Temperature-dependent imaging of living cells by AFM

Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 degrees C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature.     

Geometric parameters effect of the atomic force microscopy smart piezoelectric cantilever on the different rough surface topography quality by considering the capillary force

Nowadays, the atomic force microscopy (AFM) is widely used in the nanotechnology as a powerful nano‐robot. The surface topography in Nanoscale is by far one of the most important usages of the AFM device. Hence, in this article, the vibration motion of a piezoelectric rectangular cross‐section micro‐cantilever (MC) which oscillates in the moist environment has been examined based on the Timoshenko beam theory. After extracting the MC governing equations according to Hamilton's principle, the finite element method has been used to discretize the motion equations. The surface topography has been simulated for various roughness forms in the tapping and non‐contact modes by considering the effects of the Van der Waals, capillary and contact forces. Also, the experimental results obtained from the glass surface topography have been simulated. The results illustrate that the time delay in higher natural frequencies in the tapping mode is shorter in comparison with the non‐contact mode, especially, for the lower natural frequencies. The sensitivity analysis of the natural frequencies, topography depth and time delay have been simulated. Results indicate that the most effective parameter is the MC length. In the first mode, the first section length has the highest effect on the surface topography time delay, also, in the second vibration mode; the most effective parameter on the time delay is the MC tip length based on the simulation results.     

Probing material properties of polymeric surface layers with tapping mode AFM: Which cantilever spring constant,tapping amplitude and amplitude set point gives good image contrast and minimal surface damage?

A phase shift between the oscillatory motion and drive motion of an AFM-cantilever used for tapping mode AFM imaging can be related to adhesive and elastic properties of surface layers. In this study it was studied how optimal contrast between hard and soft surface layers can be achieved while minimizing the surface damage. This was investigated by performing classical force-distance measurements while driving the cantilever as in tapping mode imaging. The amplitude and phase response as a function of the average tip-surface separation was recorded. Five different cantilevers with a wide range of spring constants and four different tapping amplitudes were investigated and compared. Based on these experiments it is concluded that too stiff cantilever, high free tapping amplitude and low amplitude set point value often lead to surface damage, while too low spring constant and low free tapping amplitude result in poor phase image contrast. Intermediate values where little surface damage and significant image contrast are obtained were identified. In all cases it was observed that the best image contrast was obtained when the amplitude set point was chosen such that the amplitude during imaging was reduced to approximately 50% of the free amplitude.     

Topography imaging with a heated atomic force microscope cantilever in tapping mode

This article describes tapping mode atomic force microscopy (AFM) using a heated AFM cantilever. The electrical and thermal responses of the cantilever were investigated while the cantilever oscillated in free space or was in intermittent contact with a surface. The cantilever oscillates at its mechanical resonant frequency, 70.36 kHz, which is much faster than its thermal time constant of 300 micros, and so the cantilever operates in thermal steady state. The thermal impedance between the cantilever heater and the sample was measured through the cantilever temperature signal. Topographical imaging was performed on silicon calibration gratings of height 20 and 100 nm. The obtained topography sensitivity is as high as 200 microVnm and the resolution is as good as 0.5 nmHz(1/2), depending on the cantilever power. The cantilever heating power ranges 0-7 mW, which corresponds to a temperature range of 25-700 degrees C. The imaging was performed entirely using the cantilever thermal signal and no laser or other optics was required. As in conventional AFM, the tapping mode operation demonstrated here can suppress imaging artifacts and enable imaging of soft samples.     

Accurate determination of spring constant of atomic force microscope cantilevers and comparison with other methods

We present calibration results of commercial AFM cantilevers using the KRISS nanoforce calibrator (NFC) that can determine traceably spring constants with an uncertainty better than 1%, along with the results obtained from other four calibration methods: the dimensional method, the cantilever-on-cantilever method, the Sader method, and the thermal noise method. Two types (contact and tapping mode) of beam-shaped AFM cantilevers with nominal spring constants of 0.9 N m⁻¹ and 42 N m⁻¹, respectively, were investigated in this study. Because of its small uncertainty, the NFC method was used to assess the uncertainties of other four methods through comparisons between values obtained from other methods and those from the NFC method for the same cantilever. Results from other methods were generally in good agreement with those from the NFC method within the uncertainties of other methods claimed in other literatures, but values obtained from the Sader method were differed by up to 40% from the NFC values, which is 2 times worse than the known uncertainty.     

