Challenging a Preconception: Optoacoustic Spectrum Differs from the Optical Absorption Spectrum of Proteins and Dyes for Molecular Imaging.

Optoacoustic (photoacoustic) imaging has seen marked advances in detection and data analysis, but there is less progress in understanding the photophysics of common optoacoustic contrast agents. This gap blocks the development of novel agents and the accurate analysis and interpretation of multispectral optoacoustic images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. Herein, we employ MLS to analyze contrast agents (methylene blue, rhodamine 800, Alexa Fluor 750, IRDye 800CW, and indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720, and smURFP). We found that the optical absorption spectrum does not correlate with the optoacoustic spectrum for the majority of the analytes. We determined that for dyes, the transition underlying an aggregation state has more optoacoustic signal generation efficiency than the monomer transition. For proteins we found a favored optoacoustic relaxation that stems from the neutral or zwitterionic chromophores and unreported photoswitching behavior of tdTomato and HcRed. We then crystalized HcRed in its photoswitch optoacoustic state, confirming structurally the change in isomerization with respect to HcReds' fluorescence state. Finally, on the example of the widely used label tdTomato and the dye indocyanine green, we show the importance of correct photophysical (e.g., spectral and kinetic) information as a prerequisite for spectral-unmixing for in vivo imaging.

Photocontrollable Proteins for Optoacoustic Imaging.

Photocontrollable proteins revolutionized life-science imaging due to their contribution to subdiffraction-resolution optical microscopy. They might have yet another lasting impact on photo- or optoacoustic imaging (OA). OA combines optical contrast with ultrasound detection enabling high-resolution real-time in vivo imaging well-beyond the typical penetration depth of optical methods. While OA already showed numerous applications relying on endogenous contrast from blood hemoglobin or lipids, its application in the life-science was limited by a lack of labels overcoming the strong signal from the aforementioned endogenous absorbers. Here, a number of recent studies showed that photocontrollable proteins provide the means to overcome this barrier eventually enabling OA to image small cell numbers in a complete organism in vivo. In this Feature article, we introduce the key photocontrollable proteins, explain the basic concepts, and highlight achievements that have been already made.

Crystal structure of a biliverdin-bound phycobiliprotein: Interdependence of oligomerization and chromophorylation.

Small, ultra-red fluorescence protein (smURFP) introduces the non-native biliverdin (BV) chromophore to phycobiliproteins (PBPs), allowing them to be used as transgenic labels for in vivo mammalian imaging. Presently, no structural information exists for PBPs bound to the non-native BV chromophore, which limits the further development of smURFP and related proteins as imaging labels or indicators. Here we describe the first crystal structure of a PBP bound to BV. The structures of smURFP-Y56R with BV and smURFP-Y56F without BV reveal unique oligomerization interfaces different from those in wild-type PBPs bound to native chromophores. Our structures suggest that the oligomerization interface affects the BV binding site, creating a link between oligomerization and chromophorylation that we confirmed through site-directed mutagenesis and that may help guide efforts to improve the notorious chromophorylation of smURFP and other PBPs engineered to bind BV.

Characterization of Reversibly Switchable Fluorescent Proteins in Optoacoustic Imaging.

Reversibly switchable fluorescent proteins (rsFPs) have had a revolutionizing effect on life science imaging due to their contribution to sub-diffraction-resolution optical microscopy (nanoscopy). Initial studies showed that their use as labels could also be highly beneficial for emerging photo- or optoacoustic imaging. It could be shown that their use in optoacoustics (i) strongly improves the imaging contrast-to-noise ratio due to modulation and locked-in detection, (ii) facilitates fluence calibration, affording precise measurements of physiological parameters, and finally (iii) could boost spatial resolution following similar concepts as used for nanoscopy. However, rsFPs show different photophysical behavior in optoacoustics than in optical microscopy because optoacoustics requires pulsed illumination and depends on signal generation via nonradiative energy decay channels. This implies that rsFPs optimized for fluorescence imaging may not be ideal for optoacoustics. Here, we analyze the photophysical behavior of a broad range of rsFPs with optoacoustics and analyze how the experimental factors central to optoacoustic imaging influence the different types of rsFPs. Finally, we discuss how knowledge of the switching behavior can be exploited for various optoacoustic imaging approaches using sophisticated temporal unmixing schemes.

The sound of drug delivery: Optoacoustic imaging in pharmacology.

