Guide to the construction and use of an adaptive optics two-photon microscope with direct wavefront sensing

Two-photon microscopy, combined with appropriate optical labeling, has enabled the study of structure and function throughout nervous systems. This methodology enables, for example, the measurement and tracking of sub-micrometer structures within brain cells, the spatio-temporal mapping of spikes in individual neurons, and the spatio-temporal mapping of transmitter release in individual synapses. Yet the spatial resolution of two-photon microscopy rapidly degrades as imaging is attempted at depths more than a few scattering lengths into tissue, i.e., below the superficial layers that constitute the top 300 to 400 µm of neocortex. To obviate this limitation, we measure the wavefront at the focus of the excitation beam and utilize adaptive optics that alters the incident wavefront to achieve an improved focal volume. We describe the constructions, calibration, and operation of a two-photon microscopy that incorporates adaptive optics to restore diffraction-limited resolution throughout the nearly 900 µm depth of mouse cortex. Our realization utilizes a guide star formed by excitation of red-shifted dye within the blood serum to directly measure the wavefront. We incorporate predominantly commercial optical, optomechanical, mechanical, and electronic components; computer aided design models of the exceptional custom components are supplied. The design is modular and allows for expanded imaging and optical excitation capabilities. We demonstrate our methodology in mouse neocortex by imaging the morphology of somatostatin-expressing neurons at 700 µm beneath the pia, calcium dynamics of layer 5b projection neurons, and glutamate transmission to L4 neurons. ### Competing Interest Statement The authors have declared no competing interest.