Finite-element vibration analysis of tapping-mode atomic force microscopy in liquid

Investigation of morphology and mechanical properties of biological specimens using atomic force microscopy (AFM) often requires its operation in liquid environment. Due to the hydrodynamic force, the vibration of AFM cantilevers in liquid shows dramatically different dynamic characteristics from that in air. A good understanding of the dynamics of AFM cantilevers vibrating in liquid is needed for the interpretation of scanning images, selection of AFM operating conditions, and evaluation of sample's mechanical properties. In this study, a finite element (FE) model is used for frequency and transient response analysis of AFM cantilevers in tapping mode (TM) operated in air or liquid. Hydrodynamic force exerted by the fluid on AFM cantilevers is approximated by additional mass and hydrodynamic damping. The additional mass and hydrodynamic damping matrices corresponding to beam elements are derived. With this model, numerical simulations are performed for an AFM cantilever to obtain the frequency and transient responses of the cantilever in air and liquid. The comparison between our simulated results and the experimentally obtained ones shows good agreement. Based on the simulations, different characteristics of cantilever dynamics in air and liquid are discussed.     

Simulation-aided design and fabrication of nanoprobes for scanning probe microscopy

We proposed and demonstrated a flexible and effective method to design and fabricate scanning probes for atomic force microscopy applications. Computer simulations were adopted to evaluate design specifications and desired performance of atomic force microscope (AFM) probes; the fabrication processes were guided by feedback from simulation results. Through design-simulation-fabrication iterations, tipless cantilevers and tapping mode probes were successfully made with errors as low as 2% in designed resonant frequencies. For tapping mode probes, the probe tip apex achieved a 10 nm radius of curvature without additional sharpening steps; tilt-compensated probes were also fabricated for better scanning performance. This method provides AFM users improved probe quality and practical guidelines for customized probes, which can support the development of novel scanning probe microscopy (SPM) applications.     

MAC mode atomic force microscopy studies of living samples, ranging from cells to fresh tissue

Magnetic AC mode (MAC mode) atomic force microscopy (AFM), a novel type of tapping mode AFM in which the cantilever is driven directly by a magnetic field, is a powerful tool for imaging with high spatial resolution and better signal-to-noise in liquid environment. It may largely extend the application of AFM to living samples, especially those are sensitive to cantilever forces, even to multilayer tissue samples. However, there are few reports on the imaging of living cells by MAC mode AFM previously. In our present study, we explore the optimal imaging conditions of MAC mode AFM on living astrocytes and fresh arterial intima surface. We also used nude tips for PicoTREC panel (i.e., Aux in BNC, a new data collecting channel) to image living samples and discussed its difference with phase imaging. We show that living biological samples can be imaged by MAC mode AFM at details of comparable resolution as those by high resolution scanning electron microscopy. Furthermore, the combination of height, amplitude, phase and TREC panel signals provide abundant informations for the characteristics of living samples, such as topography, profile, stiffness and adhesion.     

Three-channel false colour AFM images for improved interpretation of complex surfaces: A study of filamentous cyanobacteria

Imaging signals derived from the atomic force microscope (AFM) are typically presented as separate adjacent images with greyscale or pseudo-colour palettes. We propose that information-rich false-colour composites are a useful means of presenting three-channel AFM image data. This method can aid the interpretation of complex surfaces and facilitate the perception of information that is convoluted across data channels. We illustrate this approach with images of filamentous cyanobacteria imaged in air and under aqueous buffer, using both deflection-modulation (contact) mode and amplitude-modulation (tapping) mode. Topography-dependent contrast in the error and tertiary signals aids the interpretation of the topography signal by contributing additional data, resulting in a more detailed image, and by showing variations in the probe-surface interaction. Moreover, topography-independent contrast and topography-dependent contrast in the tertiary data image (phase or friction) can be distinguished more easily as a consequence of the three dimensional colour-space.     

Microfabricated torsion levers optimized for low force and high-frequency operation in fluids

We developed a mass production fabrication process for making symmetrically supported torsion cantilevers/oscillators with highly compliant springs. These torsion probes offer advantages in atomic force microscopy (AFM) because they are small, have high optical gain, do not warp and can be made with two independent axes. Compared to traditional AFM cantilevers, these probes have higher frequency response, higher Q, lower noise, better optics (since the mirror does not bend) and two data channels. Soft small levers with sub-pN force resolution can resonate cleanly above 10 kHz in water. When fabricated with a ferromagnetic coating on the rigid reflecting pad, they can be driven magnetically or serve as high-resolution magnetometers. Asymmetric levers can be tapping mode probes or high-resolution accelerometers. The dual axis gimbaled probes with two orthogonal axes can operate on a standard AFM with single beam illumination. These probes can be used as self-referencing, drift free, cantilevers where one axis senses the substrate position and the other the sample position. These levers can be optimized for differential contrast or high-resolution friction imaging.     

