Unstained Analysis and Evaluation of Bone Quality Characteristics of Rat Femur Cross Section by AIRsight Infrared/Raman Microscopy

Significance of the Topic

The analysis of bone quality extends beyond simple density measurements to include the composition and maturity of bone tissue, which is composed of inorganic hydroxyapatite and organic collagen. Unstained mapping of these components using vibrational spectroscopy provides vital insight into bone health, remodeling, and pathology without the need for chemical staining or decalcification.

Objectives and Study Overview

This study demonstrates the use of a combined infrared and Raman microscope to map compositional and structural characteristics across an undecalcified rat femur cross section. Key objectives include evaluation of mineral to matrix ratios, carbonate substitution, crystal maturity, and specific amino acid distributions using the AIRsight instrument.

Methodology and Instrumentation

The rat femur was freeze‐sectioned perpendicular to its long axis using a microtome, with the specimen maintained in parallel to the measurement stage. Reflection mode micro‐FTIR and micro‐Raman maps were acquired over a 650 μm × 300 μm area with 50 μm steps. Infrared conditions included 8 cm-1 resolution, 200 scans, and a 50 μm aperture. Raman measurements used a 785 nm laser, 50× objective, 10 s exposure, and five accumulations.

Main Results and Discussion

The FTIR spectrum revealed amide I, II, III bands of collagen and phosphate and carbonate bands of hydroxyapatite, while Raman spectroscopy confirmed amino acids such as phenylalanine and hydroxyproline. Multivariate curve resolution separated pure spectra of collagen and mineral and generated concentration maps showing collagen concentrated on the inner cortex and hydroxyapatite distributed throughout.

Bone Quality Metrics:

• Mineral to Matrix Ratio: The PO43- to amide I ratio from FTIR increased toward the outer cortex, indicating more mature mineral deposition at the periosteal surface.
• Carbonate to Phosphate Ratio: Raman CO32-/PO43- maps showed elevated carbonate content at bone edges, reflecting active remodeling zones.
• Mineral Maturity: Secondary derivative analysis of FTIR peaks at 1030 and 1110 cm-1 highlighted a belt of higher crystallinity located interior to the outer contour.
• Amino Acid Distribution: Phenylalanine to phosphate ratios from Raman data were highest near the periosteum, likely reflecting surface organic matrix components.

Benefits and Practical Applications

The combined infrared/Raman approach on a single microscope enables flexible selection of spectroscopic modality for target analytes. Non‐destructive, label‐free mapping yields quantitative metrics of bone composition, crystallinity, and organic matrix distribution, supporting research in osteoporosis, biomaterials, and tissue engineering.

Future Trends and Opportunities

Advances in spatial resolution, automated multivariate and machine‐learning analysis, and integration of additional modalities such as Raman polarization imaging will enhance the capability to assess bone microstructure. Extending these methods to clinical biopsies and engineered tissues may lead to diagnostic and prognostic tools in orthopedics and regenerative medicine.

Conclusion

This application study illustrates how simultaneous infrared and Raman microspectroscopy combined with multivariate and derivative processing can deliver comprehensive maps of bone composition and quality in an unstained rat femur cross section. The AIRsight microscope offers a versatile platform for detailed bone analysis without sample alteration.

Reference

1. Taylor EA, Donnelly EL. Raman and Fourier transform infrared imaging for characterization of bone material properties. Bone. 2020;139.
2. Taylor EA, Lloyd AA, Salazar-Lara C, Donnelly EL. Raman and FTIR mineral to matrix ratios correlate with physical chemical properties of model compounds and native bone tissue. Appl Spectrosc. 2017.
3. Grunenwald A, Keyser C, Sautereau AM, Crubézy E, Ludes B, Drouet C. Revisiting carbonate quantification in apatite biominerals: a validated FTIR methodology. J Archaeol Sci. 2014;49.