| In this paper, the mechanism and construction of VSPFM are introduced as well as its application on the measurement of biomolecules such as DNAs and antibodies.VSPFM is a deviation from SPFM. In VSPFM, a conductive AFM tip is biased to a voltage and an electric field arises between the tip and the surface as a result of the polarization of the surface. Compared with the van der Waals attractive force, the electrostatic force (also termed polarization force) is stronger and has a longer range. Tip-sample separation is then increased as a result of the combination of polarization force with van der Waals force. The difference between SPFM and VSPFM is that the biased tip of VSPFM is driven by a mechanical vibration.By changing variables such as the bias and Asp, the tip can be located from non-contact to tapping region with the assistance of polarization force. In VSPFM non-contact mode, the difficulty of short range of imaging force in AFM is overcome because of the combination of polarization force with van der Waals force. In VSPFM tapping mode, the tip-sample interaction is even smaller than the minimum force in AFM tapping mode stable imaging with the assistance of polarization force.When tip-sample separation is below several nanometers in VSPFM, van der Waals force constitutes the dominant part of tip-sample interactions especially in the transitional region between non-contact and tapping mode, and the polarization force only assists in stabilizing the imaging. However, in normal Electrostatic Force Microscopy (EFM), electrostatic force is the main force in imaging.In the application of AFM on bimolecules, the tip pressure still exists in tapping mode and results in great deformation of these soft bimolecules, though the lateral force in tapping mode is much less than that in contact mode. Tip pressure is essentially a result of the repulsive force between the tip and the substrate. Theapparent heights of these molecules in AFM tapping mode are questionable since they are always smaller than their actual diameters. In this paper, we provide a new method for height measurement of soft samples on substrates by VSPFM, in which tip pressure can be decreased to a certain extent and the measurement will be more precise.There are two ways in height measurement by VSPFM:One is to change tip-sample separation by Asp. Firstly, the relationship between AAsp and DZ0 (vertical displacement of the tip in zero-scale scan) can be calculated and calibrated in zero-scale scan (scan size is 0 nm). Then the tip is positioned on a molecule and approaches to the surface with the decrease of Asp. From the top of the molecule to the substrate, the vertical displacement of the tip can be calculated from AAsp and is close to the true height of this molecule.The other is to change tip-sample separation by altering the parameter of lift scan height in lift mode. The height of the tip is maintained in main scan, and in interleave scan, the tip is lowered from the top of the molecule to the substrate with the decrease of lift scan height. The difference in the parameter of lift scan height approximates the true height of this molecule.The height measurement carried out by VSPFM has some advantages. The vertical movement of the tip is more controllable with the assistance of long-range polarization force, and stable images can be obtained at different tip-sample seperation. With the lift of the tip, repulsive force (tip pressure) decreases rapidly because its effective range is only several angstroms and the deformation is reduced consistently. The new method and apparatus of the measurement derived from this paper is simple and accessible compared with the existing techniques.The measured heights of colloidal gold particles by VSPFM are almost the same asthose in TM-AFM, but the results of dsDNA and antibodies behave differently even under the same conditions such as tips and samples. A possible reason is that the tip pressure in AFM tapping mode results in large deformation on soft DNA and antibodies b...
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