Noninvasive Protein Structural Flexibility Mapping by Bimodal Dynamic Force Microscopy

Abstract

Mapping of the protein structural flexibility with sub-2-nm spatial resolution in liquid is achieved by combining bimodal excitation and frequency modulation force microscopy. The excitation of two cantilever eigenmodes in dynamic force microscopy enables the separation between topography and flexibility mapping. We have measured variations of the elastic modulus in a single antibody pentamer from 8 to 18 MPa when the probe is moved from the end of the protein arm to the central protrusion. Bimodal dynamic force microscopy enables us to perform the measurements under very small repulsive loads (30-40 pN). DOI: 10.1103/PhysRevLett.106.198101 PACS numbers: 87.15.Àv, 62.25.Àg, 62.30.+d, 68.37.Ps Ultrahigh resolution, sensitive, and minimally invasive characterization techniques are needed to understand hybrid surfaces integrated by organic, inorganic, and biological structures in air or liquid. Atomic force microscopy (AFM) [1] has enabled molecular resolution images of packed arrays of biomolecules [2-5], sub-2-nm images of individual biomolecules [6], and studying biomolecular interactions in liquid [7]. Single force spectroscopy measurements are already an established tool to measure intermolecular and intrabiomolecule forces [8]; however, those measurements are not compatible with high resolution imaging.Recently, several multifrequency AFM schemes have been proposed to improve high resolution imaging, contrast, and quantitative mapping of material properties [9][10][11][12][13][14][15][16][17][18][19][20][21]. Generically, those schemes exploit the nonlinear character of the tip-surface forces to either activate or detect higher eigenmodes or harmonics and to open new channels to improve imaging and composition sensitivity. Sahin and coworkers [22] have been able to image the protein flexibility of packed two-dimensional bacteriorhodopsin layers by implementing a force time inversion method that exploits the presence of higher harmonics in torsional harmonic cantilevers.In this study, we propose a different dynamic force microscopy approach to image and measure the protein flexibility with molecular resolution. The method combines bimodal excitation with frequency modulation AFM (FM AFM) which allows separating the topography from the protein flexibility mapping. The combined experimental and theoretical findings show that high resolution imaging and protein flexibility mapping can be achieved under the application of extremely low forces ($ 40 pN). Thus, high resolution imaging of isolated proteins in liquid is achieved. The agreement obtained between the nominal height of the protein subunits as determined from diffraction techniques and those reported here implies that the proteins are imaged in liquid in a noninvasive manner.Bimodal dynamic force microscopy involves the mechanical excitation of two cantilever eigenmodes [11,23]; then the cantilever deflection as measured in the photodiode can be expressed as( 1) where z 0 is the mean deflection and Oð"Þ includes other high order terms which are usually...

Single cycle and transient force measurements in dynamic atomic force microscopy

“…The instrument is currently employed by researchers in elds ranging from biology 8,9 to semiconductor theory and devices 10 and in hybrid systems. 11 With the AFM one can routinely image single nanostructures, 12,13 map heterogeneous compositional [14][15][16][17][18] and/or nanomechanical [19][20][21] properties and/ or processes, 22,23 study molecular interactions 24,25 and larger biological systems, 26,27 identify single atoms, 28 molecules 29,30 and/or chemical composition 31 and structures, 32 study the friction induced by single atomic motion 33 and, more recently, even discriminate bond order and symmetry. 34,35 The fundamental principle however is relatively straightforward; atoms, nanostructures and surfaces are probed with high precision on the lateral and vertical axes via a nanoscopic tip mounted on a microstructure, typically a micro-cantilever, by monitoring its deection.…”

Multi-modal analysis on the intermittent contact dynamics of atomic force microscope

“…For example, an AFM vibrates with a driving frequency lower than or around the first natural frequency of the AFM cantilever and has no contact with the sample [14]. However, the driving frequency can be much higher than the first natural frequency of AFM cantilever [6,15] and the higher modes can thus participate in the motion. In the AFM imaging application, the phase shift of higher mode has been used to characterize the mechanical, electrical and magnetic interactions [15].…”

“…However, the driving frequency can be much higher than the first natural frequency of AFM cantilever [6,15] and the higher modes can thus participate in the motion. In the AFM imaging application, the phase shift of higher mode has been used to characterize the mechanical, electrical and magnetic interactions [15]. We demonstrate that the results computed by the single mode analysis can be very different from those computed by multimodes when the driving frequency is higher than the AFM cantilever first natural frequency.…”

Phase contrast and operation regimes in multifrequency atomic force microscopy

“…In the figure, the free amplitude of the second mode (sixth harmonic) A 06 has been varied in the range 0.1-5 nm since this is characteristic of standard values in bimodal AFM. 1,14,15 Fig. 2(a) Fig.…”

“…The single frequency virial that controls the phase shift might undergo transitions in sign while the average force (modal virial) remains positive (negative). Multifrequency atomic force microscopy (AFM) is an emerging 1,2 branch of AFM where two or more frequencies 3,4 are externally excited in order to map material composition, [5][6][7] enhance resolution 8 and sensitivity, [9][10][11] and quantify material properties [12][13][14] with gentle forces. The theory that controls the response of the cantilever while simultaneously exciting several frequencies and modes however is still emerging 12,15,16 and might result complex.…”