Simulation of Atomic Force Microscopy (AFM) with applications to image analysis and control design

Background and Aims The classification of renal diseases is dependent on the evaluation of kidney biopsies using light microscopy techniques. Ultrastructural details (e.g. podocyte foot processes, amyloid fibrils, Fabri's intracellular inclusions) need transmission electron microscopy (TEM) which has high costs, requires specialized personnel and is time consuming. Therefore, alternative techniques are highly desirable. Here we report our experience with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Method Paraffin-embedded kidney sections (3μm thick) were examined after dewaxing and Jones-methenamine methane staining for type IV collagen fibers. Dried urine sediments were prepared using standard methods for urine cytological analysis. Analysis was then performed using atomic force microscopy (nGauge system) on dried specimens and with Scanning Electron Microscopy (Zeiss) after gold-spattering of the surface. Reference images were obtained by standard microscopy. Results The combination of SEM and thin paraffin sections allows the observation of ultrastructural details in kidney biopsies with a very simple and fast preparation. AFM is even faster than SEM, as it does not require any preparation of the surface of the sample. An important advantage of SEM is the possibility to view the sample at low magnification, which makes it easier to identify the location of the glomeruli. It is thus possible to switch from small magnifications (100x) to very extreme magnifications (10000x). Notably, the interpretation of the images requires knowledge of the artifacts induced by paraffin inclusion. Specifically, structures and cells containing lipids (for example foamy cells) appear empty. Chromatin is also condensed in the nuclei around the periphery of the nucleus and in spike structures whit large empty areas. The recognition of nuclei in SEM and AFM is important for the subsequent interpretation of the images. Podocyte pedicles fusion can be identified relatively easily in SEM and in AFM. Both techniques add relevant ultrastructural details of urine sediment cells, from epithelial cells to isolated tubular cells. Also, casts show many ultrastructural features which are not evident in classical optical microscopy. It is also possible to measure the thickness of the basement membrane with great precision both in SEM and with AFM. The major limitation of this approach is the limited experience of pathologists with these methods and the absence of an atlas of normal and pathological kidney. Conclusion Considering the affordable costs of SEM and AFM instruments and the simple preparation of samples for their analysis, these should be considered a valid alternative to TEM. SEM and AFM are alternative methods, simple and relatively cheap and very fast to obtain ultrastructural details of renal biopsies. We thank ANED and AST for the great support given to research.

Biological atomic force microscopy (AFM) is a fast growing and advancing field. This review's objective is to overview the state of the art and to retrace achievements of biological AFM as presented by past and present research, and wishes to give a (subjective) outlook where AFM may go in the upcoming years. The following areas of interest are discussed: High-resolution imaging, cell imaging, single molecule force spectroscopy, cell mechanical measurements, combined AFM instrumentation, and AFM instrumentation. Of all these topics, particular representative examples are shown, each of them standing for a variety of achievements by many research groups.

Recent development of atomic force microscopy (AFM) applications in imaging living cells is reviewed, focusing on technical progress and application advancements made in the following major areas: (i) high-resolution imaging of cellular structures, (ii) real-time monitoring of cellular dynamic processes, and (iii) detecting micromechanical properties of the cell. Technical and experimental difficulties frequently encountered in AFM applications in above areas are presented and possible strategies for overcoming these obstacles are discussed. Significant advances in the AFM study of living cells can be achieved from further development of AFM technology, sample preparation, and innovative applications.

