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Atomic force microscopy based approaches have led to remarkable advances in the field of mechanobiology. However, linking the mechanical cues to biological responses requires complementary techniques capable of recording these physiological characteristics. In this study, we present an instrument for combined optical, force, and electrical measurements based on a novel type of scanning probe microscopy cantilever composed of a protruding volcano-shaped nanopatterned microelectrode (nanovolcano probe) at the tip of a suspended microcantilever. This probe enables simultaneous force and electrical recordings from single cells. Successful impedance measurements on mechanically stimulated neonatal rat cardiomyocytes in situ were achieved using these nanovolcano probes. Furthermore, proof of concept experiments demonstrated that extracellular field potentials (electrogram) together with contraction displacement curves could simultaneously be recorded. These features render the nanovolcano probe especially suited for mechanobiological studies aiming at linking mechanical stimuli to electrophysiological responses of single cells.
Assuntos
Fenômenos Mecânicos , Microscopia de Varredura por Sonda , Animais , Microeletrodos , Microscopia de Força Atômica , Miócitos Cardíacos , RatosRESUMO
The accuracy and repeatability of atomic force microscopy (AFM) imaging significantly depend on the accuracy of the piezoactuator. However, nonlinear properties of piezoactuators can distort the image, necessitating sensor-based closed-loop actuators to achieve high accuracy AFM imaging. The advent of high-speed AFM has made the requirements on the position sensors in such a system even more stringent, requiring higher bandwidths and lower sensor mass than traditional sensors can provide. In this paper, we demonstrate a way for high-speed, high-precision closed-loop AFM nanopositioning using a novel, miniaturized micro-electro-mechanical system position sensor in conjunction with a simple PID controller. The sensor was developed to respond to the need for small, lightweight, high-bandwidth, long-range and sub-nm-resolution position measurements in high-speed AFM applications. We demonstrate the use of this sensor for closed-loop operation of conventional as well as high-speed AFM operation to provide distortion-free images. The presented implementation of this closed-loop approach allows for positioning precision down to 2.1 Å, reduces the integral nonlinearity to below 0.2%, and allows for accurate closed loop imaging at line rates up to 300 Hz.
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In current nanopore-based label-free single-molecule sensing technologies, stochastic processes influence the selection of translocating molecule, translocation rate and translocation velocity. As a result, single-molecule translocations are challenging to control both spatially and temporally. Here we present a method using a glass nanopore mounted on a three-dimensional nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at a controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio by two orders of magnitude compared with free translocations. We demonstrate the method's versatility by assessing DNA-protein complexes, DNA rulers and DNA gaps, achieving down to single-nucleotide gap detection.
Assuntos
Nanoporos , DNA , Nanotecnologia , Razão Sinal-RuídoRESUMO
Improving the imaging speed of atomic force microscopy (AFM) requires accurate nanopositioning at high speeds. However, high speed operation excites resonances in the AFM's mechanical scanner that can distort the image, and therefore typical users of commercial AFMs elect to operate microscopes at speeds below which scanner resonances are observed. Although traditional robust feedforward controllers and input shaping have proven effective at minimizing the influence of scanner distortions, the lack of direct measurement and use of model-based controllers have required disassembling the microscope to access lateral scanner motion with external sensors in order to perform a full system identification experiment, which places excessive demands on routine microscope operators. Further, since the lightly damped instrument dynamics often change from experiment to experiment, model-based controllers designed from offline system identification experiments must trade off high speed performance for robustness to modeling errors. This work represents a new way to automatically characterize the lateral scanner dynamics without addition of lateral sensors, and shape the commanded input signals in such a way that disturbing dynamics are not excited. Scanner coupling between the lateral and out-of-plane directions is exploited and used to build a minimal model of the scanner that is also sufficient to describe the nature of the distorting resonances. This model informs the design of an online input shaper used to suppress spectral components of the high speed command signals. The method presented is distinct from alternative approaches in that neither an information-complete system identification experiment nor microscope modification are required. Because the system identification is performed online immediately before imaging, no tradeoff of performance is required. This approach has enabled an increase in the scan rates of unmodified commercial AFMs from 1-4 lines s(-1) to over 40 lines s(-1).
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In this paper, we present a detailed investigation into the suitability of atomic force microscopy (AFM) cantilevers with integrated deflection sensor and micro-actuator for imaging of soft biological samples in fluid. The Si cantilevers are actuated using a micro-heater at the bottom end of the cantilever. Sensing is achieved through p-doped resistors connected in a Wheatstone bridge. We investigated the influence of the water on the cantilever dynamics, the actuation and the sensing mechanisms, as well as the crosstalk between sensing and actuation. Successful imaging of yeast cells in water using the integrated sensor and actuator shows the potential of the combination of this actuation and sensing method. This constitutes a major step towards the automation and miniaturization required to establish AFM in routine biomedical diagnostics and in vivo applications.
