Compact submicron-precision exact-constraint six-axis stage for sensor-free multicolor fluorescence synchronous imaging

Precise spatial registration of multiple detection focal planes is essential for high-resolution multicolor fluorescence synchronous imaging, yet it remains challenging in compact systems due to mechanical disturbances and alignment complexity. Here, we present a compact submicron-precision exact-constraint (SPEC) six-axis positioning stage for sensor-free registration of detection focal planes in a three-color fluorescence micro-optical sectioning tomography (fMOST) system. By integrating an exact-constraint mechanism with partial motion decoupling, optimized preload, and a natural frequency of 982 Hz, the stage achieves robust anti-vibration performance under 180 Hz vibratome sectioning. A data-driven open-loop compensation method eliminates systematic and hysteresis errors, yielding translational accuracy better than 1 μm and rotational accuracy within 30 μrad. Combined with a dual-step registration pipeline, the system attains sub-pixel alignment accuracy (<0.33 μm ) across three channels. High-fidelity multicolor imaging of fluorescently labeled mouse brain slices demonstrates precise subcellular co-localization of neurons, astrocytes, and nuclei without post-processing registration. This compact, cost-effective, and sensor-free SPEC platform offers a versatile solution for reliable multicolor 3D fluorescence imaging in neuroscience and histopathology.