<p>The composition, topography, adhesiveness and nanomechanical properties of biomaterials are all factors that affect biological processes, e.g. biofilm formation, cell differentiation, and morphogenesis [1-4]. Atomic Force Microscopy (AFM) is a highly versatile tool, ideal for the characterization of samples properties ranging from single molecules to complex biological systems, on the nm scale.</p> <p>JPK BioAFMs, like the NanoWizard<sup>®</sup> ULTRA Speed 2, enable fast imaging of challenging biological samples and the visualization of dynamic processes with high spatio-temporal resolution under near physiological conditions, e.g. the kinetics of collagen type I fibrillogenesis was imaged in situ revealing the formation of the 67 nm D-banding hallmark.</p> <p>Our distinctive Quantitative Imaging mode (QI™) measures various sample properties such as topography, nanomechanics and adhesion on the nanometer scale. Complex data like contact point, Young´s modulus or recognition images can also be extracted at the same resolution. To demonstrate the capability and flexibility of the QI™ mode, various biological samples like living cells have been investigated and their topographical and mechanical properties determined. QI™, based on fast force mapping, can also be used to determine the mechanical properties of different bacterial strains. We will discuss the application of HighSpeed AFM for the characterisation of dynamic biofilms with high spatiotemporal resolution, information which can then be directly correlated with advanced optical microscopy for immuno-characterisation of the sample.</p> <p>Investigating large, sticky and rough samples such as tissues and hydrogels using AFM has always been a challenge due to the limited z-axis of the AFM. The HybridStage™, equipped with an extended xyz scanner unit up to 300x300x300 µm<sup>3</sup>, an additional motorized unit for large sample movements in the mm range and optical tiling, is ideal for investigating such samples. This combination enables multi-region AFM probing over a large, uneven sample area and provides additional correlative optical data sets.</p> <p>Adhesion dynamics between cells and biomaterials play a crucial role in, e.g. the applicability of potential implant materials. The AFM based Single Cell Force Spectroscopy platform enables quantitative measurement of the interactions between individual cells and any substrate.</p> <p>A number of important research topics in the field of biomedicine, relate directly to the increased antimicrobial resistance of various biofilms to commonly prescribed drugs, and have identified adhesion, as a leading factor in biofilm formation, colony progression, and pathogenesis of microbial agents. JPK BioAFM has developed novel techniques for studying single-molecule forces and adhesion profiles at the cell/cell or cell/substrate interface. We will provide an overview of how to functionalize various surface substrates for the attachment of bacteria.</p> <p>We will also provide information on working with AFM under Biosafety Level L2/L3 conditions.</p> <p> </p> <p>[1]      Elter, P. et al., Eur Biophys J, <strong>2011</strong>, 40(3):317-27</p> <p>[2]      Engler AJ. Et al., Cell;<strong> 2006</strong>, 126(4):677-89</p> <p>[3]      Cisneros, DA. et al., Small,<strong> 2007</strong>, 3(6):956-63</p> <p>[4]         Koser DA. Et al., Nat. Neurosci.; <strong>2016</strong>, 19:1592-1598</p>

This chapter presents an overview of Atomic Force Microscopy (AFM) principles followed by details on AFM instrumentation. In particular, the frequency modulation method of the non-contact AFM (NC-AFM) used in ultra-high vacuum conditions is explained in detail. Then, applications of NC-AFM for an atomic-scale range characterization of semiconductor and isolator surfaces as well as supported nanostructures are introduced.

Prototype cantilevers are presented that enable quantitative surface force measurements using contact-mode atomic force microscopy (AFM). The "hammerhead" cantilevers facilitate precise optical lever system calibrations for cantilever flexure and torsion, enabling quantifiable adhesion measurements and friction measurements by lateral force microscopy (LFM). Critically, a single hammerhead cantilever of known flexural stiffness and probe length dimension can be used to perform both a system calibration as well as surface force measurements in situ, which greatly increases force measurement precision and accuracy. During LFM calibration mode, a hammerhead cantilever allows an optical lever "torque sensitivity" to be generated for the quantification of LFM friction forces. Precise calibrations were performed on two different AFM instruments, in which torque sensitivity values were specified with sub-percent relative uncertainty. To examine the potential for accurate lateral force measurements using the prototype cantilevers, finite element analysis predicted measurement errors of a few percent or less, which could be reduced via refinement of calibration methodology or cantilever design. The cantilevers are compatible with commercial AFM instrumentation and can be used for other AFM techniques such as contact imaging and dynamic mode measurements.

