;Spatial resolution
The spatial resolution of an AFM-IR instrument is related to the contact area between the probe and sample. The contact area is given by a = 3PR/4E* and 1/E* = (1-n)/ E+ (1-n)/ E, where P is the force employed to the probe, n and nrepresent the Poisson ratios of the sample and probe, respectively, and E and E are the elastic moduli of the sample and probe materials respectively. Typically, an AFM-IR has a lateral spatial resolution of 10–400 nm, for example, 100 nm, λ/150, and λ/400. Recently, Ruggeri et al. have demonstrated the acquisition of infrared absorption spectra and chemical maps at the single molecule level in the case of protein molecules with ca. 10 nm diameter and a molecular weight of 400 kDa.
;Instrumentation
In AFM-IR, an AFM probe is used to measure the absorption response of the sample to infrared radiation. The general approach for AFM/FTIR is shown in Figure 10.
There are a few different experimental setups when the infrared radiation is projected onto the sample as shown below: top, side, and bottom illumination setups (Figure 11).
In the first developed setup of AFM-IR, a sample is mounted onto an infrared-transparent zinc selenide prism for excitation purposes (Figure 12), then an optical parametric oscillator (OPO)-based tunable IR lased is radiated on the molecules to be probed by the instrument. Similar to conventional ATR spectroscopy, IR beam illuminates the sample through total internal reflection mechanism (Figure 12). The sample will heat up while absorbing radiation which causes a rapid thermal expansion of the sample surface. This expansion will increase the resonant oscillations of the AFM cantilever in a characteristic ringdown pattern (ringdown patterns means the decay of cantilever oscillation exponential in nature). Through Fourier transformation analysis, the signal could be isolated to obtain the amplitudes and frequencies of the oscillations. The amplitudes of the cantilever provide information of local absorption spectra, whereas the oscillation frequencies depend on the mechanical stiffness of the sample (Figure 12).