Abstract

Two-photon microscopy has been widely used in neuroscience due to its deeper penetration depth compared with confocal microscopy. However, as imaging depth increases, its imaging quality will also degrade dramatically because of the accumulated aberration and scattered light. Here we report a high-resolution two-photon microscopy deep into the scattering medium, by structure illuminating the sample in the focal volume and demodulating the fluorescent signal thereafter. Our results show that compared with conventional two-photon microscopy the maximum imaging depth increases by hundreds micron in the mouse brain, and even in the surficial layer the imaging resolution is improved by around 15%.

Two-photon microscopy has been widely used in neuroscience due to its deeper penetration depth compared with confocal microscopy. However, as imaging depth increases, its imaging quality will also degrade dramatically because of the accumulated aberration and scattered light. Adaptive optics is one of the most powerful tools to recover diffraction-limited resolution deep into the brain. However, it takes additional time for wavefront aberration measurement and compensation. Here we report a high-resolution two-photon microscopy deep into the scattering medium, by structure illuminating the sample in the focal volume and demodulating the fluorescent signal thereafter. The special structure-illumination pattern is design to make the focal point scanning sinusoidally only in the small focal volume. Here we first theoretically analyze the image formation of the structure illuminated two-photon microscopy (SITP) with D-shaped pupils and one annular one circular pupils, respectively. Their intensity points spread functions are investigated and the three-dimensional (3D) optical transfer functions (OTF) are provided. Then, the inertial-free scanning capability of SITP is demonstrated. After that, the ability of rejecting out-of-focus background and reducing noise is demonstrated. Our experimental results show that compared with conventional two-photon microscopy the maximum imaging depth increases by hundreds micron in the mouse brain, and even in the surficial layer the imaging resolution is improved by around 15%. Moreover, no additional time will be taken during to optical aberration and scattered light compensation / rejection.

© 2017 Optical Society of America