BioScience AFM
of small samples like soft biomolecules in liquid where precise tip-sample force control is essential. Figure 9 shows examples of this mode from the previously described purple membrane protein bacterio rhodopsin and a soft lambda phage DNA.
Force-controlled AFM . Traditional AFM imaging modes, in particular contact mode, have some well-known drawbacks for challenging samples that exhibit steep edges or are simply too soſt , sticky, or loosely attached to the surface. T ese problems originate from the relatively high lateral/shear forces that are typically applied with very rough or heterogeneous samples and may result in distortion of some sample features. Some of these eff ects are overcome in dynamic AM-AFM with the trade-off of sometimes lower resolution.
QI mode solves this problem by virtually eliminating all lateral forces and simultaneously allowing the user to directly control the force applied at every pixel of the image. T e QI method is a force-distance-curve-based AFM mode ensuring that not more than a user set force is applied to the sample. A novel tip movement algorithm records a complete force curve at every pixel while only performing lateral movement between pixels. T e QI mode produces a complete map with a high spatial resolution recorded with an imaging speed of a few minutes using a linear velocity movement of the cantilever within the entire force curve. T is opens many possibilities for real force curve analysis and extraction of various material properties. An example of such a force curve and the parameters that can be extracted from it is given in Figure 1e .
Figure 9 : Phase-modulation AFM of biomolecules in buffer. (a) Phase- modulation height image of bacteriorhodopsin in 10 mM TRIS (150 mM KCl) buffer. (b) Height channel from a phase-modulation scan combined with fast scanning on lambda phage DNA sample in 10 mM HEPES (2 mM Ni 2+ ) buffer, acquired at a line rate of 10 Hz. Z -scales in (a) and (b) are 0.4 and 4 nm, respectively.
(AM-AFM). In AM-AFM, the cantilever is excited with a fixed amplitude and frequency around its resonance with the tip-sample interaction forces being detected as an amplitude damping [ 18 ]. It has been shown, however, that phase response in phase modulation AFM (PM-AFM) is much faster compared to AM-AFM. This in turn allows for excellent force control in addition to the application of really small cantilever amplitudes of often 0.1–0.2 nm to minimize tip-sample interaction. This makes PM-AFM the preferred dynamic AFM mode for high-resolution imaging
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Acquiring an entire force-distance curve at every pixel enables several operations for extraction of quanti- tative data. The acquired data can be used to calculate the nano-mechanical properties using either standard or customized-fit algorithms. Typical parameters that can be extracted include the work of adhesion, the actual adhesion, and contact point determination. Specific adhesion events can be detected and Young’s modulus can be calculated using an appropriate contact mechanics model. The contact point height determination enables the recreation of the so-called zero-force image (having no indentation) or simply the topography and indentation information from the sample at different forces within the range of the applied set point ( Figures 10 a and 10 b). The high sensitivity and versatility of this method becomes even more obvious when applied to single molecules. This is illustrated with the resolution of the subtrimeric structure of the purple membrane protein bacteriorhodopsin ( Figures 10c–e ). A single measurement set shows the differences in sample topography at three different forces and allows the user to adapt the measurement settings in real time. This also enables the 3D tomographic reconstruction of the sample at a later stage and the correlation of the information coming from different channels.
Conclusions
Biological AFM has come a long way since the invention of the scanning probe technique in 1986. The
www.microscopy-today.com • 2015 November
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