Daniel Razansky

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.

Multifunctional Nanocarriers for diagnostics, drug delivery and targeted treatment across blood-brain barrier: perspectives on tracking and neuroimaging

Nanotechnology has brought a variety of new possibilities into biological discovery and clinical practice. In particular, nano-scaled carriers have revolutionalized drug delivery, allowing for therapeutic agents to be selectively targeted on an organ, tissue and cell specific level, also minimizing exposure of healthy tissue to drugs. In this review we discuss and analyze three issues, which are considered to be at the core of nano-scaled drug delivery systems, namely functionalization of nanocarriers, delivery to target organs and in vivo imaging. The latest developments on highly specific conjugation strategies that are used to attach biomolecules to the surface of nanoparticles (NP) are first reviewed. Besides drug carrying capabilities, the functionalization of nanocarriers also facilitate their transport to primary target organs. We highlight the leading advantage of nanocarriers, i.e. their ability to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells surrounding the brain that prevents high-molecular weight molecules from entering the brain. The BBB has several transport molecules such as growth factors, insulin and transferrin that can potentially increase the efficiency and kinetics of brain-targeting nanocarriers. Potential treatments for common neurological disorders, such as stroke, tumours and Alzheimers, are therefore a much sought-after application of nanomedicine. Likewise any other drug delivery system, a number of parameters need to be registered once functionalized NPs are administered, for instance their efficiency in organ-selective targeting, bioaccumulation and excretion. Finally, direct in vivo imaging of nanomaterials is an exciting recent field that can provide real-time tracking of those nanocarriers. We review a range of systems suitable for in vivo imaging and monitoring of drug delivery, with an emphasis on most recently introduced molecular imaging modalities based on optical and hybrid contrast, such as fluorescent protein tomography and multispectral optoacoustic tomography. Overall, great potential is foreseen for nanocarriers in medical diagnostics, therapeutics and molecular targeting. A proposed roadmap for ongoing and future research directions is therefore discussed in detail with emphasis on the development of novel approaches for functionalization, targeting and imaging of nano-based drug delivery systems, a cutting-edge technology poised to change the ways medicine is administered.

Volumetric real-time multispectral optoacoustic tomography of biomarkers

Multispectral optoacoustic tomography (MSOT) has recently been developed to enable visualization of optical contrast and tissue biomarkers, with resolution and speed representative of ultrasound. In the implementation described here, MSOT enables operation in real-time mode by capturing single cross-sectional images in <1 ms from living small animals (e.g., mice) and other tissues of similar dimensions. At the core of the method is illumination of the object using multiple wavelengths in order to resolve spectrally distinct biomarkers over background tissue chromophores. The system allows horizontal placement of a mouse in the imaging chamber and three-dimensional scanning of the entire body without the need to immerse the mouse in water. Here we provide a detailed description of the MSOT scanner components, system calibration, selection of image reconstruction algorithms and animal handling. Overall, the entire protocol can be completed within 15–30 min for acquisition of a whole-body multispectral data set from a living mouse.

Multispectral photoacoustic imaging of fluorochromes in small animals

Fluorochromes have become essential reporter molecules in biological research. We show that the depth-resolved distribution of fluorochromes in small animals can be imaged with 25 fmol sensitivity and 150 microm spatial resolution by means of multispectral photoacoustic imaging. The major advantage of the multispectral approach is the sensitive differentiation of chromophores and fluorochromes of interest based on self-reference measurements, as evidenced in this study by resolving a commonly used fluorochrome (Alexa Fluor 750) in mouse. The suggested method is well suited for enhancing visualization of functional and molecular information in vivo and longitudinally.

Video rate optoacoustic tomography of mouse kidney perfusion

Optoacoustic tomography can visualize optical contrast in tissues while capitalizing on the advantages of ultrasound, such as high spatial resolution and fast imaging capabilities. We report a novel multispectral optoacoustic tomography system for deep tissue small animal imaging. The previously undocumented capacity of whole-body optoacoustic tomography at a video rate has been demonstrated by visualizing mouse kidney perfusion using Indocyanine Green in vivo.

