Separating DNA with different topologies by atomic force microscopy in comparison with gel electrophoresis.

Atomic force microscopy, which is normally used for DNA imaging to gain qualitative results, can also be used for quantitative DNA research, at a single-molecular level. Here, we evaluate the performance of AFM imaging specifically for quantifying supercoiled and relaxed plasmid DNA fractions within a mixture, and compare the results with the bulk material analysis method, gel electrophoresis. The advantages and shortcomings of both methods are discussed in detail. Gel electrophoresis is a quick and well-established quantification method. However, it requires a large amount of DNA, and needs to be carefully calibrated for even slightly different experimental conditions for accurate quantification. AFM imaging is accurate, in that single DNA molecules in different conformations can be seen and counted. When used carefully with necessary correction, both methods provide consistent results. Thus, AFM imaging can be used for DNA quantification, as an alternative to gel electrophoresis.

Theoretical Models for Surface Forces and Adhesion and Their Measurement Using Atomic Force Microscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Colloidal drug probe : method development and validation for adhesion force measurement using atomic force microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between the drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from a dry powder inhaler (DPI) formulation. Initially model silica probes of approximately 4 μm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-Ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres preattached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Determination of strain-rate-dependent mechanical behavior of living and fixed osteocytes and chondrocytes using atomic force microscopy and inverse finite element analysis

The aim of this paper is to determine the strain-rate-dependent mechanical behavior of living and fixed osteocytes and chondrocytes, in vitro. Firstly, Atomic Force Microscopy (AFM) was used to obtain the force-indentation curves of these single cells at four different strain-rates. These results were then employed in inverse finite element analysis (FEA) using Modified Standard neo-Hookean Solid (MSnHS) idealization of these cells to determine their mechanical properties. In addition, a FEA model with a newly developed spring element was employed to accurately simulate AFM evaluation in this study. We report that both cytoskeleton (CSK) and intracellular fluid govern the strain-rate-dependent mechanical property of living cells whereas intracellular fluid plays a predominant role on fixed cells’ behavior. In addition, through the comparisons, it can be concluded that osteocytes are stiffer than chondrocytes at all strain-rates tested indicating that the cells could be the biomarker of their tissue origin. Finally, we report that MSnHS is able to capture the strain-rate-dependent mechanical behavior of osteocyte and chondrocyte for both living and fixed cells. Therefore, we concluded that the MSnHS is a good model for exploration of mechanical deformation responses of single osteocytes and chondrocytes. This study could open a new avenue for analysis of mechanical behavior of osteocytes and chondrocytes as well as other similar types of cells.

Colloidal drug probe : Method development and validation for adhesion force measurement using Atomic Force Microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between a model drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from dry powder inhaler (DPI) formulations. Model silica probes of approximately 4 lm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres pre-attached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/ hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Combined Atomic Force Microscopy and Modeling Study of The Evolution of Octadecylamine Films on a Mica Surface

The time evolution of the film thickness and domain formation of octadecylamine molecules adsorbed oil a mica surface is investigated Using atomic force microscopy. The adsorbed Film thickness is determined by measuring the height profile across the mica-amine interface of a mica surface partially immersed in a 15 mM solution of octadecylamine in chloroform. Using this novel procedure, adsorption of amine on mica is found to occur in three distinct stages, with morphologically distinct domain Formation and growth occurring during each stage. In the first stage, where adsorption is primarily in the thin-film regime, all average Film thickness of 0.2 (+/- 0.3) nm is formed for exposure times below 30 s and 0.8 (+/- 0.2) nm for 60 s of immersion time. During this stage, large sample spanning domains are observed. The second stage, which occurs between 60-300 s, is associated with it regime of rapid film growth, and the film thickness increases from about 0.8 to 25 nm during this stage. Once the thick-film regime is established, further exposure to the amine solution results in all increase in the domain area, and it regime of lateral domain growth is observed. In this stage, the domain area coverage grows from 38 to 75%, and the FTIR spectra reveal an increased level of crystallinity in the film. Using it diffusion-controlled model and it two-step Langmuir isotherm, the time evolution of the film growth is quantitatively captured. The model predicts the time at which the thin to thick film transition occurs as well its the time required for complete film growth at longer times. The Ward-Tordai equation is also solved to determine the model parameters in the monolayer (thin-film) regime, which occurs during the initial stages of film growth.

