Fig 1.
(A) The sample surface is illuminated with a tunable IR laser. When the wavelength of the laser corresponds to an IR absorption band of the sample, the IR absorption will create heat and entail a rapid thermal expansion of the absorbing regions. This rapid thermal expansion induces oscillations (B) in the AFM tip at its resonant frequencies (C). Because the absorption is proportional to the cantilever oscillation, an absorption spectrum can be derived. (D) Absorption spectrum acquired on cancellous bone in which the phosphate band (920–1200 cm-1) and the amide I band (1592–1712 cm-1) characterize the mineral and collagen components, respectively. Adapted with permission from Anasys Instruments.
Table 1.
Infrared parameters commonly used to analyze bone tissue.
Fig 2.
Temporal and spatial reproducibility.
(A) The temporal reproducibility was assessed on PMMA (n = 7). The 5 repeated measurements for each of the 16 locations were averaged (black) and the corresponding standard deviation was derived for each location (red). The low standard deviation validates a good temporal reproducibility. (B) Example of a group of five repeated measurements showing a good temporal reproducibility. (C) The spatial reproducibility was assessed on seven samples. The 16 spectra (locations) per sample were averaged (black) and the corresponding standard deviation was derived (red). The low standard deviation validates a good spatial reproducibility.
Fig 3.
Comparison of normalized FTIR and AFM-IR spectra.
Spectra acquired on (A) PMMA and (B) cancellous bone. In the cancellous bone, the newly formed bone was located on the trabecular edge (in the first 20 microns) and more mature bone was closer to the center of the trabecula. No major shifts were observed between the two techniques, but differences in shapes and ratios are evident. In the amide band of the bone spectra (B), the amide II peak was significantly smaller in the AFM-IR spectra and a shoulder was evident around 1600 cm-1 not present in the FTIR spectra. In the mineral band for the newly formed bone, the shoulder corresponding to the peak 1128 cm-1 was more prominent.
Fig 4.
AFM-IR images show a non-mineralized layer (osteoid) not observed with FTIR.
Images acquired on the same cancellous bone samples with AFM-IR (right column) and FTIR (left and middle columns). The images in the left column were acquired with FTIR; the rectangles indicate the areas where the corresponding AFM-IR images were acquired. The FTIR images obtained within the rectangular area are enlarged in the middle column to match the size of the AFM-IR images, shown in the right column. The first row is the mineral-to-matrix ratio, the second row shows maps of the hydroxyapatite crystals acquired at 1030 cm-1, and the third row shows maps of the collagen acquired at 1660 cm-1. Color scales indicate the relative IR intensity for the technique used (left and middle columns share the same color scales).
Fig 5.
AFM image showing a line scan acquisition.
Example of line scan measurements (yellow dotted line) performed from the trabecular surface where the bone tissue is the youngest (the white arrow represents increasing tissue age from the surface), to the interior where the bone tissue is more mature. An ultrastructure is evident with layers of different orientations. Also fibers are visible at the interior, possibly mineralized fibers that appeared when the section was cut and deposited onto the substrate.
Fig 6.
Average (n = 8) of the spectroscopic parameters recorded by AFM-IR as a function of the distance from the trabecular surface.
Repeated measurements were acquired every 1 um as a line orthogonal to the trabecular surface. The mineral-to-matrix ratio increased and the acid phosphate substitution ratio decreased with bone maturity. The crystallinity and the collagen crosslink ratio remained constant.
Fig 7.
Example of a single line scan recorded by AFM-IR.
Evolution of the corresponding infrared parameters as a function of the distance from the trabecular edge resulting from repeated measurements acquired as a line orthogonal to the trabecula edge. * p<0.05 compared to the previous data point. The p-values were calculated using a one-sided Wilcoxon Mann Whitney test.
Fig 8.
AFM-IR images of the four spectroscopic parameters acquired on the same area of a cancellous bone sample.
Maps were derived from images acquired at six wavenumbers: 1128 cm-1, 1096 cm-1, 1030 cm-1, 1020 cm-1 (mineral band), 1690 cm-1, and 1660 cm-1 (amide). Those maps, particularly the mineral-to-matrix ratio and the collagen crosslink ratio, show layers of high and low intensities. Color scales are the AFM-IR intensity ratios.
Fig 9.
Height and infrared intensities acquired on a cancellous bone thin section (300 nm) before and after demineralization of the section using EDTA.
The topographic images (A and B) were acquired with the AFM in contact mode. The mineral (C and D) and collagen (E and F) images were acquired by AFM-IR at wavenumbers 1030 cm-1 and 1660 cm-1, respectively. The ratio of these two images is the mineral-to-matrix ratio (G and H). Before EDTA treatment the images had an alternating (layered or striated) pattern when both mineral and collagen were present in the section. The two components are entangled, and individual contributions cannot be distinguished. To uncouple the contributions, the collagen component was isolated by dissolving the mineral crystals. After EDTA treatment, the mineral image (D) shows a null intensity, indicating that the demineralization was accomplished. In the collagen image after demineralization (F) an alternating pattern still exists in the absence of mineral, suggesting that the collagen (either structure or density) is a significant contributor to the periodic pattern although the collagen structure may be affected by the mineral dissolution. The collagen pattern is also reflected in the mineral-to-matrix image.