany 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 bioin- spired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoa- coustic imaging of GPCR-mediated signaling molecules. INTRODUCTION Due to this relationship, optoacoustic imaging is a highly Photoacoustic (optoacoustic) imaging is based on the scalable modality regarding spatial resolution and imag- photoacoustic effect – the conversion of absorbed light ing depth. Recently, we have introduced raster-scan opto- energy into heat, causing the generation of acoustic acoustic mesoscopy (RSOM) in epi-illumination mode, of- waves that can be captured by ultrasound detectors1. fering imaging depths of several millimeters and a lateral 5 Over recent years, the optoacoustic imaging technique and axial resolution of 20-30 µm and ~5 µm, respectively , has been increasingly used in biomedicine because of its thereby bridging the gap between macroscopic and mi- advantageous combination of optical and ultrasound im- croscopic optoacoustic imaging modalities. aging, decoupling the underlying mechanisms of contrast Up to now, contrast generation for optoacoustic imaging and spatial resolution. On the one hand, optoacoustic im- has mostly relied on utilizing endogenous chromophores aging relies on absorption-based contrast, allowing for or externally administered photoabsorbers such as or- rich optical differentiation of photoabsorbing molecules ganic dyes or nanoparticles2. With respect to genetically such as exogenous contrast agents or endogenous tissue encoded contrast agents for optoacoustic imaging, over- chromophores2. On the other hand, optoacoustic imaging expression of (reversibly switchable) fluorescent proteins achieves penetration depths of millimeters or even centi- that absorb in the visible or infrared range have been ex- meters and high ultrasonic resolutions because of the plored, in which either cyclized amino acids or cofactors weak acoustic scattering in tissue. such as biliverdin act as the chromophore 6–9. In general, the acoustic signals measured in photoacous- Alternatively, biosynthetic pigments such as melanin and tics contain a broad range of ultrasonic frequencies. The violacein have been utilized as contrast agents, which are size of the imaged photoabsorbers governs the frequency beneficial because of their generally higher photobleach- content of the photoacoustic signal: smaller absorbers ing resistance, enzymatic amplification of the genetic sig- generate higher photoacoustic frequencies than larger nal and potentially higher local concentrations as com- absorbers. For example, a spherical absorber with 20 µm pared to chromoproteins10,11. The advantages of melanin diameter generates frequencies up to ~100 MHz, while a are that it is synthesized in a monoenzymatic reaction and 10 µm absorber produces frequencies as high as ~200 that it yields strong and broad absorption over the visible MHz3. range extending into the near-infrared12. Moreover, mela- Furthermore, high frequencies experience stronger nin exhibits a fluorescence quantum yield of less than 1% acoustic attenuation than lower frequencies4, leading to thereby maximizing heat generation by non-radiative de- 12 an inverse relationship of highest measurable frequency cay, a prerequisite for photoacoustic signal generation . and thus achievable spatial resolution with imaging depth. For cell types that endogenously produce and sequester melanin such as melanophores, it represents an excellent bioRxiv preprint doi: https://doi.org/10.1101/455022; this version posted October 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. contrast agent for optoacoustic imaging that has, for in- CNiFER sensors28 that detect a specific GPCR ligand stance, enabled volumetric tracking of melanophore mi- such as glutamate via a downstream fluorescent calcium gration during zebrafish development13. If melanin pro- indicator signal in xenografted human embryonic kidney duction is not compartmentalized however, it has been (HEK) cells, photoacoustics could directly image melano- shown to be toxic in standard cell lines14. In melano- some relocalization via changes in the photoacoustic sig- phores, melanin is stored in specialized organelles that nal amplitudes. are transported inside the cell along its microtubular cyto- For gold and melanin-like nanoparticles, it has been skeletal network, referred to as melanosomes. demonstrated that an aggregation of the nanoparticles Certain animals such as chameleons and cuttlefish are can lead to an increase of photoacoustic signal amplitude able to adjust their skin color to the environment by trans- due to overlapping transient thermal fields surrounding locating the melanosomes within melanophores, which is the nanoparticles29,30. Changes of photoacoustic frequen- mediated by neuronal and hormonal inputs15. This impres- cies have also been measured in the context of detecting sive behavior is used for camouflage to hide from preda- aggregation and morphological changes of red blood cells tors and in the case of the cuttlefish may also be used as in microscopic settings in vitro31,32. 16 means of communication . Forms of camouflage have In the given context, we thus reasoned that dynamic pig- been reported also in the common laboratory fish model ment relocalization could also be detectable by shifts in zebrafish (Danio rerio). If a zebrafish is on a dark back- the photoacoustic frequencies above 10 MHz because ground, its melanophores disperse their melanosomes, the average size of the imaged photoabsorbing objects is leading to a darker appearance of the melanized fish skin. altered by the melanophore dynamics. A demonstration Conversely, aggregation of melanosomes around the nu- on intracellular pigment dynamics in vivo would establish cleus of the melanophores triggered by bright background the frequency content of photoacoustic signals as a new 17 colors results in a lighter appearance of the animal . observable for the high-resolution photoacoustic sensing Classical neurohumoral molecules are involved in regu- of dynamic molecular processes in live animals. lating this melanosome relocalization within melano- For the optoacoustic 3D monitoring of melanophore dy- phores. These include the pituitary-derived melanin-stim- namics, we selected the RSOM modality as it combines a ulating hormone (α-MSH), which induces melanosome number of advantages over traditional optical imaging dispersion, and the hypothalamus-derived melanin-con- systems. First, RSOM achieves the necessary spatial res- 17–19 centrating hormone (MCH) , which induces melano- olution to resolve single melanophores and to track some aggregation. Other hormones that influence mela- changes in the absorptive volume of the imaged cells. Se- 20,21 nosome translocation include melatonin , catechola- cond, RSOM relies on widefield laser illumination and 15,22 23 mines such as noradrenaline , and serotonin . The acoustic focusing (as opposed to optical focusing), being mechanism of action of these molecules is realized via less prone to pigment shadowing than pure optical modal- binding to G-protein–coupled receptors (GPCR) ex- ities. Third, the photoacoustic frequencies detectable via pressed on the cell surface of melanophores, which mod- RSOM (> 200 MHz) are high enough to capture frequency ulates the intracellular levels of cyclic adenosine 3’-5’-
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