Nanoscale Studies of Stratum Corneum with Integrated AFM and IR Spectroscopy

By AZoNano Staff Writers

Topics Covered

Introduction
Overview of nanoIR
AFM-IR Analysis of stratum corneum
Summary
About Anasys Instruments

Introduction

The outermost layer of the skin, which serves as a barrier to foreign substances and maintains the homeostasis of the human body, is called the stratum corneum (SC). These functions are critical for ensuring an individual's well-being.

The SC includes a brick-and-mortar structure of corneocytes and lipids that is generated from the underlying skin layers. In this application note, the lipid distribution within a specimen of SC is studied at substantially higher spatial resolution than is possible with conventional Fourier transform infrared (FT-IR) microscopy, using instrumentation (Anasys nanoIR™) that combines a tunable IR laser source with an atomic force microscope (AFM).

Overview of nanoIR

The nanoIR from Anasys Instruments eliminates the limits on spatial resolution inherent to infrared microscopy. The nature of the IR absorbance detection results in the simultaneous measurement of nanoscale chemical compositions, mechanical properties, and surface morphologies.

The nanoIR also has integrated nanoscale thermal property mapping, making it a true multifunctional tool. Figure 1 shows the nanoIR instrument.

Figure 1. The nanoIR instrumentation platform; the system is typically mounted onto a 4 ft x 3 ft (1.2 m x 0.9 m) vibration isolation table

Using a proprietary design, the system's IR source is continuously tunable from 1200 to 3600cm-1 covering a broad range of the mid-IR spectrum.

As the sample absorbs radiation, it heats up, leading to rapid thermal expansion that excites resonant oscillations of the AFM cantilever. The induced oscillations decay in a characteristic ringdown as shown in Figure 2.

Figure 2. Schematic showing the technique behind nanoIR

The amplitudes and frequencies of the oscillations are extracted by Fourier transformation of the ringdown. Measuring the amplitudes of the cantilever oscillation as a function of the source wavelength creates local absorption spectra; the oscillation frequencies of the ringdown are related to the mechanical stiffness of the sample.

Survey regions of a sample via AFM imaging can be quickly surveyed by the nanoIR instrument. As shown in Figure 3 for polystyrene, AFM-IR spectra have excellent correlation with the bulk FT-IR spectra. This permits the researcher to import an individual nanoIR spectrum into commercial IR databases where they can be digitally searched to identify the materials at specific locations in the sample. Alternatively, the IR source can be tuned to a single wavelength and the intensity of rapid thermal expansions are collected in real-time as the specimen moves under the stationary AFM tip.

Figure 3. A comparison of the spectrum generated by the nanoIR (red) and conventional FT-IR (blue) of polystyrene

Further to its ability to provide high-resolution IR spectra, the nanoIR instrument provides information on the mechanical properties of the sample. This is accomplished by monitoring the frequency of the fundamental or higher resonant modes of the cantilever.

This is similar to the contact resonance method used for a number of years in the AFM community. The contact resonant frequency of the cantilever correlates to the stiffness of the sample and can be used to map the modulus of the sample qualitatively.

The nanoIR platform can also perform nanoscale thermal analysis utilizing novel AFM cantilevers that incorporate a resistive heating element. This enables identification or mapping of the amorphous/crystalline content, stress, extent of cure, or other material properties which can be characterized by the transition temperature of the material.

This combination of measurement capabilities creates a multifunctional tool in nanoIR that provides nanoscale structure, chemical, mechanical and thermal properties.

AFM-IR of Stratum Corneum - Procedure & Results

A Kapton tape strip was used to obtain a sample of SC from a subject's skin surface. The sample was then transferred from the tape directly onto the ZnSe prism. After removing the tape, some clusters of SC cells remain adhered to the prism surface, such that they could be scanned with the AFM.

The high lipid content in SC can be seen readily from local AFM-IR spectra as show in Figure 5. The methylene antisymmetric and symmetric stretching absorptions are near 2920 and 2852cm-1 , respectively, which suggest the presence of long-chain aliphatic materials (lipids).

The band height ratio between 2920cm-1 and 3290 cm-1 is observed to vary depending on the location. Here, the weaker band at 3290 cm-1 (blue curve, Figure 5) suggests a lower total protein content relative to the hydrophobic lipid-like components. These results agree favorably with the literature on pigskin.

This SC specimen may look nondescript on the surface, but interesting and chemically distinct structures can be elucidated by AFM-IR. Along with the amide-I and amide-II bands, the medium-strong signal at 1732cm-1 is seen near the center of this sample of SC (red curve, Figure 4). This is where the lipids are thought to be concentrated. On the periphery, the SC would still have lipids but at a lower level, as suggested by the weaker 1732cm-1 signals.

Figure 4. Point-and-click spectral acquisition over a large area of a SC specimen on a ZnSe prism; the area in the dashed box is enlarged and displayed in Fig 5 (low wavenumber spectra are normalized to 1640 cm-1)

Figure 5. Enlarged area as indicated by the dashed box in Fig 4; high wavenumber spectra showing variations near the lipid-rich area (spectra normalized to 2920 cm-1)

IR imaging using the AFM-IR technique offers an insightful visualization of the SC specimen. As expected, the strong amide absorption at 1650 cm-1 is observed at nearly every point of measurement within the SC. However, stronger absorptions at 1732 cm-1 are detected mostly in the area identified by the AFM-IR spectra as seen in Figures 4 and 5.

Summary

High resolution IR analysis of a human stratum corneum specimen is performed without cryomicrotomy using this novel AFM-IR technique. Simultaneous collection of topographic and IR images at 1650 and 1732cm-1, along with localized IR spectra enable the spatial visualization of the distribution of proteins and lipids in the specimen.

This technique can potentially be expanded to examine the distributions of cosmetics and topical medicines in stratum corneum, as well as their effects on the protein and lipid structure and distribution.

About Anasys Instruments

Anasys Instruments provides innovative AFM and related accessories which offer chemical, mechanical, and thermal analysis at the sub-100nm scale. The Company’s technology and products are being used to address metrology and analysis challenges in the polymers, pharmaceuticals, data-storage, and advanced-materials markets.

Anasys has been awarded numerous awards which establish Anasys as leaders in innovative technology, including the inaugural MICRO/NANO 25 Award in 2007, the R&D 100 Award in 2010 for the nanoIR and Microscopy Today’s 2011 Innovation Award for their breakthrough AFM-IR platform.

This information has been sourced, reviewed and adapted from materials provided by Anasys Instruments.

For more information on this source, please visit Anasys Instruments.

Date Added: Mar 7, 2014 | Updated: Mar 10, 2014
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