Infrared Nanospectroscopy (AFM-IR)

The term was first used to denote a method that combined a tuneable free electron laser with an atomic force microscope (AFM, a type of SPM) equipped with a sharp probe that measured the local absorption of infrared light by a sample with nanoscale spatial resolution.

[8][9][10][16][19][20] Then, the use of modern pulsed optical parametric oscillators and quantum cascade lasers, in combination with top-illumination, have enabled to investigate samples on any substrate and with increase sensitivity and spatial resolution.

As most recent advances, AFM-IR has been proved capable to acquire chemical maps and nanoscale resolved spectra at the single-molecule scale from macromolecular self-assemblies and biomolecules with circa 10 nm diameter,[18][17][21][22] as well as to overcome limitations of IR spectroscopy and measure in aqueous liquid environments.

The earliest measurements combining AFM with infrared spectroscopy were performed in 1999 by Hammiche et al. at the University of Lancaster in the United Kingdom,[1] in an EPSRC-funded project led by M Reading and H M Pollock.

[2] Both groups used a conventional Fourier transform infrared spectrometer (FTIR) equipped with a broadband thermal source, the radiation was focused near the tip of a probe that was in contact with a sample.

Both of these early experiments used a broadband source in conjunction with an interferometer; these techniques could, therefore, be referred to as AFM-FTIR although Hammiche et al. coined the more general term photothermal microspectroscopy or PTMS in their first paper.

The original technique dubbed AFM-IR that induced resonant motion in the probe using a Free Electron Laser has developed by exploiting the foregoing permutations so that it has evolved into various forms.

The pioneering experiments of Hammiche et al and Anderson had limited spatial resolution due to thermal diffusion - the spreading of heat away from the region where the infrared light was absorbed.

Consequently, the spatial resolution achieved by the early AFM-IR approaches was around one micron or more, due to the low modulation frequencies of the incident radiation created by the movement of the mirror in the interferometer.

All subsequent experiments by Hammiche, Pollock, Reading and their co-workers were made using this type of interface including the instrument constructed by Hill et al. for nanoscale imaging using a pulsed laser.

In the first paper on AFM-based infrared by Hammiche et al.,[1] the relevant well-established theoretical considerations were outlined that predict that high spatial resolution can be achieved using rapid modulation frequencies because of the consequent reduction in the thermal diffusion length.

Dazzi and his colleagues used a wavelength-tuneable, free electron laser at the CLIO facility[Note 2] in Orsay, France to provide an infrared source with short pulses.

Like earlier workers,[2][6] they used a conventional AFM probe to measure thermal expansion but introduced a novel optical configuration: the sample was mounted on an IR-transparent prism so that it could be excited by an evanescent wave.

Absorption of short infrared laser pulses by the sample caused rapid thermal expansion that created a force impulse at the tip of the AFM cantilever.

[33][34][35][36][37][38][39] By measuring the cantilever oscillation amplitude as a function of wavenumber, Dazzi's group was able to obtain absorption spectra from nanoscale regions of the sample.

A key advantage of the use of a tuneable laser source, with a narrow wavelength range, is the ability to rapidly map the locations of specific chemical components on the sample surface.

To achieve this, Dazzi's group tuned their free electron laser source to a wavelength corresponding to the molecular vibration of the chemical of interest, then mapped the cantilever oscillation amplitude as function of position across the sample.

[8] Hill also made use of an optical parametric oscillator as the infrared source in the manner of Hammiche et al.[24] This novel combination of topside illumination,[4] OPO source[24] and measuring thermal expansion[2][6][8] proved capable of nanoscale spatial resolution for infrared imaging and spectroscopy (the figures show a schematic of the UEA apparatus and results obtained with it).

The preliminary results of Reading et al.[40] show that directing a broadband QCL though an interferometer can give an easily detectable response from a conventional AFM probe measuring thermal expansion.

Since free electron lasers are rare and available only at select institutions, a key to enabling a commercial AFM-IR was to replace them with a more compact type of infrared source.

The CEO of Anasys Instruments recognised this achievement by calling it " an exciting major advance" in a letter written to the university and included in the final report of EPSRC project EP/C007751/1.

[28][43] In all of these examples a spectrum is acquired that spans the entire mid-IR range for each pixel, this is considerably more powerful than measuring the absorption of a single wavelength as is the case for AFM-IR when using either the method of Dazzi et al. or Hill et al. Reading and his group demonstrated how, because thermal probes can be heated, localized thermal analysis[4][28][29] can be combined with photothermal infrared spectroscopy using a single probe.

Theory predicts improvements in spatial resolution could be achieved by confining data analysis to the early part of the thermal response to a step change increase in the intensity of the incident radiation.

Sensitivity improvements were achieved using specialized cantilever probes with an internal resonator[49] and by wavelet based signal processing techniques.

[8][16][33][51] The advent of quantum cascade lasers (QCL) and the use of the electromagnetic field enhancement between metallic probes and substrates have improved the sensitivity and spatial resolution of AFM-IR down to the measurement of large (>0.3 μm) and flat (~2–10 nm) self-assembled monolayers, where still hundreds of molecules are present.

So the AFM-IR technique can measure the infrared absorption by the amplitude of the cantilever oscillation response and the mechanical properties of the sample via the contact resonance frequency and quality factor.

Since Ruggeri et al. pioneering work[16] on the aggregation pathways of the Josephin domain of ataxin-3, responsible for type-3 spinocerebellar ataxia, an inheritable protein-misfolding disease, AFM-IR was used to characterize molecular conformations in a wide spectrum of applications in protein and life sciences.

[81] This approach has delivered new mechanistic insights into the behaviour of disease-related proteins and peptides, such as Aβ42,[17] huntingtin[21] and FUS,[53] which are involved in the onset of Alzheimer's, Huntington's and Amyotrophic lateral sclerosis (ALS).