Performance improved for two-photon multi-focus microscopy based on a spatial light modulator by eliminating the zero-order beam

Two-photon microscopy (TPM) has a wide range of applications in the biomedical field. Two-photon multi-focus microscopy (TPMM) greatly improves the imaging speed by combining TPM with multi-focus technology. Therefore, TPMM based on spatial light modulator (SLM) has greater advantages in generating multi-focus point (MFP) with uniform intensity and flexible position than to other schemes. However, the zero-order beam (ZOB) produced by SLM in TPMM causes imperfection of the imaging system. For example, some sample areas are scanned repeatedly or illuminated unexpectedly. In this article, we proposed a TPMM system with improved performance by eliminating the ZOB. Apart from the target MFP, we utilize a phase-only SLM to generate another corrective beam (CB) with controllable amplitude, phase, and position precisely. The CB can produce destructive interference completely with the ZOB generated by the dead areas of the SLM. The method has a larger field of view, higher efficiency, and better uniformity in generating MFP compared to the method of overlaying the blaze grating. Simulations and experiments demonstrate the advantages of this approach.