Real-time 3D optoacoustic tracking of cell-sized magnetic microrobots circulating in the mouse brain vasculature.

Mobile microrobots hold remarkable potential to revolutionize health care by enabling unprecedented active medical interventions and theranostics, such as active cargo delivery and microsurgical manipulations in hard-to-reach body sites. High-resolution imaging and control of cell-sized microrobots in the in vivo vascular system remains an unsolved challenge toward their clinical use. To overcome this limitation, we propose noninvasive real-time detection and tracking of circulating microrobots using optoacoustic imaging. We devised cell-sized nickel-based spherical Janus magnetic microrobots whose near-infrared optoacoustic signature is enhanced via gold conjugation. The 5-, 10-, and 20-µm-diameter microrobots are detected volumetrically both in bloodless ex vivo tissues and under real-life conditions with a strongly light-absorbing blood background. We further demonstrate real-time three-dimensional tracking and magnetic manipulation of the microrobots circulating in murine cerebral vasculature, thus paving the way toward effective and safe operation of cell-sized microrobots in challenging and clinically relevant intravascular environments.

In situ characterization of microparticulate optoacoustic contrast agents in an intracardiac perfusion mouse model.

Extrinsically administered light-absorbing agents may greatly enhance the sensitivity and imaging performance of optoacoustic tomography (OAT). Beyond the use of targeted contrast agents in functional and molecular imaging applications, tracking of highly absorbing microparticles has recently been shown to facilitate super-resolution volumetric angiography and mapping of blood flow. However, in vivo characterization of new types of microparticulate absorbing agents is often hindered due to their potential toxicity, incompatible dimensions, or sub-optimal extinction spectrum shadowed by strong background absorption of hemoglobin. Herein, we used an intracardiac perfusion mouse model to individually track the perfusion of absorbing particles through the cerebral vasculature by acquiring a sequence of high-frame-rate 3D OAT images. The particles were injected in the left ventricle of the heart after substitution of blood by an artificial cerebrospinal fluid post mortem, which has further contributed to minimizing the background OAT signals induced by hemoglobin absorption. The presented approach can greatly aid the development of new microparticulate contrast agents with optimized performance for various OAT imaging applications.

Publisher Correction: Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Deep tissue volumetric optoacoustic tracking of individual circulating tumor cells in an intracardially perfused mouse model.

Widespread metastasis is the major cause of death from melanoma and other types of cancer. At present, the dynamic aspects of the metastatic cascade remain enigmatic. The feasibility to track circulating melanoma cells deep within living intact organisms can greatly impact our knowledge on tumor metastasis, but existing imaging approaches lack the sensitivity, spatio-temporal resolution or penetration depth to capture flowing tumor cells over large fields of view within optically-opaque biological tissues. Vast progress with the development of optoacoustic tomography technologies has recently enabled two- and three-dimensional imaging at unprecedented frame rates in the order of hundreds of Hertz, effectively mapping up to a million image voxels within a single volumetric snapshot. Herein, we employ volumetric optoacoustic tomography for real-time visualization of passage and trapping of individual B16 melanoma cells in the whole mouse brain. Detection of individual circulating melanoma cells was facilitated by substituting blood with an artificial cerebrospinal fluid that removes the strong absorption background in the optoacoustic images. The approach can provide new opportunities for studying trafficking and accumulation of metastatic melanoma cells in different organs.

Monitoring of Stimulus Evoked Murine Somatosensory Cortex Hemodynamic Activity With Volumetric Multi-Spectral Optoacoustic Tomography.

Sensory stimulation is an attractive paradigm for studying brain activity using various optical-, ultrasound- and MRI-based functional neuroimaging methods. Optoacoustics has been recently suggested as a powerful new tool for scalable mapping of multiple hemodynamic parameters with rich contrast and previously unachievable spatio-temporal resolution. Yet, its utility for studying the processing of peripheral inputs at the whole brain level has so far not been quantified. We employed volumetric multi-spectral optoacoustic tomography (vMSOT) to non-invasively monitor the HbO, HbR, and HbT dynamics across the mouse somatosensory cortex evoked by electrical paw stimuli. We show that elevated contralateral activation is preserved in the HbO map (invisible to MRI) under isoflurane anesthesia. Brain activation is shown to be predominantly confined to the somatosensory cortex, with strongest activation in the hindpaw region of the contralateral sensorimotor cortex. Furthermore, vMSOT detected the presence of an initial dip in the contralateral hindpaw region in the delta HbO channel. Sensorimotor cortical activity was identified over all other regions in HbT and HbO but not in HbR. Pearson's correlation mapping enabled localizing the response to the sensorimotor cortex further highlighting the ability of vMSOT to bridge over imaging performance deficiencies of other functional neuroimaging modalities.

Isolated Murine Brain Model for Large-Scale Optoacoustic Calcium Imaging.

Real-time visualization of large-scale neural dynamics in whole mammalian brains is hindered with existing neuroimaging methods having limited capacity when it comes to imaging large tissue volumes at high speeds. Optoacoustic imaging has been shown to be capable of real-time three-dimensional imaging of multiple cerebral hemodynamic parameters in rodents. However, optoacoustic imaging of calcium activity deep within the mammalian brain is hampered by strong blood absorption in the visible light spectrum as well as a lack of activity labels excitable in the near-infrared window. We have developed and validated an isolated whole mouse brain preparation labeled with genetically encoded calcium indicator GCaMP6f, which can closely resemble in vivo conditions. An optoacoustic imaging system coupled to a superfusion system was further designed and used for rapid volumetric monitoring of stimulus-evoked calcium dynamics in the brain. These new imaging setup and isolated preparation's protocols and characteristics are described here in detail. Our new technique captures calcium fluxes as true three-dimensional information across the entire brain with temporal resolution of 10 ms and spatial resolution of 150 µm, thus enabling large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with any existing neuroimaging techniques.

Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain.

Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy.

Wavelength-dependent optoacoustic imaging probes for NMDA receptor visualisation.

The cellular localisation and binding specificity of two NMDAR-targeted near-IR imaging probes has been examined by microscopy, followed by exemplification of MSOT to monitor simulated glutamate bursts in cellulo and a preliminary study in mice observing the signal in the brain.

Short and long-term phototoxicity in cells expressing genetic reporters under nanosecond laser exposure.

Nanosecond-duration laser pulses are exploited in a plethora of therapeutic and diagnostic applications, such as optoacoustic imaging. However, phototoxicity effects of pulsed radiation in living cells, in particular those expressing genetic reporters, are not well understood. We established a three-dimensional fluorescent protein expressing cellular model in order to reliably investigate the extent and major exposure parameters responsible for both photobleaching and phototoxicity under pulsed laser exposure, unveiling a variety of possible effects on living cells, from reversible photobleaching to cytotoxicity and cell death. Significant losses of fluorescence levels were identified when exposing the cells to illumination conditions considered safe under common standards for skin exposure in diagnostic imaging applications. Thus, the use of photolabile fluorescent proteins and their in vivo exposure parameters have to be designed carefully for all applications using pulsed nanosecond radiation. In particular, loss of signal due to bleaching may significantly alter signals in longitudinal measurements, making data quantification challenging.