The inability to directly visualize large-scale neural dynamics across the entire mammalian brain in the millisecond temporal resolution regime is among the main limitations of existing neuroimaging methods. Recent advances in optoacoustic imaging systems have led to the establishment of this technology as an alternative method for real-time deep-tissue observations. Particularly, functional optoacoustic neurotomography (FONT) has recently been suggested for three-dimensional imaging of both direct calcium activity and cerebral hemodynamic parameters in rodents. However, the lack of suitable calcium indicators featuring optical absorption peaks within the so-called near-infrared window has hampered the applicability of FONT for imaging neuronal activity deep within the mammalian brain. To surmount this challenge, we developed and validated an intracardially perfused murine brain model labelled with genetically encoded calcium indicator GCaMP6f that closely simulates in vivo conditions. Penetration of light through skull and skin is greatly facilitated after blood is substituted by artificial cerebrospinal fluid (ACSF). The new preparation enabled here the observation of stimulus-evoked calcium dynamics within the mouse brain at penetration depths and spatio-temporal resolution scales not attainable with other neuroimaging techniques.
Fluorescence imaging is widely employed in biological discovery due to its excellent molecular sensitivity and contrast. However, due to light scattering wide-field fluorescence images are blurred resulting in very low spatial resolution and low image contrast. The existing scanning optical microscopy techniques are commonly restricted to sub-millimeter field-of-view or otherwise slow imaging speeds, limiting their applicability for imaging of fast biological dynamics occurring on larger spatial scales. Herein, we developed a rapid scanning wide-field multifocal structured illumination microscopy method based on a beam-splitting grating and an acousto-optic deflector synchronized with a high speed camera. The multi-beam pattern is focused by a condensing lens and a macroscopic objective to generate multifocal structured illumination profile on the imaged sample that is rapidly scanned at kHz rates. Experimental results show that the proposed method can achieve real-time fluorescence microscopy over a centimeter-scale field of view. Owing to the low numerical aperture of the diffracted beams, the illumination has a large depth of focus and hence is generally not affected by the sample’s curvature, which allowed here imaging of perfusion in the entire mouse cerebral cortex noninvasively. The new approach can be readily incorporated into traditional wide-field microscopes to attain optimal tradeoff between spatial resolution and field of view. It further establishes a bridge between conventional wide-field macroscopy and laser scanning confocal microscopy, thus anticipated to find broad applicability in a variety of applications looking at large-scale fluorescent-based biodynamics.
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.
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