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We advance two-photon microscopy for near-diffraction-limited imaging up to 850 μm below the pia in awake mice. Our approach combines direct wavefront sensing of light from a guidestar (formed by descanned fluorescence from Cy5.5- conjugated dextran in brain microvessels) with adaptive optics to compensate for tissue-induced aberrations in the wavefront. We achieve high signal-to-noise ratios in recordings of glutamate release from thalamocortical axons and calcium transients in spines of layer 5b basal dendrites during active tactile sensing.
Two-photon laser scanning microscopy is indispensable for imaging through the mammalian brain with subcellular resolution1. However, resolution and efficiency decrease with depth as a result of scattering and optical aberrations that are created by tissues. To mitigate such effects, the use of high-energy excitation pulses2 trades increased depth for the risk of nonlinear photodamage, while the use of an underfilled objective aperture3 sacrifices spatial resolution for increased depth. Alternatively, adaptive optics (AO)4 can improve multiphoton imaging by synthesizing a distortion to the wavefront of the excitatory beam that compensates for tissue-induced aberrations in the wavefront. The desired excitation wavefront can be determined using direct5–7 or indirect8–10 methods of wavefront sensing. Direct sensing of the wavefront from a descanned guidestar signal leads to wavefront correction with high photon and time efficiency6,7. A previous application of this approach, in which exogenous dye was injected into mouse cortex to form the guidestar, enabled the determination of spine geometry up to 600 μm below the pia and functional imaging of spines down to 500 μm below the pia7 (Supplementary Fig. 1), that is, in granular but not infragranular layers, in an acute preparation. An unmet challenge is to develop a robust imaging paradigm to resolve axons and spines throughout infragranular layers, which encompass all output neurons from the neocortex to other brain areas, including premotor neurons.