Multispectral optoacoustic tomography (MSOT) scanner for whole-body small animal imaging

A major difficulty arising from whole-body optoacoustic imaging is the long acquisition times associated with recording signals from multiple spatial projections. The acquired signals are also generally weak and the signal-to-noise-ratio is low, problems often solved by signal averaging, which complicates acquisition and increases acquisition times to an extent that makes many in vivo applications challenging or even impossible. Herein we present a fast acquisition multispectral optoacoustic tomography (MSOT) scanner for whole-body visualization of molecular markers in small animals. Multi-wavelength illumination offers the possibility to resolve exogenously administered fluorescent probes, biomarkers, and other intrinsic and exogenous chromophores. The system performance is determined in phantom experiments involving molecular probes and validated by imaging of small animals of various scales.

High-sensitivity compact ultrasonic detector based on a pi-phase-shifted fiber Bragg grating

A highly sensitive compact hydrophone, based on a pi-phase-shifted fiber Bragg grating, has been developed for the measurement of wideband ultrasonic fields. The grating exhibits a sharp resonance, whose centroid wavelength is pressure sensitive. The resonance is monitored by a continuous-wave (CW) laser to measure ultrasound-induced pressure variations within the grating. In contrast to standard fiber sensors, the high finesse of the resonance--which is the reason for the sensors high sensitivity--is not associated with a long propagation length. Light localization around the phase shift reduces the effective size of the sensor below that of the grating and is scaled inversely with the resonance spectral width. In our system, an effective sensor length of 270 μm, pressure sensitivity of 440 Pa, and effective bandwidth of 10 MHz were achieved. This performance makes our design attractive for medical imaging applications, such as optoacoustic tomography, in which compact, sensitive, and wideband acoustic detectors are required.

Real-time imaging of cardiovascular dynamics and circulating gold nanorods with multispectral optoacoustic tomography.

Macroscopic visualization of functional and molecular features of cardiovascular disease is emerging as an important tool in basic research and clinical translation of new diagnostic and therapeutic strategies. We showcase the application of Multispectral Optoacoustic Tomography (MSOT) techniques to noninvasively image different aspects of the mouse cardiovascular system macroscopically in real-time and in vivo, an unprecedented ability compared to optical or optoacoustic (photoacoustic) imaging approaches documented so far. In particular, we demonstrate imaging of the carotid arteries, the aorta and the cardiac wall. We further demonstrate the ability to dynamically visualize circulating gold nanorods that can be used to enhance contrast and be extended to molecular imaging applications. We discuss the potential of this imaging ability in cardiovascular disease (CVD) research and clinical applications.

Optical Imaging of Cancer Heterogeneity with Multispectral Optoacoustic Tomography

PURPOSE To investigate whether multispectral optoacoustic tomography (MSOT) can reveal the heterogeneous distributions of exogenous agents of interest and vascular characteristics through tumors of several millimeters in diameter in vivo. MATERIALS AND METHODS Procedures involving animals were approved by the government of Upper Bavaria. Imaging of subcutaneous tumors in mice was performed by using an experimental MSOT setup that produces transverse images at 10 frames per second with an in-plane resolution of approximately 150 μm. To study dynamic contrast enhancement, three mice with 4T1 tumors were imaged before and immediately, 20 minutes, 4 hours, and 24 hours after systemic injection of indocyanine green (ICG). Epifluorescence imaging was used for comparison. MSOT of a targeted fluorescent agent (6 hours after injection) and hemoglobin oxygenation was performed simultaneously (4T1 tumors: n = 3). Epifluorescence of cryosections served as validation. The accumulation owing to enhanced permeability and retention in tumors (4T1 tumors: n = 4, HT29 tumors: n = 3, A2780 tumors: n = 2) was evaluated with use of long-circulating gold nanorods (before and immediately, 1 hour, 5 hours, and 24 hours after injection). Dark-field microscopy was used for validation. RESULTS Dynamic contrast enhancement with ICG was possible. MSOT, in contrast to epifluorescence imaging, showed a heterogeneous intratumoral agent distribution. Simultaneous imaging of a targeted fluorescent agent and oxy- and deoxyhemoglobin gave functional information about tumor vasculature in addition to the related agent uptake. The accumulation of gold nanorods in tumors seen at MSOT over time also showed heterogeneous uptake. CONCLUSION MSOT enables live high-spatial-resolution observations through tumors, producing images of distributions of fluorochromes and nanoparticles as well as tumor vasculature.

In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography

We report a technique for fluorescence tomography that operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The method uses multi-projection illumination and photon transport description in opaque tissues. We demonstrate whole-body three-dimensional visualization of the morphogenesis of GFP-expressing salivary glands and wing imaginal discs in living Drosophila melanogaster pupae in vivo and over time.