Nanoscale Chemical Imaging of Soft Biological Tissues

In one of our earlier applications, we demonstrated PiFM’s capability in mapping the concentration of Li in Li_XFePO₄. The results agreed qualitatively to the results obtained via X-ray absorption spectroscopy (XAS) microscopy on similar samples. In this blog, we highlight the capability of PiFM on fixed human skin samples that were penetrated by topically applied dexamethasone and show that there is similarly a good correlation between XAS microscopy and PiFM, with PiFM offering much higher spatial resolution.

FTIR spectrum of dexamethasone; (b) combined PiFM images (top) acquired at 1708 1/cm for dexamethasone (red) and at 1558 1/cm for corneocytes (green) and X-ray transmission image of the same region. — FTIR spectrum of dexamethasone; (b) combined PiFM images (top) acquired at 1708 cm⁻¹ for dexamethasone (red) and at 1558 cm⁻¹ for corneocytes (green) and X-ray transmission image of the same region. *Region A is further investigated by PiFM in Figure 2.*

A cross-section of prepared skin slices of 300 nm thickness was deposited on a Si₃N₄window for XAS microscopy; the same area was then subsequently analyzed by PiFM. Given the FTIR spectrum of dexamethasone (Fig. 1a), PiFM images were acquired at 1708 cm⁻¹ and 1559 cm⁻¹ to highlight dexamethasone molecules and corneocytes (via amide II band), respectively; amide I band was avoided since dexamethasone has a strong absorption at around 1660 cm⁻¹. PiFM images for dexamethasone and corneocytes were colored to red and green, respectively, and combined to map the two different components; the resulting chemical map is shown in Fig. 1b (top) and compared to the X-ray transmission image (below) at 530.2 eV, the maximum absorption energy for dexamethasone. From both PiFM and X-ray transmission images, we can see that the drug is primarily concentrated toward the top region of the stratum corneum, highlighted by the black rectangular box; this result is similar to previous work on X-ray microscopy [REF: K. Yamamoto et al., Eur. J. Pharm. Biopharm. 18, 30 (2017)].

Detailed view on the stratum corneum of fixed human skin: (a) AFM topography visualizing the structure of corneocytes; (b) chemical PiFM contrast related to amides probed at 1558 cm−1; (c) PiFM contrast related to dexamethasone at 1708 cm−1; (d) two PiFM images combined to map chemical components. — Detailed view on the stratum corneum of fixed human skin: (a) AFM topography visualizing the structure of corneocytes; (b) chemical PiFM contrast related to amides probed at 1558 cm⁻¹; (c) PiFM contrast related to dexamethasone at 1708 cm⁻¹; (d) two PiFM images combined to map chemical components.

Figure 2 shows PiFM images at higher magnification along with the topography. The top images (12 mm × 12 mm) show the stratified structure of the stratum corneum clearly in all imaging modes; the bottom portion of these images coincide with the region A defined in Fig. 1b. Fig. 2c shows clearly how the drug is concentrated in the uppermost layer of the stratum corneum as seen in Fig. 1. The bottom row of images (3 mm × 3 mm) in Figure 2 zoom into the region defined by white boxes in the top images.

Figure 2b’ shows that no amide II signals are seen in between the corneocytes, most likely due to the thin lipid lamellae that are known to occur between the corneocytes. However, Fig. 2c’ shows that the topically applied drug penetrates into these lipid lamellae between the corneocytes and fills them partially; this is even more clearly visible in Fig. 2d’ (combined PiFM image, red for dexamethasone and green for corneocytes), which is magnified by 2X for easier observation.

With its spatial resolution that goes beyond X-ray microscopy and the lack of concern for radiation damage on soft tissue samples, PiFM is an attractive technique to acquire simultaneous chemical and morphological information in true nanoscale.

Credit: The samples were provided by E. Rühl, Freie Universität Berlin (Germany).