Nanoscale iR spectroscopy

20th December 2018

Tapping AFM-IR has extended the range of applications that can be addressed.
Fig 1. Concept of tapping AFM-IR operation
Example showing tapping AFM-IR spectra.
Fig 3.(a) AFM topography map, (b) tapping AFM-IR spectrum of collagen fibre (c) Subsequent tapping AFM-IR map at 1655cm (d) IR map overlaid on AFM topography map.

Jérémie Mathurin & Anirban Roy explore nanoscale IR spectroscopy for biological applications

Infrared (IR) spectroscopy is one of the most recognised analytical measurement techniques in academic, government and industrial R&D laboratories for the characterisation of polymeric materials. The spatial resolution of conventional bulk IR spectroscopy is limited to 3-10 µm by diffraction limit, depending on the method used. On the other hand, atomic force microscopy (AFM) is a widely used nanoscale imaging technique that provides the user with a high spatial resolution topographic map of a sample surface. Until now, the major drawback of AFM has been its inability to chemically characterise the material underneath the tip. AFM-IR is a photothermal technique that combines AFM and IR spectroscopy to unambiguously identify the chemical composition of a sample with tens of nanometre spatial resolution. So far, it has been successfully used in contact mode in a variety of applications. However, the contact mode is unsuitable for soft or loosely adhered samples, such as the less than 200nm polymeric nanoparticles (NPs) that are of wide interest for biomedical applications.

When the sample absorbs photons from a pulsed, tunable, monochromatic IR laser light source, it heats up and rapidly expands, inducing an impulse to the AFM probe in contact with the sample. This causes an oscillation of the AFM cantilever at its contact resonant frequencies. The amplitude of each of the contact resonant frequencies has been proven to be proportional to the IR absorbance. As a consequence, by tuning the laser through a range of wavenumbers, an IR spectrum can be collected that correlates with the conventional Fourier transform infrared (FT-IR) spectra. The spatial resolution of the measurement is no longer limited by the diffraction-limited spot size of the IR beam, but instead is determined by the diameter of the AFM tip, which is in the order of 10nm. The use of fast, tunable, pulsed IR laser sources with variable repetition rates, such as quantum cascade lasers (QCLs), has significantly improved the speed and sensitivity of photothermal AFM-IR, and has also enabled the measurement of IR spectra in AFM tapping mode.

AFM-IR spectra are typically collected with the AFM probe in direct contact with the surface of the sample. This is not a problem when collecting point spectra where the AFM tip is held at a fixed location on the sample, unless the sample is particularly soft or mobile. However, during IR image acquisition, where the IR source wavelength is fixed and the AFM tip is scanned across the sample surface, the contact mode can be more problematic for soft or loosely adhered samples. This is because the tip can deform or remove the sample as it moves across the surface, leading to sample and/or tip deformation and inaccurate results.

This problem was solved for conventional AFM by the development of tapping mode measurements, where the tip is not continuously in contact with the sample but instead taps, making intermittent contact with the surface. This allows highly reproducible imaging of a broader range of samples even if they are very soft or loosely adhered. Tapping mode is typically performed by driving the AFM cantilever at its fundamental free resonance and bringing the AFM tip down to the sample until it touches the surface by carefully monitoring the amplitude value in a feedback control mechanism. The tip is then scanned across the sample surface and the topography of the sample is recorded by maintaining a constant oscillation amplitude. In tapping AFM-IR mode of operation, the AFM cantilever is typically oscillating at its fundamental frequency (or first overtone) and the IR laser pulse rate is set to the difference between the fundamental and the first overtone. Due to nonlinear tip-sample interaction at the surface, photothermal IR (AFM-IR) signal can be detected at the sum or difference of these frequencies, as explained in Fig.1.

Fig. 2 shows tapping AFM-IR spectra and an IR absorption (i.e., chemical) image collected on a thin film sample of purple membrane deposited on a gold substrate. The yellow dotted line drawn across the IR absorbance image collected with the laser tuned to the protein amide I absorbance band at 1,660cm-1 shows that sub-10nm spatial resolution has been achieved in this measurement.

Fig. 3 demonstrates tapping AFM-IR measurements performed on a single collagen fibre. Spectral measurement shows expected amide I and II bands at 1,665 and 1,555cm-1, respectively. Protein residues are highlighted in the tapping AFM-IR image at amide I band. Both the AFM topography and chemical map shows the characteristic D-bands structures with ~67nm periodicity consistent with reported value.

As mentioned before, contact mode AFM-IR is typically unsuitable for soft or loosely adhesive samples, such as polymeric nanoparticles (NPs) of less than 200nm, which are of wide interest for biomedical applications. Fig. 4 shows that tapping AFM-IR allows accurate visualisation of both the location of the NPs’ shells and that of the incorporated material. Nowadays, poly(lactic acid) (PLA) and poly(D,L-lactic-co-glycolic acid) (PLGA) polymers are the most employed biomaterials to prepare drug nanocarriers. Tapping AFM-IR allows simultaneous imaging spherical PLA/PLGA NPs without distorting or displacing them in spite of their loose interaction with the AFM substrate. Besides the improved topography, the superiority of the tapping mode over traditional contact was striking when investigating the chemical composition of the PLA NPs by recording the IR signals of their components. PLA NPs were prepared using polyvinyl alcohol (PVA), the most commonly used surfactant, which confers on them colloidal stability. The general assumption is that these NPs possess a core (PLA or PLGA)-shell (PVA) structure. The tapping AFM-IR method proved unambiguously the existence of a hydrophilic surfactant corona around the NPs core with high resolution. These studies pave the way toward the use of tapping AFM-IR as a powerful tool to control the quality of NP formulations based on individual NP detection and component quantification.

Tapping AFM-IR has not only improved the resolution of nanoscale IR spectroscopy, it has also extended the range of applications that can be addressed, providing new nanoscale chemical information.

Jérémie Mathurin is with the University of Paris Sud & Anirban. Roy is with Bruker






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