Closed-loop correction of illumination-focal-plane mismatch enables consistent multicolor sectioning in OS-SIM

In multicolor optical-sectioning structured illumination microscopy (OS-SIM), wavelength-dependent illumination-focal-plane mismatch shifts the effective sectioning planes of different channels relative to a common detection focal plane, producing inconsistent sectioning performance. We present a closed-loop strategy that (i) localizes each channel's illumination-focal-plane position from the central peak of its axial modulation response, (ii) aligns the planes with channel-specific equal-focal-length 4f modules, and (iii) verifies residual mismatch by PZT-driven objective scanning. In our three-color system, the blue illumination focal plane was displaced 13--14 µm from both the red and green planes before correction; after correction, the median absolute peak-position differences |R-G|, |R-B|, and |G-B| fell to 0.25, 0.20, and 0.11 µm, respectively. Fluorescent-bead and brain-slice imaging confirmed improved interchannel sectioning consistency and fewer associated artifacts. The approach directly corrects illumination-focal-plane mismatch, a physical source of interchannel sectioning inconsistency, rather than relying solely on detection-side chromatic aberration correction or postprocessing registration.