Molecular imaging by means of multispectral optoacoustic tomography (MSOT).

Optical imaging is a powerful modality in biological discovery. The mainstream of optical interrogations, however, largely relies on microscopy, which imposes depth limitations on resolving novel classes of optical reporter agents developed for in vivo use, such as fluorescent proteins and probes, other chromophoric molecules, and nanoparticles with specificity to cellular and subcellular activity. We review herein emerging optoacoustic (also termed photoacoustic) technologies that allow the visualization of optical reporter agents with never-seen-before visualization performance, enabling volumetric quantitative molecular imaging in entire organs, small animals, or human tissues. Multiwavelength/ multispectral optoacoustic (photoacoustic) methods, in particular, allow for highly specific molecular imaging through several millimeters to centimeters of tissue with resolutions in the 20-200 μm range, combining high contrast versatility with resolution that is largely independent from photon scattering in tissues. The principles of operation, key operational characteristics, and examples of in ViVo imaging in fish and mice are described, showcasing performance that forecasts optoacoustic imaging as a method of choice for biological visualization and selected clinical segments. Optical imaging operates on contrast mechanisms that offer highly versatile ability to visualize cellular and subcellular function and structure. Correspondingly, fluorescence microscopy and imaging are overwhelmingly utilized in biomedical research, for example in immunohistochemistry, in vitro assays, or cellular imaging in ViVo. The compelling advantages of fluorescence are reflected in the recent development of powerful classes of fluorescent tags that can stain functional and molecular processes in vivo. A widely acknowledged technology is the 2008 Nobel-prize awarded fluorescent protein, which offers perhaps the most versatile tool for biological imaging.1 Fluorescent proteins are reporter molecules that attain the ability to tag cellular motility and subcellular processes, from gene expression and signaling pathways to protein function and interactions, merging optimally with postgenomic “-omics” investigations and interrogating biology at the systems level. Promising new developments include the introduction of truly near-infrared shifted FPs, with excitation and emission spectra above 650 nm.2 Such performance opens exciting possibilities for whole body animal imaging, as it allows high sensitivity imaging through several centimeters of tissue, due to the low photon attenuation by tissue in the 650-950 nm range, i.e. the nearinfrared (NIR) spectral region. In parallel, a plethora of extrinsically administered probes are being developed, also operating in the NIR region.3,4 Fluorescent probes are optical reporter agents that can probe tissue constituents and their function by staining in ViVo certain classes of cells, receptors, proteases, and other moieties of cellular or subcellular activity. During the past decade, a large number of experimental and commercially available fluorescent agents is increasingly offered, from fluorescent dyes with preferential accumulation to tissues of interest to activatable photoproteins and fluorogenic-substrate-sensitive fluorochromes3 with molecular specificity. Collectively, these developments offer a highly potent toolbox for biological imaging.5 So far, these contrast mechanisms were proven efficient in a number of small-animal applications, but many of these agents attain strong potential for clinical translation as well. In addition, voltage sensitive dyes, fluorescence resonance energy transfer approaches, and lifetime measurements further allow the sensing of ions, protein-protein interactions, or the effects of the biochemical environment on the fluorochrome.6,7 Using fluorescence therefore, previously invisible processes associated with tissue and disease growth and treatment can be sensed and visualized in real-time and longitudinally. Naturally, fluorescence is widely used in basic biological discovery and drug discovery, and it is even considered for clinical studies of cancer and inflammation and neurodegenerative and cardiovascular disease, to name a few examples.