Light microscopy has been a key tool for biological and medical research for centuries, but the limited penetration depth due to light scattering has restricted its in vivo imaging ability to superficial regions. Nowadays, adaptive optics and active wavefront shaping techniques are increasingly used to compensate sample-induced aberrations in nonlinear optical microscopy. However, in most cases, the wavefront control element, such as deformable mirror, is imaged onto the pupil plane of the microscope objective. This configuration limits the field of view over which spatially irregular aberrations can be corrected. A better choice is to place the wavefront control element, in a plane conjugate to the primary source of aberrations.
Here we demonstrate a novel design of a variable-conjugation plane adaptive optics two-photon microscope for deep-tissue bioimaging and systematically investigate all the trade-offs in the design. We use a liquid crystals spatial light modulator for precise control of the initial wavefront. The design of the microscope allows not only to extend the corrected field of view but also to easily adjust the position of the conjugate plane for different imaging depths in a three-dimensional scattering sample. We demonstrate the feasibility of the microscope and the efficiency of aberration cancellation at different depths of up to more than 1 mm. The enhancement of the intensity in the focal spot over the whole volume has been carefully investigated for variety of samples.