Optical alignment device for two-photon microscopy

Two-photon excitation fluorescence microscopy has revolutionized our understanding of brain structure and function through the high resolution and large penetration depth it offers. Investigating neural structures in vivo requires gaining optical access to the brain, which is typically achieved by replacing a part of the skull with one or several layers of cover glass windows. To compensate for the spherical aberrations caused by the presence of these layers of glass, collar-correction objectives are typically used. However, the efficiency of this correction has been shown to depend significantly on the tilt angle between the glass window surface and the optical axis of the imaging system. Here we expand these observations and characterize the effect of the tilt angle on the collected fluorescence signal with thicker windows (double cover glass) and compare these results with an objective devoid of collar-correction. Finally, we present a simple optical alignment device designed to rapidly minimize the tilt angle in vivo and align the optical axis of the microscope perpendicularly to the glass window, thereby significantly improving the imaging quality. The performance of the device is demonstrated in an in vivo setting and a tilt-correction procedure is described, enabling the accurate alignment (<0.2°) of the cover glass with the imaging plane in only few iterations.