Facility-deployable sequential Raman and second-harmonic generation microscopy with quantified spatial registration on a commercial confocal platform

Correlated Raman and second-harmonic generation (SHG) microscopy can link broadband chemical information with structural contrast from non-centrosymmetric materials, but practical implementation in shared imaging facilities is constrained by instrument complexity, uncertain spatial alignment, and limited reproducibility documentation. We describe a facility-deployable workflow for sequential spontaneous Raman microspectroscopy and SHG imaging on a commercial WITec alpha300R confocal platform equipped with a modular SHG channel. Both modalities use the same objective and nominal scan coordinates, and switching is performed without deliberate sample repositioning. Spatial correspondence was therefore treated as a measured performance characteristic rather than assumed to be exact or simultaneous. An edge-based benchmark yielded a residual lateral registration offset of 0.50 µm under the tested conditions. Additional characterization included a Raman lateral resolution of 0.49 ± 0.02 µm, Raman and SHG signal-to-noise behavior as a function of acquisition settings, and inter-session/multi-user spectral reproducibility (coefficient of variation, 4.60%). Bacterial colonies, human lung airway sections, and collagen-rich tendon were used solely as validation specimens for spectral mapping, sequential chemical-structural imaging, and polarization-sensitive SHG response. The principal contribution is a documented integration, calibration, registration-validation, and acquisition workflow that can be reproduced in shared facilities using a commercial base platform and equivalent modular hardware, together with explicit discussion of drift, acquisition time, and data-handling trade-offs.