Enhanced endocytosis of nano-curcumin in nasopharyngeal cancer cells: An atomic force microscopy study

Recent studies in drug development have shown that curcumin can be a good competent due to its improved anticancer, antioxidant, anti-proliferative, and anti-inflammatory activities. A detailed real time characterization of drug (curcumin)-cell interaction is carried out in human nasopharyngeal cancer cells using atomic force microscopy. Nanocurcumin shows an enhanced uptake over micron sized drugs attributed to the receptor mediated route. Cell membrane stiffness plays a critical role in the drug endocytosis in nasopharyngeal cancer cells. (C) 2011 American Institute of Physics. [doi:10.1063/1.3653388]

Note: Design and development of an integrated three-dimensional scanner for atomic force microscopy

A compact scanning head for the Atomic Force Microscope (AFM) greatly enhances the portability of AFM and facilitates easy integration with other tools. This paper reports the design and development of a three-dimensional (3D) scanner integrated into an AFM micro-probe. The scanner is realized by means of a novel design for the AFM probe along with a magnetic actuation system. The integrated scanner, the actuation system, and their associated mechanical mounts are fabricated and evaluated. The experimentally calibrated actuation ranges are shown to be over 1 mu m along all the three axes. (c) 2013 AIP Publishing LLC.

Design and Evaluation of Torsional Probes for Multifrequency Atomic Force Microscopy

Multifrequency atomic force microscopy is a powerful nanoscale imaging and characterization technique that involves excitation of the atomic force microscope (AFM) probe and measurement of its response at multiple frequencies. This paper reports the design, fabrication, and evaluation of AFM probes with a specified set of torsional eigen-frequencies that facilitate enhancement of sensitivity in multifrequency AFM. A general approach is proposed to design the probes, which includes the design of their generic geometry, adoption of a simple lumped-parameter model, guidelines for determination of the initial dimensions, and an iterative scheme to obtain a probe with the specified eigen-frequencies. The proposed approach is employed to design a harmonic probe wherein the second and the third eigen-frequencies are the corresponding harmonics of the first eigen-frequency. The probe is subsequently fabricated and evaluated. The experimentally evaluated eigen-frequencies and associated mode shapes are shown to closely match the theoretical results. Finally, a simulation study is performed to demonstrate significant improvements in sensitivity to the second-and the third-harmonic spectral components of the tip-sample interaction force with the harmonic probe compared to that of a conventional probe.

Direct Measurement of Three-Dimensional Forces in Atomic Force Microscopy

Direct measurement of three-dimensional (3-D) forces between an atomic force microscope (AFM) probe and the sample benefits diverse applications of AFM, including force spectroscopy, nanometrology, and manipulation. This paper presents the design and evaluation of a measurement system, wherein the deflection of the AFM probe is obtained at two points to enable direct measurement of all the three components of 3-D tip-sample forces in real time. The optimal locations for measurement of deflection on the probe are derived for a conventional AFM probe. Further, a new optimal geometry is proposed for the probe that enables measurement of 3-D forces with identical sensitivity and nearly identical resolution along all three axes. Subsequently, the designed measurement system and the optimized AFM probe are both fabricated and evaluated. The evaluation demonstrates accurate measurement of tip-sample forces with minimal cross-sensitivities. Finally, the real-time measurement system is employed as part of a feedback control system to regulate the normal component of the interaction force, and to perform force-controlled scribing of a groove on the surface of polymethyl methacrylate.

Quantitative characterization of adhesion and stiffness of corneal lens of Drosophila melanogaster using atomic force microscopy

Atomic force Microscopy (AFM) has become a versatile tool in biology due to its advantage of high-resolution imaging of biological samples close to their native condition. Apart from imaging, AFM can also measure the local mechanical properties of the surfaces. In this study, we explore the possibility of using AFM to quantify the rough eye phenotype of Drosophila melanogaster through mechanical properties. We have measured adhesion force, stiffness and elastic modulus of the corneal lens using AFM. Various parameters affecting these measurements like cantilever stiffness and tip geometry are systematically studied and the measurement procedures are standardized. Results show that the mean adhesion force of the ommatidial surface varies from 36 nN to 16 nN based on the location. The mean stiffness is 483 +/- 5 N/m, and the elastic modulus is 3.4 +/- 0.05 GPa (95% confidence level) at the center of ommatidia. These properties are found to be different in corneal lens of eye expressing human mutant tau gene (mutant). The adhesion force, stiffness and elastic modulus are decreased in the mutant. We conclude that the measurement of surface and mechanical properties of D. melanogaster using AFM can be used for quantitative evaluation of `rough eye' surface. (C) 2015 Elsevier Ltd. All rights reserved.