The measurement of small forces by atomic force microscopy (AFM) is of increasing importance in many applications. For example, in analytical applications where individual molecules are probed, or nanoindentation measurements as a source of information about materials properties on a nanometer scale. The fundamentals of AFM force measurement, and some of these applications, are briefly reviewed. In most cases absolute, not relative, measurements of forces are needed for valid comparisons with theory and other measurement techniques (such as optical tweezers). We review methods of AFM force calibration and the major uncertainties involved. The force range considered in this work is roughly from 10 pN to around 500 nN. We describe some issues of the repeatability of force measurements that can be important in common AFM instruments. In most cases the aspect that then limits the accuracy with which forces can be measured is the uncertainty in the stiffness (more specifically the normal force constant) of the atomic force microscope cantilever at the center of the instrument. It is known that commercially available microfabricated atomic force microscope cantilevers have a wide range of force constant, for cantilevers of nominally the same type and even the same production batch. Calibration is necessary, and many methods have been used over the years. We compare the accuracy that can be achieved and the ease of use of these different methods, including theoretical (dimensional), thermal, static and dynamic methods and their variants. A device developed at NPL should help overcome many of the problems of force constant calibration, at least for the most common AFM configurations. This is a microfabricated silicon device, which, because of its very small mass, is not susceptible to vibration as a larger device would be. A new calibration method based on electrical and Doppler measurements allows the calibration of the force constant of this device traceable to the SI newton. It can then be sent to AFM users for straightforward calibration of AFM force constants. We conclude with a brief discussion of the special problems of calibration of lateral forces, such as those obtained in frictional force measurements.

In this paper, we present a new architecture for analog amplitude demodulation in high-speed atomic force microscopy (AFM) applications. It features single-cycle capabilities and the possibility to work with elevated cantilever resonance frequencies up to 9 MHz. This latter feature is particularly important in view of the upcoming next generation of high resonance frequency cantilevers. As a proof-of-principle, we show a prototype of the proposed single-sideband upconversion architecture to be able to work with a conventional MultiMode 8 AFM system (Bruker) and a FastScan-A cantilever in air with f res≈ 1.6 MHz. Overall, the presented lock-in amplifier with its improved demodulation speed will foster the research activities towards faster imaging speeds in AFM applications and thereby allow to further establish it as a tool for real-time studies of the dynamics of molecular and even atomic processes.

The necessity for development of new drug delivery systems for poorly soluble drugs and therapeutics which targets directly the pathogenic area requires the design of complex systems in nanoscale. Atomic Force Microscopy (AFM) since its discovery offers a unique tool to develop novel drug delivery systems. Its capability to investigate, characterise surfaces and measure forces with spatial resolution at nano-scale respectively contributes to develop and analyse pharmaceutical systems and biomedical devices with complex structures and chemistries. Specifically, polymeric nanoparticles and liposomal drug formulation have been studied extensively by using AFM where their size and morphology where revealed [1]. However, apart from topographical information, AFM can provide details on the local compositions of the sample. There are many modes that can be used to achieve this but the most common is to monitor the phase shift of the oscillating cantilever in tapping mode Such ‘Phase imaging’ can be used to detect nanoscale variation in composition, adhesion, friction, viscoelasticity, and other properties of the materials. Changes in the phase lag often indicate changes in the properties of the sample surface. Phase imaging has proved an extremely useful tool for pharmaceutical characterization. Phase imaging has been used to reveal polymeric forms from single crystal measurements [2] to confirm phase separation of two copolymers for drug delivery [3], to establish the stability of the formulation on different environments [4] and to identify formation of amorphous domains during milling of crystalline salbutamol [5]. Moreover, the ability of AFM to operate on different conditions of temperature humidity and in liquid allows studying the kinetics of range of phenomena. An interesting work was published from Miyazaki et al. where the crystal growth rate of Nifedipine, dispersed in PEG polymer matrix, was quantified [6]. Also, the dissolution process of aspirin on different facets of the crystal was studied by Danesh et al . showing their differences in dissolution rate [7]. The ability of AFM to measure force measurements have been used to investigate interactions among the different compounds in the formulation. To achieve this, the AFM cantilever is modified by attaching a particle directly onto the probe tip and is brought in contact with the substrate of interest, the force required to separate them is the measure of their adhesion. This application of AFM has been used extensively to aerosol drug formulation where the API is associated with a carrier which is released in the site of actions. For instance, AFM has been used to investigate the adhesion force of salbutamol with different excipients [8]. Moreover, localised surface energy and young’s modulus of various compounds have been measured. For example, Young’s modulus were derived from both PLA and PLA/everolimus directly from stent surface on different temperatures. The results showed that everolimus does not affect the mechanical properties of PLA up to 1/1 (w/w) drug loading of the mixture. Also, a significant drop of the Young’s modulus in solution was observed at 36°C, suggests that in vivo the Tg of the polymer is below body temperature [9]. Furthermore, force measurements have been used to identify the encapsulation the efficiency of liposomes as the rigidity of encapsulating liposomes is higher [10].

