Tapping AFM-IR Provides High-Resolution Chemical Imaging

Infrared (IR) spectroscopy is one of the most accepted analytical measurement methods for the characterization of materials in government, academic, and industrial R&D laboratories. The spatial resolution of traditional bulk IR spectroscopy is restricted by diffraction to approximately 3–10 µm, based on the technique employed.

Atomic force microscopy (AFM) is an extensively employed nanoscale imaging method that offers the user with a high spatial resolution topographic map of a sample surface. So far, the main disadvantage of AFM has been its inability to chemically characterize the material beneath the tip. AFM-IR, a photothermal technique, integrates AFM and IR spectroscopy to clearly find the chemical composition of a sample with a spatial resolution of tens of nanometers.

Until now, it has been effectively used in contact mode in various applications. However, contact mode has shown to be incompatible for soft or loosely adhesive samples, for example, the polymeric nanoparticles (NPs) below 200 nm that are of wide interest in biomedical applications. This article discusses how Tapping AFM-IR overcomes such drawbacks, introducing the power of both IR spectroscopy and AFM topography mapping to a much broader range of applications.

On absorbing photons from a pulsed tunable monochromatic IR laser light source, the sample heats up and quickly expands, producing an impulse to the AFM probe in contact with the sample. This leads to oscillation of the AFM cantilever at its contact resonant frequencies. The amplitude of each of the contact resonant frequencies has been established to be in proportion with the IR absorbance. As a result, an IR spectrum comparable to traditional Fourier transform infrared (FT-IR) spectra can be obtained by tuning the laser via a range of wave numbers.

The diffraction-limited spot size of the IR beam is not restricted by the spatial resolution of the measurement anymore, but instead, it is decided by several factors including the diameter of the AFM tip, ranging from 10 to 30 nm. The use of rapid, tunable pulsed IR laser sources with variable repetition rates, such as quantum cascade lasers(QCLs), has considerably enhanced the speed and sensitivity of photothermal AFM-IR and has also allowed the measurement of IR spectra in AFM tapping mode.^1,2

Principles of Tapping AFM-IR

AFM-IR spectra are usually obtained with the AFM probe directly contacting the sample surface. This is problematic only when the sample is specifically soft or mobile, or else, this is not an issue when obtaining point spectra where the AFM tip is kept at a fixed location on the sample. However, at the time of IR image acquisition, where the IR source wavelength is fixed and the AFM tip is scanned over the surface of the sample, the contact mode can be more challenging for soft or loosely bonded samples.

Expanding the AFM-IR technique to incorporate softer samples was made possible by the development of tapping mode-based IR measurements, where the tip is not in continuous contact with the sample but instead taps, making sporadic contact with the surface. This enables highly reproducible imaging of a wider range of samples, although they are very soft or loosely bonded. Tapping mode is usually carried out by driving the AFM cantilever at its primary free resonance and lowering the AFM tip down to the sample such that the amplitude is restricted by contact with the sample surface. The tip is subsequently scanned over the sample surface and the topography of the sample is recorded by maintaining a steady oscillation amplitude.

Tapping AFM-IR is usually carried out in a way comparable to regular tapping mode by tapping at the fundamental free resonance of the cantilever, except that the IR laser aimed at the tip-sample location is pulsed at higher repetition rates. When an IR absorption band of the sample is excited by a specific laser wavenumber, it will cause heating and an oscillating photothermal expansion of the sample. The highest signal would be obtained by directly pulsing the laser at a cantilever resonant frequency. Here, the quality factor of the corresponding AFM cantilever mode would improve the oscillation amplitude.

In order to obtain local measurements, a heterodyne approach is employed, which involves setting the repetition rate of the laser to the difference frequency of the fundamental and second flexural eigenmodes of the AFM cantilever. The non-linearity of the force interaction in tapping mode combines the tapping oscillation with the sample photothermal expansion at the sum frequency, producing amplitude at the second mode of the cantilever. An alternative method to this measurement is to carry out the tapping oscillation at the second mode of the cantilever and detect the IR absorption using the fundamental mode. In both cases, the signal is dominated by the local signal during the tip-sample contact, and only the spatial resolution is limited by the AFM tip radius. Measurements taken using this method have shown a spatial resolution of 10 nm.

Figure 1. Basic principles of Tapping AFM-IR

Sub-10 nm Spatial Resolution Tapping AFM-IR Chemical Imaging of a Block Copolymer

A tapping AFM height image of a thin film of a block copolymer of polystyrene (PS) and poly(2-vinyl pyridine) (P2VP) is illustrated in Figure 2a. While it is obvious that there are two domains present, the chemical composition of the individual domains cannot be determined from the AFM height image alone. On tuning the pulsed QCL to the fixed wave number of 1588 cm⁻¹ (where P2VP has a strong IR absorption band), an improved signal intensity is detected when the AFM tip makes contact with a P2VP domain in the sample and the QCL repetition rate is matched with the difference frequency between the first and second AFM cantilever modes.

The sample is subsequently scanned in AFM tapping mode, maintaining the QCL repetition rate and wave number fixed. When the AFM tip intermittently contacts a P2VP domain, it results in a strong resonant response. As the AFM tip shifts to a PS domain location, the signal becomes much weaker for two reasons:

1. IR laser radiation at 1588 cm⁻¹ is not absorbed as strongly as the P2VP domains by the PS domains
2. The mechanical stiffness of the PS domains are different when compared to the P2VP domains, which results in a shift in the second contact resonance peak frequency from its value when the tip intermittently contacts the P2VP domains (which means the signal is less improved due to the fact that the frequency difference between the two cantilever resonance modes does not match the QCL repetition rate anymore).

