Contrast improvement in two-photon microscopy with instantaneous differential aberration imaging

Two-photon microscopy (TPM) has been widely used for thick tissue imaging. However, its penetration depth is fundamentally limited by the loss of signal contrast. Differential aberration imaging (DAI) can reject out-of-focus fluorescence in TPM by subtracting an aberrated image from an unaberrated one. This technique is simple and effective but compromises imaging speed because two images must be taken sequentially. Here we report a new strategy for two-photon DAI based on near-instantaneous temporal multiplexing, enabling high-speed imaging with pixel rates limited only by fluorescence lifetime and laser repetition rate. Our technique can be implemented with standard two-photon microscopes since it does not require active optical elements and it is based on a synchronized sampling strategy that does not require specialized hardware. We demonstrate and characterize the resultant contrast improvement when imaging fluorescently-labeled mouse brain at video-rate.