Particles with sizes in the order of a few micrometers can significantly enhance the capabilities of optoacoustic imaging systems by improving visualization of arbitrarily oriented vascular structures and achieving resolution beyond the acoustic diffraction barrier. Particle tracking may also be used for mapping the blood flow in two and three dimensions. However, a trade-off exists between the particle absorption properties and size, whereas large sized microparticles also tend to arrest in the capillary network. We analyzed the flow of microparticles in an intracardiac perfusion mouse model in which blood is effectively substituted by artificial cerebrospinal fluid (ACSF). This enables mitigating the strong blood absorption background in the optoacoustic images thus facilitating the visualization of microparticles. A sequence of three-dimensional optoacoustic images of the mouse brain is then acquired at a high frame rate of 100 Hz after injection of the particles in the left heart ventricle. By visualizing the flow of particles of different sizes in microvascular structures it is possible to establish optimal trade-offs between the particle size, their optoacoustic signal and perfusion properties.
Neuronal activity occurs simultaneously and in a highly coordinated fashion in many different areas across the brain. Real-time visualization of large-scale neural dynamics in whole mammalian brains is hindered with the existing neuroimaging methods that are limited in their ability to image large tissue volumes at high speeds. Genetically encoded calcium indicators (GECIs) that modulate their fluorescence intensity as a function of intracellular calcium concentrations are powerful tools for the observation of large neuronal networks. Optoacoustic imaging has been shown capable of real-time three-dimensional imaging of multiple cerebral hemodynamic parameters in rodents. However, optoacoustic imaging of calcium activity deep in mammalian brain is hampered by strong blood absorption in the visible light spectrum as well as lack of activity labels excitable in the near-infrared window. We developed and validated an isolated whole mouse brain preparation labelled with genetically encoded calcium indicator GCaMP6f, which can closely resemble in vivo conditions and exhibit functional activity for several hours to several days. An optoacoustic imaging system coupled to a superfusion system was further devised and used for rapid volumetric monitoring of calcium dynamics in the brain evoked using an epilepsy-inducing drug. The new technique captures calcium fluxes as true 3D information across the entire brain with temporal resolution of 10ms and spatial resolution of 150µm, thus enabling large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques. The system could be readily adapted to work with future generations of far-red- and near-infrared GECIs.