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Técnicas Biossensoriais/métodos , Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Saccharomyces cerevisiae/ultraestrutura , Ar , Reagentes de Ligações Cruzadas , Desenho de Equipamento , ÁguaRESUMO
A versatile atomic force microscope (AFM), which can be installed in a scanning electron microscope (SEM), is introduced. The flexible design of the instrument enables correlated analysis for different experimental configurations, such as AFM imaging directly after nanoindentation in vacuum. In order to demonstrate the capabilities of the specially designed AFM installed inside a SEM, slip steps emanating around nanoindents in single crystalline brass were examined. This example showcases how the combination of AFM and SEM imaging can be utilized for quantitative dislocation analysis through the measurement of the slip step heights without the hindrance of oxide formation. Finally, an in situ nanoindentation technique is introduced, illustrating the use of AFM imaging during indentation experiments to examine plastic deformation occurring under the indenter tip. The mechanical indentation data are correlated to the SEM and AFM images to estimate the number of dislocations emitted to the surface.
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High speed atomic force microscopy can provide the possibility of many new scientific observations and applications ranging from nano-manufacturing to the study of biological processes. However, the limited imaging speed has been an imperative drawback of the atomic force microscopes. One of the main reasons behind this limitation is the excitation of the AFM dynamics at high scan speeds, severely undermining the reliability of the acquired images. In this research, we propose a piezo based, feedforward controlled, counter actuation mechanism to compensate for the excited out-of-plane scanner dynamics. For this purpose, the AFM controller output is properly filtered via a linear compensator and then applied to a counter actuating piezo. An effective algorithm for estimating the compensator parameters is developed. The information required for compensator design is extracted from the cantilever deflection signal, hence eliminating the need for any additional sensors. The proposed approach is implemented and experimentally evaluated on the dynamic response of a custom made AFM. It is further assessed by comparing the imaging performance of the AFM with and without the application of the proposed technique and in comparison with the conventional counterbalancing methodology. The experimental results substantiate the effectiveness of the method in significantly improving the imaging performance of AFM at high scan speeds.
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Microscopia de Força Atômica/instrumentação , Algoritmos , Desenho de Equipamento , Fatores de TempoRESUMO
The fracture resistance of biomineralized tissues such as bone, dentin, and abalone is greatly enhanced through the nanoscale interactions of stiff inorganic mineral components with soft organic adhesive components. A proper understanding of the interactions that occur within the organic component, and between the organic and inorganic components, is therefore critical for a complete understanding of the mechanics of these tissues. In this paper, we use Atomic Force Microscope (AFM) force spectroscopy and dynamic force spectroscopy to explore the effect of ionic interactions within a nanoscale system consisting of networks of Dentin Matrix Protein 1 (DMP1) (a component of both bone and dentin organic matrix), a mica surface, and an AFM tip. We find that DMP1 is capable of dissipating large amounts of energy through an ion-mediated mechanism, and that the effectiveness increases with increasing ion valence.
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The topography of freshly fractured bovine and human bone surfaces was determined by the use of atomic force microscopy (AFM). Fracture surfaces from both kinds of samples exhibited complex landscapes formed by hydroxyapatite mineral platelets with lateral dimensions ranging from â¼90 nm × 60 nm to â¼20 nm × 20 nm. Novel AFM techniques were used to study these fracture surfaces during various chemical treatments. Significant topographical changes were observed following exposure to aqueous solutions of ethylenediaminetetraacetic acid (EDTA) or highly concentrated sodium fluoride (NaF). Both treatments resulted in the apparent loss of the hydroxyapatite mineral platelets on a timescale of a few seconds. Collagen fibrils situated beneath the overlying mineral platelets were clearly exposed and could be resolved with high spatial resolution in the acquired AFM images. Time-dependent mass loss experiments revealed that the applied agents (NaF or EDTA) had very different resulting effects. Despite the fact that the two treatments exhibited nearly identical results following examination by AFM, bulk bone samples treated with EDTA exhibited a â¼70% mass loss after 72 h, whereas for the NaF-treated samples, the mass loss was only of the order of â¼10%. These results support those obtained from previous mechanical testing experiments, suggesting that enhanced formation of superficial fluoroapatite dramatically weakens the protein-hydroxyapatite interfaces. Additionally, we discovered that treatment with aqueous solutions of NaF resulted in the effective extraction of noncollagenous proteins from bone powder.