Compressive sensing for fast 3-D and random-access two-photon microscopy

3-D two-photon excitation (TPE) microscopy has been a critical tool for biological study since its introduction. Yet, the speed is largely limited by its point detector, e.g., photomultiplier tube (PMT), which requires a point-scanning imaging sequence. In this Letter, we present a multi-focus compressive sensing (CS) method for 3-D and random-access TPE microscopy based on a digital micromirror device (DMD). This new platform combines CS with a unique holography-based DMD random-access scanner to enhance the imaging speed by three to five times for imaging arbitrarily selected regions in 3-D specimens without sacrificing the resolution. In the experiments, 1–20 randomly selected foci are generated by modulating the wavefront of a femtosecond laser via binary holography, where the combined intensity is recorded by a PMT. By exploiting CS algorithms, 3-D images at arbitrarily selected sites can be reconstructed. Simulations and imaging experiments on different samples have been performed to verify the principle and identify the optimal processing parameters, including the number of laser foci and sampling ratios. The results show that high-resolution images can be obtained by using a 25% sampling ratio and five foci. The new CS-based TPE imaging method may find important applications in biological studies, e.g., neuronal imaging and optogenetics.