In order to selectively improve the PS domains in the block copolymer film, the laser wavenumber is adjusted to 1492 cm⁻¹, where PS has a strong IR absorbance, and the QCL repetition rate is modified such that it matches the difference between the AFM fundamental resonant frequency and the frequency-shifted second AFM cantilever mode. Figure 2c illustrates an overlay of the Tapping AFM-IR images obtained at 1588 and 1492 cm⁻¹. The 10 nm spatial resolution achieved on this sample (demonstrated in Figure 2c and 2d) is mainly because of the exquisite sensitivity of the tapping AFM method to small mechanical stiffness differences.

The collection of complete AFM-IR spectra (in either contact mode or tapping mode) is important to verify the assignments of the polymer domains to the proper specific chemical species. Figure 2b demonstrates that the green Tapping AFM-IR spectrum (obtained in a green domain) is consistent with enriched P2VP content, whereas the red spectrum (obtained in a red domain) is consistent with higher PS content.

Figure 2. Chemical characterization of PS-P2VP block co-polymer sample by Tapping AFM-IR. (a) Tapping AFM height image. (b) Tapping AFM-IR spectra clearly identifying each chemical component. (c) Tapping AFM-IR overlay image highlighting both components (PS@ 1492 and P2VP@ 1588). (d) Profile cross-section highlighting the achievable spatial resolution, 10 nm. Sample courtesy of Dr Gilles Pecastaings and Antoine Segolene, University of Bordeaux, France.

Sub-10 nm Spatial Resolution Chemical Imaging and Spectroscopy of a Biological Membrane

Illustrated in Figure 3 are the Tapping AFM-IR absorbance images and spectra of a 5-nm-thick film of purple membrane deposited on a template-stripped Au substrate. The observed difference in the ratio of relative intensities of the amide I band at 1660 cm⁻¹ and the amide II band at 1542 cm⁻¹ is probably because of an orientation difference of the polypeptide chains, as the exciting QCL radiation is polarized normal to the surface.

The Tapping AFM-IR absorbance image was obtained with QCL wavenumber tuned to 1660 cm⁻¹. Island regions of the protein component of the purple membrane are visibly clear in the IR absorbance image. A plot of the 1660 cm⁻¹ band intensity obtained from the dotted line on the IR absorbance image represents that a spatial resolution of around 4 nm is attained.

Figure 3. Tapping AFM-IR spectra and absorbance images of a 5-nm-thick film of Halobacterium salinarum (purple membrane) deposited on a template-stripped Au substrate.

Sub-10 nm Spatial Resolution Chemical Imaging and Spectroscopy of a Graphene Wedge

Illustrated in Figure 4 are the scattering scanning nearfield optical (s-SNOM) reflection, absorption, and photothermal Tapping AFM-IR images collected at 930 cm⁻¹ of a graphene wedge deposited on a flat silicon substrate. The surface plasmon polaritron signal close to the edge can be seen apparently in the photothermally detected image because of the mechanical resonance improvement of this region of the sample.

Figure 4. Measurement of graphene wedge on silicon with s-SNOM and Tapping AFM-IR shows plasmonic effects at the edge.

Biomedical Application of Tapping AFM-IR to Polymeric Nanoparticles

As discussed earlier, contact mode AFM-IR is usually not ideal for soft or loosely bonding samples, such as polymeric nanoparticles (NPs) of size below 200 nm, which are of great interest for biomedical applications. Figure 5 illustrates that Tapping AFM-IR enables accurate visualization of the location of the NPs’ shells as well as the location of the incorporated material. Currently, the most used biomaterials to prepare drug nanocarriers are poly(D,L-lactic-co-glycolicacid) (PLGA) and poly(lactic acid) (PLA) polymers. Tapping AFM-IR enables spherical PLA/PLGA NPs to be imaged simultaneously without distorting or dislocating them, despite their loose interaction with the AFM substrate.

Figure 5. Comparison of contact mode AFM-IR (A) and Tapping AFM-IR (B, C) chemical maps on PLA NPs, and a schematic illustration of the NPs drying process on the AFM substrate (D). The 3D overlaid (topography and IR absorption) views clearly illustrate the topographic variations induced by the AFM acquisition mode. For A and B, red represents the strong absorption of the ester carbonyl band of the PLA core at 1760 cm⁻¹ and for C, red represents the strong absorption of the C H–bending and of the PVA corona at 1415 cm⁻¹. In D, the cores and the shells are schematically represented in red and blue, respectively. Adapted from Mathurin, et al., Analyst, 2018, DOI: 10.1039/c8an01239c.

In addition to the enhanced topography, the dominance of tapping over conventional contact was prominent when examining the chemical composition of the PLA NPs by recording the IR signals of their components. PLA NPs were prepared using polyvinylalcohol (PVA), the most widely used surfactant, which offers colloidal stability. The universal assumption is that these NPs have a core (PLA or PLGA)-shell (PVA) structure. The Tapping AFM-IR technique explicitly confirmed the presence of a hydrophilic surfactant corona around the NPs core with high resolution. These studies open the door for the use of Tapping AFM-IR to control the quality of NP formulations on the basis of individual NP detection and component quantification.

Apart from improving the resolution of nanoscale IR spectroscopy by an order of magnitude, Tapping AFM-IR has also widened the range of applications that can be dealt with, offering new nanoscale chemical information for applications such as metal oxide frameworks, polymeric materials, biomedical, catalysis, fibers, among many others.

References

1. Centrone et al., Analyst, 2018, 143, 3808-3813, DOI: 10.1039/c8an00838h.
2. Methune, et al., Analyst, 2018, DOI: 10.1039/c8an01239c.

This information has been sourced, reviewed and adapted from materials provided by Bruker Nano Surfaces.

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