Rituximab, a monoclonal antibody against the CD20 molecule expressed on B cells, has revolutionized the treatment of B cell lymphomas. The use of rituximab significantly improves the long-term survival rates of lymphoma patients. However, there are still many patients who develop resistance to rituximab. Hence, the urgent need is enhancing the potency of anti-CD20 antibodies beyond that achieved with rituximab to provide effective therapies for more patients. Traditional studies about the killing mechanisms of rituximab are commonly based on optical microscopy, which cannot reveal the detailed situations due to the limit of 200-nm resolution. Quantitatively investigating the actions of rituximab at the nanoscale is still scarce. The advent of atomic force microscopy (AFM) provides a powerful and versatile platform for in situ investigating the nanoscale behaviors of individual live cells. The applications of AFM in life sciences in the past decade have yielded a lot of novel insights into cell biology and related diseases. In this article, the typical applications (e.g., ultra-microstructures, mechanical properties, and molecular recognition) of AFM in investigating the three killing mechanisms of rituximab at the nanoscale are summarized; the challenges facing AFM single-cell assay and its future directions are also discussed.

The atomic force microscope (AFM) is a probe-based microscope that uses nanoscale and structural imaging where high resolution is desired. AFM has also been used in mechanical, electrical, and thermal engineering applications. This unique technique provides vital local material properties like the modulus of elasticity, hardness, surface potential, Hamaker constant, and the surface charge density from force versus displacement curve. Therefore, AFM was used to measure both the diameter and mechanical properties of the collagen nanostraws in human costal cartilage. Human costal cartilage forms a bridge between the sternum and bony ribs. The chest wall of some humans is deformed due to defective costal cartilage. However, costal cartilage is less studied compared to load bearing cartilage. Results show that there is a difference between chemical fixation and non-chemical fixation treatments. Our findings imply that the patients' chest wall is mechanically weak and protein deposition is abnormal. This may impact the nanostraws' ability to facilitate fluid flow between the ribs and the sternum. At present, AFM is the only tool for imaging cells' ultra-structure at the nanometer scale because cells are not homogeneous. The first layer of the cell is called the cell membrane, and the layer under it is made of the cytoskeleton. Cancerous cells are different from normal cells in term of cell growth, mechanical properties, and ultra-structure. Here, force is measured with very high sensitivity and this is accomplished with highly sensitive probes such as a nano-probe. We performed experiments to determine ultra-structural differences that emerge when such cancerous cells are subject to treatments such as with drugs and electric pulses. Jurkat cells are cancerous cells. These cells were pulsed at different conditions. Pulsed and non-pulsed Jurkat cell ultra-structures were investigated at the nano meter scale using AFM. Jurkat cell mechanical properties were measured under different conditions. In addition, AFM was used to measure the charge density of cell surface in physiological conditions. We found that the treatments changed the cancer cells' ultra-structural and mechanical properties at the nanometer scale. Finally, we used AFM to characterize many non-biological materials with relevance to biomedical science. Various metals, polymers, and semi-conducting materials were characterized in air and multiple liquid media through AFM - techniques from which a plethora of industries can benefit. This applies especially to the fledging solar industry which has found much promise in nanoscopic insights. Independent of the material being examined, a reliable method to measure the surface force between a nano probe and a sample surface in a variety of ionic concentrations was also found in the process of procuring these measurements. The key findings were that the charge density increases with the increase of the medium's ionic concentration.

