ENHANCING OSTEOCYTE STAINING IN HUMAN BONE VIA ULTRASMALL LIGAND-FUNCTIONALIZED CORE-SHELL FLUORESCENT PROBE
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Direct optical visualization of osteocytes and the lacunar-canalicular network (LCN) is essential for understanding bone microstructure and osteocyte-mediated regulation. However, imaging within native human bone remains challenging due to mineral density, optical opacity, and strong matrix autofluorescence. Here, we establish a multimodal hierarchical imaging methodology to visualize osteocytes, the LCN, and native bone microarchitecture ex vivo in human bone. This approach integrates scanning electron microscopy (SEM) with optical microscopy techniques including second harmonic generation (SHG), multiphoton microscopy (MPM), and confocal microscopy using ultrasmall fluorescent core-shell silica nanoparticles (C’Dots). We hypothesized that C’Dots label both viable and non-viable osteocytes in human bone, and that integrin-targeted c(RGDyC)-C’Dots achieve higher osteocyte-labeling efficiency than unfunctionalized C’Dots. 1.2-mm-thick human tibial plateau specimens were labeled by C’Dots through whole-mount staining. SEM enabled rapid millimeter-scale visualization of the specimen microstructure. Simultaneous acquisition of SHG and C’Dot fluorescence provided micrometer-scale structural and cellular information. Colocalization of C’Dots with viability dye validated their live and dead osteocyte labeling ability. Airyscan detection-based super-resolution confocal microscopy enabled visualization of the LCN and individual canaliculi (~200 nm). Furthermore, integrin-targeted c(RGDyC)-C’Dots achieved a 71% greater number of labeled osteocytes and 38% greater fluorescence intensity (p < 0.05). Comparison with standard rhodamine 6G staining showed that c(RGDyC)-C’Dots achieved comparable osteocyte-LCN labeling while producing stronger osteocyte-associated fluorescence than unfunctionalized C’Dots. Together, this study establishes C’Dot-based osteocyte labeling to human bone ex vivo thereby introducing a clinically translated minimally invasive optical imaging platform for visualizing osteocyte networks and microstructural alterations in native human bone.