Higher Harmonic Atomic Force Microscopy: Imaging of Biological Membranes in Liquid

The contribution of higher harmonics to the movement of a dynamic force microscope cantilever interacting with a sample in liquid was investigated. The amplitude of the second harmonic has been found to be an order of magnitude higher in liquid than in air, reflecting an increased sensitivity to local variations in elasticity and interaction geometries. A theoretical model of the tip-sample interactions in liquid was introduced and shown to be consistent with experimental findings. Second harmonic amplitude images were recorded on soft biological samples yielding a lateral resolution of $\sim 0.5\mathrm{nm}$.

Figure 1

Comparison between simulation and experimental data obtained for a silicon nitride tip interacting with a mica surface in buffer solution. (a) Simulated amplitude vs. distance curve. (b) Measured amplitude vs. distance curve (Instrument: Pico Plus AFM, Agilent Tec., Tempe Az, USA). (c) Simulated second harmonic amplitude vs. distance curve. (d) Experimental second harmonic amplitude vs. distance curve obtained using an external Lock-in amplifier (SR 830 DSP, Stanford Research Systems, Sunnyvale Cal., USA). (e) Frequency spectra (normalized by the driving frequency) obtained from simulation for $A_{sp}/A_0=0.9$. (f) Measured frequency spectra (normalized by the driving frequency, recorded using a NI PCI-6013 data acquisition device).

Figure 2

Dependence of the second harmonic amplitude ($A_2$) on interaction parameters. (a) $A_2$ as a function of effective tip radius. (b) $A_2$ as a function of the samples Young module.

Figure 3

High resolution on a bacterial $S$-layer. Second harmonic images were recorded using an external Lock-in amplifier (SR 830 DSP, Stanford Research Systems, Sunnyvale Cal., USA). (a) Simultaneous recorded topography (left panel) and second harmonic image (right panel). Substructures within the unit cell can be clearly observed (resolution $\sim 0.5\mathrm{nm}$). Scansize: $210\times 175{\mathrm{nm}}^2$; color code: 0–0.9 nm, 0–0.5 V. (b) Frequency spectra recorded on positions 1 and 2 of (a). The spectral contributions reflect the different properties of the tip-protein (pos. 2) and tip-hole (pos. 1) interactions (note that spectra are shifted with respect to the normalized driving frequency for better visibility). Inset: Contribution of the second harmonic amplitude in linear representation. (c) Average of 55 unit cells (left panel) from a (right panel) and a sketch of the expected lattice structure (right panel) 16.

Figure 4

Measurements on human rhinovirus serotype 2 (HRV2). (a) Topography image of HRV2 layer on mica. (b) Simultaneously recorded second harmonic amplitude image clearly revealing substructures of the viral capsids (circle). Scansize: $350\times 350{\mathrm{nm}}^2$; color code: 0–13 nm, 0–0.35 V.