Compressive sensing-based multi-focus line-scanning two-photon microscopy for fast 3D imaging

Fast 3D volume imaging methods have been playing increasingly important roles in biological studies. In this article, we present the design and characterization of a multi-focus line-scanning two-photon microscope. Specifically, a digital micromirror device (DMD) is employed to generate a randomly distributed focus array on a plane (i.e., x-z) via binary holography. Next, a galvanometric mirror scans the focus array in a direction normal to the plane (i.e., y-axis) over the imaging volume. For sparse samples, e.g., neural networks in a brain, 1-3 foci are used together with compressive sensing algorithm to achieve a volume imaging rate of 15.5 volumes/sec over 77 × 120 × 40 µm³. High-resolution optical cross-sectional images on selected planes and regions can be generated by sequentially scanning the laser focus generated on the x-z plane with good imaging speeds (e.g., 107 frames/sec over 80 × 120 × 40 µm³). In the experiments, microbeads, pollens, and mouse brain slices have been imaged to characterize the point spread function and volume image rate and quality at different sampling ratios. The results show that the multi-focus line-scanning microscope presents a fast and versatile 3D imaging platform for deep tissue imaging and dynamic live animal studies.