Studying local mechanical properties of metallic alloys and composites and their defect structures such as grain boundaries, cracks, and dislocations allows a better understanding and design of these structural materials. With a nanoindenting atomic force microscope (NI-AFM), which uses diamond tips, such measurements are possible on a nanometer scale. Measurements on the microstructure of superalloys are presented, where different precipitates with sizes in the range of 100 nm are characterized by their different hardness values. In addition, this new technique was used to characterize the nanohardness of the plastic zone and stress fields of cracks and grain boundaries. Therefore, crack tips of in-situ loaded bending specimens of intermetallic alloys (NiAl) were investigated. The measurements show a change in nanohardness and elasticity from the distance of interfaces. In material science the high-resolution capabilities of the atomic force microscopy (AFM) are used for studying metallographic sections, where different phases produce topographic contrasts in the image. A extremly small height difference in the microstructure is sufficient for a height contrast in the image, which allows a quantitative determination of particle size distributions and volume contents of precipitates [1, 2]. The plastic deformation behavior of metals is strongly influenced by the distribution and movement of defects as dislocations, grain boundaries and cracks. In-situ loading experiments in an AFM on specimens loaded in 4-point bending demonstrate the good imaging properties of an AFM for such defects [3]. Mechanical properties such as Young’s modulus of elasticity and hardness values were obtainable in the past by various indentation techniques, where a load displacement curve is recorded during the indentation process. These techniques were mainly applied for studying the mechanical properties of thin films. A combination of a nanoindenting instrument with an AFM now allows the local mechanical properties of different phases and particles in metallic alloys to be measured, since the indents can be positioned very exactly with the AFM. Such a combined nanoindenting AFM (NI-AFM) requires a special force measurement technique. Piezo hysteresis and creep of the used piezo scanner have to be eliminated. In addition, indenting should be performed by a tip which is centrally fixed and not at the end of a bending beam (cantilever) [4], because otherwise the location of the indent changes during loading. These properties are fulfilled by a capacitive transducer, which is used for load generation and measurement instead of the standard laser deflection unit. A diamond tip is used for the indentation experiments and topographic imaging in a conventional AFM mode. The resulting force indentation curves contain two pieces of information: the universal hardness H and the reduced modulus of elasticity Er. The possibilities of the NI-AFM for studying local mechanical properties are demonstrated on two different examples: first the hardness of the superalloy CMSX-6 was characterized; second, local changes of mechanical properties near a crack tip in a NiAl single crystal were examined. 1 Experimental setup and sample preparation The nanoindentation measurements shown in this paper were performed with an add-on-force transducer from Hysitron Inc. This transducer, mounted on a conventional AFM, controls the z movement of the tip (indentation, applied load, and feedback signal) while the piezos of the AFM control the lateral x-y movement. Further technical details on the transducer are described in [5]. Specimens of the superalloy CMSX-6 were examined with a Nanoscope II AFM from Digital Instruments. Since the x-y movement is achieved by scanning the whole specimen, only small specimens with a maximum diameter of 20 mm can be analyzed. Crack tips in NiAl single crystals were investigated with a bending apparatus specially constructed for the Explorer AFM from TopoMetrix. (This bending apparatus is described in [6].) Because the whole transducer assembly is mounted on the x-y scanner, the combined system is more sensitive to mechanical noise. However, large specimens can be examined with

Identification of Transcription Factor AP-1 Bound to Untranslated 5′ Regulatory Region DNA of mdr1 Gene with Atomic Force Microscopy.

