Bessel foci used for two-photon fluorescence excitation have enabled high-speed volumetric imaging. At high numeric aperture, their imaging performances are compromised by substantial side-ring excitation and suffer from reduced image contrast. Here, we describe axially extended Bessel-droplet foci with suppressed side rings and more resistance to optical aberrations. Applying novel phase patterns to generate Bessel-droplet foci of variable NAs at high power throughput, we achieved continuous volume imaging by scanning them interferometrically along the axial direction. With Bessel-droplet foci, we demonstrated high-resolution volumetric imaging of synaptic anatomy and function as well as lymphatic circulations in the mouse brain in vivo.
To visualize neuronal structure and function in the physiological context, optical microscopy that is non-invasive and capable of resolving sub-cellular structures has become the method of choice. Structured illumination microscopy (SIM) is a widefield fluorescence imaging technique that optically sections 3D samples, but its applications have been usually limited to in vitro samples. To apply SIM to in vivo imaging, we modified optical-sectioning SIM reconstruction algorithm and incorporated adaptive optics. We demonstrated fast, high-resolution in vivo imaging with optical sectioning for structural and functional interrogations of the brain in vivo.
An enabling technology for the monitoring of neural activity, multiphoton microscopy with Bessel focus scanning is a high-speed volumetric imaging method with subcellular lateral resolution. Similar to many other optical microscopy techniques, however, its axially extended Bessel-like focus experiences sample-induced optical aberrations, which lead to reduced resolution and image contrast at depth. In this study, we demonstrated an adaptive optical Bessel focus scanning multiphoton microscope with pupil-segmentation-based wavefront sensing and highly efficient sample-plane aberration correction. Applying it to mouse brain imaging in vivo, we observed up to threefold signal enhancement for functional spine imaging at depth.
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