Simultaneous multiregion aberration correction in spatial light modulator-based two-photon multifocal microscopy

Abstract

Abstract. Two-photon multifocal microscopy (TPMM) enables faster imaging through parallel excitation, but aberrations in thick tissue degrade image quality and often vary across different regions of the field of view (FOV). We present a single spatial light modulator (SLM) framework for simultaneous multiregion aberration correction in SLM-based TPMM. The workflow includes three steps: multiregion aberration sensing, excitation-path correction, and emission-path correction. For excitation correction, we use an aberration-iterated and structure-optimized Gerchberg–Saxton strategy to embed region-specific aberrations into hologram optimization while maintaining multifocal uniformity. For emission correction, we reconstruct each scan position by integrating fluorescence from each excitation focus over a sufficiently large area, which cancels detection-path aberrations without adding complex hardware. We validate the method on fluorescent beads embedded in mouse brain tissue and on >100  μm-thick potato tissue, correcting 16 regions (41  μm×41  μm each) using a 60×/1.2 NA water-immersion objective. Compared with conventional two-photon microscopy (TPM)-equivalent imaging, full correction improves signal-to-noise ratio by 7.8× and lateral resolution by 1.3×, and yields clearer structures across multiple regions. This approach provides flexible large-FOV TPMM imaging with a relatively simple optical setup.