A Multicolor 3D-STORM High-Resolution Visualization Protocol for Collagen Mineralization in Self-Assembled Recombinant Type I Collagen Fibrils.

This protocol describes a multicolor three-dimensional stochastic optical reconstruction microscopy (3D-STORM) method for nanoscale visualization of collagen mineralization in a recombinant type I collagen self-assembled fibril model. The method enables simultaneous imaging of collagen, non-collagenous proteins (e.g., chondroitin sulfate), and calcium phosphate mineral phases. Sample preparation involves amino‑silanization and collagen self‑assembly, followed by mineralization using a calcium phosphate medium that forms amorphous calcium phosphate (ACP) at an early stage (30 min) and matures into hydroxyapatite (HAP) by 6 h. Multiplexed immunofluorescence labeling is then performed, and samples are first assessed by confocal microscopy before 3D-STORM image acquisition using an oxygen-scavenging imaging buffer. Data processing and analysis are carried out using publicly available software. Compared to conventional electron or confocal microscopy, this protocol combines molecular specificity with nanoscale resolution (typical lateral precision 20-30 nm, axial 50-60 nm), allowing three‑dimensional visualization of intrafibrillar versus extrafibrillar mineralization patterns. Representative results show clear visualization of collagen networks, associated non-collagenous proteins, and mineral phases within three-dimensional space. Quantitative metrics including Pearson's correlation coefficient (0.89 ± 0.04) and Manders' overlap coefficient (0.91 ± 0.03) are provided in the Results section. This protocol offers a powerful tool for researchers in biomaterials science, biomineralization, and bone tissue engineering who require nanoscale insight into mineralization dynamics.