Hybrid Confocal Fluorescence and Photoacoustic Microscopy for In‐Depth and Noninvasive Imaging of Biomolecule‐Functionalized 3D Scaffolds

ABSTRACT

The precise integration of biomolecules within three‐dimensional (3D) scaffolds is essential for tissue engineering and regenerative medicine. However, conventional characterization techniques, such as scanning electron microscopy, are destructive or limited to surface analysis, while confocal fluorescence microscopy is often hindered by scaffold autofluorescence. Here, we introduce a nondestructive hybrid imaging approach combining confocal fluorescence and frequency‐domain photoacoustic microscopy for volumetric characterization of biomolecule‐functionalized scaffolds. The method was demonstrated on honeycomb‐inspired 3D scaffolds fabricated by multiphoton lithography using a zirconium‐based hybrid photoresist and functionalized with Fmoc‐Asp‐OFm amyloid peptide fibrils via gold‐ion coating. Confocal fluorescence microscopy effectively resolves the scaffold architecture, whereas photoacoustic imaging selectively reveals the spatial distribution of peptide fibrils on the surface and within the scaffold volume, unaffected by autofluorescence. By decoupling structural and biomolecular information, this complementary approach enables nondestructive volumetric assessment of functionalized 3D microstructures, offering valuable capabilities for biomaterials characterization and quality control.