By measuring and correcting sample-induced aberrations, adaptive optics (AO) enables noninvasive imaging of subcellular structures in living organisms with two-photon (2P) fluorescence microscopy. We will introduce CoCoA-2P, a self-supervised machine-learning algorithm capable of simultaneously estimating aberrations and recovering 3D structural information from a single 2P image stack without requiring external training datasets. We will showcase the applications of CoCoA-2P for high-resolution in vivo structural imaging of the mouse brain and eye lenses.
Optical microscopy with adaptive optics (AO) allows high-resolution noninvasive imaging of subcellular structures in living organisms. As alternatives to hardware-based AO methods, supervised deep-learning approaches have recently been developed to estimate optical aberrations. However, these approaches are often limited in their generalizability due to discrepancies between training and imaging settings. Moreover, a corrective device is still required to compensate for aberrations in order to obtain high-resolution images. Here we describe a deep self-supervised learning approach for simultaneous aberration estimation and structural information recovery from a single 3D image stack acquired by widefield microscopy. The approach utilizes coordinate-based neural representations to represent highly complex structures. We experimentally validated our approach with directwavefront-sensing-based AO in the same samples and showed the approach is applicable to in vivo mouse brain imaging
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.
Uncovering the structure and function of cortical vascular network down to the level of capillaries can provide useful insights on brain physiology and pathology. Ideally, the probing method should allow concurrent observation of vascular morphology and hemodynamics, with sufficient spatiotemporal resolution to resolve individual capillaries and track blood cell motion in the scattering mammalian brain. By employing an all-optical scanning two-photon fluorescence microscope, we realized kilohertz full-frame recording of cortical blood vessels beyond 700 µm deep in the mouse brain and measured blood flow speeds up to 15 mm/s
Microendoscopy incorporating a gradient index (GRIN) lens has emerged as a powerful tool for in vivo imaging. The lack of optical sectioning capability of widefield microendoscopy and the intrinsic optical aberrations of the GRIN lens itself, however, limit the achievable image contrast and resolution in three-dimensional (3D) tissues. In this study, we applied HiLo to widefield microendoscopy for optical sectioning. We also utilized adaptive optics (AO) to measure and correct GRIN lens aberrations. Together, HiLo and AO enabled subcellular-resolution microendoscopy imaging with optical sectioning and allowed us to image fine neuronal processes and synapses 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.
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