Optoacoustic (photoacoustic) imaging offers unique opportunities for visualizing biological function in vivo by achieving high-resolution images of optical contrast much deeper than any other optical technique. The method detects ultrasound waves that are generated inside tissue by thermo-elastic expansion, i.e., the conversion of light absorption by tissue structures to ultrasound when the tissue is illuminated by the light of varying intensity. Listening instead of looking to light offers the major advantage of image formation with a resolution that obeys ultrasonic diffraction and not photon diffusion laws. While the technique has been widely used to explore contrast from endogenous photo-absorbing molecules, such as hemoglobin or melanin, the use of exogenous agents can extend applications to a larger range of biological and possible clinical applications, such as image-guided surgery, disease monitoring, and the evaluation of drug delivery, biodistribution, and kinetics. This review summarizes recent developments in optoacoustic agents, and highlights new functions visualized and potent pharmacology applications enabled with the use of external contrast agents.

Alginate beads as a highly versatile test-sample for optoacoustic imaging.

Test-samples are necessary for the development of emerging imaging approaches such as optoacoustics (OA); these can be used to benchmark new labeling agents and instrumentation, or to characterize image analysis algorithms or the inversion required to form the three-dimensional reconstructions. Alginate beads (AlBes) loaded with labeled mammalian or bacterial cells provide a method of creating defined structures of controllable size and photophysical characteristics and are well-suited for both in vitro and in vivo use. Here we describe a simple and rapid method for efficient and reproducible production of AlBes with specific characteristics and show three example applications with multispectral OA tomography imaging. We show the advantage of AlBes for studying and eventually improving photo-switching OA imaging approaches. As highly defined, homogeneous, quasi point-like signal sources, AlBes might hold similar advantages for studying other agents, light-fluence models, or the impact of detection geometries on correct image formation in the near future.

Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

A practical guide to photoswitching optoacoustics tomography.

Photochromic proteins and photoswitching optoacoustics (OA) are a promising combination, that allows OA imaging of even small numbers of cells in whole live animals and thus can facilitate a more wide-spread use of OA in life-science and preclinical research. The concept relies on exploiting the modulation achieved by the photoswitching to discriminate the agents' signal from the non-modulating background. Here we share our analysis approaches that can be readily used on data generated with commercial OA tomography imaging instrumentation allowing-depending on the used photoswitching agent and sample-routine visualizations of as little as several hundreds of transgene labeled cells per imaging volume in the live animal.

Evaluation of the possibility of selective modulation of retinal glucose transporters in diabetic complications: An experimental study.

PURPOSE: To identify the possibility of modulating retinal glucose transporters in diabetic conditions to prevent retinal complications of diabetic retinopathy. MATERIALS AND METHODS: In silico and in vitro binding assays were performed to assess the effect of genistein and positive controls (pioglitazone and estradiol) on nuclear receptor estrogen receptor beta and peroxisome proliferator-activated receptor gamma (PPARγ). In vivo effects of compounds were tested on diabetic rats. Structural and functional analysis of retina was performed at 28th day followed by gene expression analysis of glucose transporters and nuclear receptors. Pioglitazone and genistein levels were analyzed by liquid chromatography with tandem mass spectrometry. RESULTS: Genistein showed equi-affinity toward PPARγ in in silico experiments contrary to in vitro findings. In multidose study, their therapeutic effects were observed by analyzing the retinal function. Retinal gene expression studies revealed that both test agents significantly up regulated PPARγ, GLUT4, and down regulated GLUT1. Genistein showed significant up regulation of GLUT4 and down regulation of GLUT1 as compared to PGZ which has been well correlated with the Electroretinography (ERG) outcome. CONCLUSION: This study showed the possibility of selective upregulation of GLUT4 (independent of PPARγ activation) in the retina of diabetic rats using genistein. Selective modulation of retinal glucose transporters as therapeutic target in ocular diabetic complications can be possibly explored.

Multiplexed whole-animal imaging with reversibly switchable optoacoustic proteins.

We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning-based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.

Structure-Based Mutagenesis of Phycobiliprotein smURFP for Optoacoustic Imaging.

Photo- or optoacoustics (OA) imaging is increasingly being used as a non-invasive imaging method that can simultaneously reveal structure and function in deep tissue. However, the most frequent transgenic OA labels are current fluorescent proteins that are not optimized for OA imaging. Thus, they lack OA signal strength, and their absorption maxima are positioned at short wavelengths, thus giving small penetration depths and strong background signals. Here, we apply insights from our recent determination of the structure of the fluorescent phycobiliprotein smURFP to mutate a range of residues to promote the nonradiative decay pathway that generates the OA signal. We identified hydrophobic and aromatic substitutions within the chromophore-binding pocket that substantially increase the intensity of the OA signal and red-shift the absorption. Our results demonstrate the feasibility of structure-based mutagenesis to repurpose fluorescent probes for OA imaging, and they may provide structure-function insights for de novo engineering of transgenic OA probes.