Correlative SHG-AFM imaging workflow for label-free quantitative analysis of tissue structure-function relationships

We present a correlative second harmonic generation (SHG) and atomic force microscopy (AFM) imaging workflow for quantifying micromechanical properties of tissue structures. SHG imaging guides AFM force mapping regions of interest, enabling spatially resolved analysis of fibrillar collagen morphology and stiffness. The workflow uses unfixed, unlabeled tissue sections and commercial software for image segmentation and alignment. Fully hardware-compatible and easily deployable across standard systems, it supports high-throughput, reproducible analysis suitable for core facilities and clinical labs. Compared to brightfield-guided mapping, SHG improves anatomical specificity and efficiency, enabling micromechanical phenotyping of collagen-based extracellular matrices without staining or fixation.