Scanning electron microscopy (SEM) and ion beam milling (FIB) techniques are mature nanoscale measurement technologies, while Atomic Force Microscopy (AFM) is a developing technology with intense interest in the scientific community for basic research and development. We discuss the recent integration of these techniques into a single instrument enabled by technological innovation in AFM instrument design, and applications of this instrument for characterization of FIB milled trenches. An AFM integrated into the SEM/FIB has specific design constraints that need to be met involving the AFM scanner, probe design, and feedback loop so that it geometrically fits into the chamber without interfering with the electron and ion beams. An ultraflat scanning stage was thus developed for precise sample motion that enable large ranges (85 microns or greater) in x,y, z. SPM probes that do not interfere with the electron beam were manufactured from fused silica in a cantilevered geometry (see Fig 1) where there is full visualization of the tip, without any loss of SPM functionality. These probes are combined with a tuning fork based feedback mechanism to maintain accurate tip-sample control; this mechanism does not involve lasers or optical beam deflection that interferes with the electron and ion beams. The many benefits of this combined approach to three-dimensional nanoscale characterization are illustrated in the imaging of a FIB milled trench. Figure 2a shows an AFM image profiling a 25-µm-deep trench milled in silicon with FIB imaged with the large Z range of the system. In this measurement, the FIB beam was used to mill this feature, and it was followed immediately by AFM imaging without having to remove the specimen from the FIB chamber and search in the AFM. Furthermore, the true depth of the trench was measured by the AFM in a crosssectional profile (Figure 2b) while a 3D reconstruction revealed the geometry of the sidewall (Figure 2c). Sidewall imaging with such long and exposed tips now are feasible with this instrument. The red arrow in this image clearly shows a Pt decoration on this structure, and the white arrow shows an undercut. Additionally, the ability of AFM to quickly and easily follow FIB milling with high Z resolution provides a straightforward and convenient method to check on FIB milling results. This capability is demonstrated in Figure 3 where several FIB-milled features in Si were monitored during the milling process. The grayscale SEM image in Figure 3a can easily locate these features and measure their widths but cannot provide direct information on their depths. The AFM delivers a 3D reconstruction (Figure 3b), and a cross sectional profile that together provide a true topographic map of specimen features. These depth profiles demonstrate the linearity of the etching process with time. This instrument incorporates important innovations in the design and technology of atomic force microscopy enabling its integration with SEM and FIB into a single powerful capability.

Cartilage is a complex avascular tissue composed of cells ("chondrocytes") embedded in an extracellular matrix (ECM) consisting of 70%-80% water. The primary components of the ECM are negatively charged aggrecans and collagen II fibrils, which possess a characteristic, ordered three-dimensional structure. The components interact to ensure that the cartilage is able to absorb shock and can function to protect the bone ends. Atomic force microscopy (AFM) can be used to examine structure-function relationships of cartilage at both micrometer and nanometer scales. When imaged at the micrometer scale with microspheres, only the ECM and chondrocytes can be distinguished. Correspondingly, mechanical testing of cartilage at the micrometer scale results in unimodal distribution of the stiffness because the bulk elastic property of the ECM is probed. In contrast, bare AFM tips are able to reveal the molecular components of the ECM at the nanometer scale. Mechanical testing at the nanometer scale reveals a bimodal distribution of the stiffness and reflects the distinct stiffness of the collagen network and the proteoglycan moiety. In this protocol, the corresponding AFM image and force map are used to reveal the distinct morphology of the collagen fibers and proteoglycan gel. Although, in principle, these experiments can be performed using any AFM, an AFM with tube scanners that have manual screws for tilting the sample is preferable because cartilage has macroscopically rough surface features. By manually tilting the probe over the sample, an optimal angle for tip approach can be achieved.