Chronic in vivo optical imaging of the spinal cord is an effective way to study the biological processes during and after spinal cord injury (SCI) in mouse models. It normally relies on an implanted spinal chamber to provide continuous optical access to the spinal cord. However, the chronic window consists of multiple layers of transparent materials with various optical properties and irregular thickness, which induce large optical aberration. Therefore, the image quality of multiphoton microscopy as well as the precision of femtosecond laser axotomy were dramatically degraded. In this work, we developed an adaptive optics (AO) microscope system integrating stimulated Raman scattering (SRS) and twophoton excited fluorescence (TPEF). Using our system, the aberrations induced by the spinal cord window were measured and compensated accordingly, enabling both high-resolution imaging and precise laser axotomy of the mouse spinal cord.
Two-photon microscopy has become the method of choice for in vivo brain imaging in neuroscience research during the past decades owing to its inherent sectioning capability and large penetration depth in scattering tissues. By integrating with a gradient refractive index (GRIN) lens that implanted into the brain, two-photon microendoscopy further extends the imaging depth down to subcortical regions. However, the imaging resolution and field of view (FOV) are compromised due to the intrinsic aberrations of the inserted GRIN lens. Here, we developed an adaptive optics (AO) two-photon microendoscopy based on direct wavefront sensing that can measure and correct the aberrations of GRIN lens during in vivo brain imaging. Using our system, the diffraction-limited resolution was restored and the fine structures such as dendritic spines of hippocampal CA1 neurons can be clearly resolved over a much extended FOV.
Non-invasive retinal imaging has greatly facilitated the research of eye disease and neurodegenerative disorders in the central nervous system (CNS). Two-photon microscopy is a powerful tool for in vivo imaging of mouse retina because it provides intrinsic optical sectioning capability and the infrared laser is less likely to excite the photoreceptors. However, the dilated mouse eye has large optical aberrations, which must be corrected to achieve high-resolution or even diffraction-limited imaging. Here, we developed an adaptive optics (AO) two-photon microscope for in vivo imaging of retinal neurons through the eyeball of living mouse. We used the two-photon excited fluorescence signal of retina as the guide star to measure and correct the aberration of mouse eye. After AO correction, the fluorescence signal was increased by at least fivefold and the fine structures such as axons of retinal ganglion cells (RGC) were clearly resolved. To take advantage of the non-invasive high-resolution imaging, we demonstrated functional calcium imaging of RGC responding to the light stimulations.
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