Application of multiphoton label-free imaging to phenotype bone development and bone pathologies

Abstract Long bones such as the femur develop through endochondral ossification (EO), where cartilage is gradually replaced by mineralised bone through a series of cellular events. Traditional imaging methods for studying the bone development process either requires exogenous dyes/labels or involve ionising radiation, limiting in-vivo application. Multiphoton microscopy offers a label-free, non-ionising approach with high chemical sensitivity for real-time tissue analysis. The study demonstrates the novel application of an integrated multimodal imaging platform, combining second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and two-photon autofluorescence (2-PaF), to investigate bone development and disease. By applying the multimodal approach to chick embryonic femurs at three developmental stages, we reveal collagen phenotypic changes associated with skeletal maturation and demonstrate its potential for identifying structural and biochemical alterations relevant to human bone disease. SHG enabled detailed characterisation of collagen fibre architecture, and additionally CARS imaging, tuned to the 2845 cm⁻¹ Raman band corresponding to the symmetric CH₂ stretching vibration of neutral lipids, identified lipid-rich structures consistent with intracellular lipid droplets. These structures are hypothesised to represent the cellular component within the developing bone cortex, an interpretation supported by stainings, which demonstrated a comparable trend in quantitative changes in cell nuclei density across developmental stages. 2-PaF was primarily included to demonstrate the multimodal imaging capability of the system and to provide a general overview of the tissue morphology through intrinsic autofluorescence-based visualisation. SHG analysis revealed a progressive increase in the number of collagen fibres/mm² and average fibre length, while fibre straightness showed a decreasing trend that later stabilised with no significant change. In contrast, average fibre width increased between D11 and D14 and plateaued thereafter. Overall, CARS imaging revealed a gradual reduction in lipid droplets/mm², consistent with dynamic changes in cellular composition during bone maturation. To explore the translational applicability of this approach, SHG imaging was applied to human bone core biopsies from a small cohort of osteoarthritic (OA; n = 3) and osteoporotic (OP; n = 3) patients. Preliminary qualitative observations revealed apparent differences in collagen organisation between samples, highlighting the potential of SHG imaging to detect variations in bone matrix architecture. While these findings are descriptive and exploratory, the successful application of multimodal, label-free imaging to human bone tissue provides a foundation for future studies investigating skeletal tissue maturation, matrix remodelling, regenerative therapies, and tissue-engineering strategies.