Novel operation mode for eliminating influence of inclination angle and friction in atomic force microscopy

The accuracy of topography imaging in contact force mode of atomic force microscopy (AFM) depends on the one-to-one corresponding relationship between the cantilever deflection and the tip–sample distance, whereas such a relationship cannot be always achieved in the presence of friction and incline angle of sample surface. Recently, we have developed a novel operation mode in which we keep the van der Waals force as constant instead of the applied normal force, to eliminate the effect of inclination angle and friction on topography imaging in the contact force mode. We have improved our AFM to enable the new operation mode for validation. Comparative experiments have been performed and the results have shown that the effect of friction and inclination angle on topography imaging in contact mode of AFM can be eliminated or at least decreased effectively by working in the new operation mode we present.     

Improving tapping mode atomic force microscopy with piezoelectric cantilevers

This article summarizes improvements to the speed, simplicity and versatility of tapping mode atomic force microscopy (AFM). Improvements are enabled by a piezoelectric microcantilever with a sharp silicon tip and a thin, low-stress zinc oxide (ZnO) film to both actuate and sense deflection. First, we demonstrate self-sensing tapping mode without laser detection. Similar previous work has been limited by unoptimized probe tips, cantilever thicknesses, and stress in the piezoelectric films. Tests indicate self-sensing amplitude resolution is as good or better than optical detection, with double the sensitivity, using the same type of cantilever. Second, we demonstrate self-oscillating tapping mode AFM. The cantilever's integrated piezoelectric film serves as the frequency-determining component of an oscillator circuit. The circuit oscillates the cantilever near its resonant frequency by applying positive feedback to the film. We present images and force-distance curves using both self-sensing and self-oscillating techniques. Finally, high-speed tapping mode imaging in liquid, where electric components of the cantilever require insulation, is demonstrated. Three cantilever coating schemes are tested. The insulated microactuator is used to simultaneously vibrate and actuate the cantilever over topographical features. Preliminary images in water and saline are presented, including one taken at 75.5 μm/s—a threefold improvement in bandwidth versus conventional piezotube actuators.     

Attachment of carbon nanotubes to atomic force microscope probes

In atomic force microscopy (AFM) the accuracy of data is often limited by the tip geometry and the effect on this geometry of wear. One way to improve the tip geometry is to attach carbon nanotubes (CNT) to AFM tips. CNTs are ideal because they have a small diameter (typically between 1 and 20nm), high aspect ratio, high strength, good conductivity, and almost no wear. A number of methods for CNT attachment have been proposed and explored including chemical vapour deposition (CVD), dielectrophoresis, arc discharge and mechanical attachment. In this work we will use CVD to deposit nanotubes onto a silicon surface and then investigate improved methods to pick-up and attach CNTs to tapping mode probes. Conventional pick-up methods involve using standard tapping mode or non-contact mode so as to attach only those CNTs that are aligned vertically on the surface. We have developed improved methods to attach CNTs using contact mode and reduced set-point tapping mode imaging. Using these techniques the AFM tip is in contact with a greater number of CNTs and the rate and stability of CNT pick-up is improved. The presence of CNTs on the modified AFM tips was confirmed by high-resolution AFM imaging, analysis of the tips dynamic force curves and scanning electron microscopy (SEM).     

Origin of phase shift in atomic force microscopic investigation of the surface morphology of NR/NBR blend film

Atomic force microscopy (AFM) was used to study the morphology and surface properties of NR/NBR blend. Blends at 1/3, 1/1 and 3/1 weight ratios were prepared in benzene and formed film by casting. AFM phase images of these blends in tapping mode displayed islands in the sea morphology or matrix-dispersed structures. For blend 1/3, NR formed dispersed phase while in blends 1/1 and 3/1 phase inversion was observed. NR showed higher phase shift angle in AFM phase imaging for all blends. This circumstance was governed by adhesion energy hysteresis between the device tip and the rubber surface rather than surface stiffness of the materials, as proved by force distance measurements in the AFM contact mode.     

Variable-force tapping atomic force microscopy as a tool in the characterization of organic devices

A common method for characterizing the phase separation of materials in mixtures is tapping mode atomic force microscopy (AFM). However, AFM results are influenced by surface-energy effects and the employed tapping force, and it might therefore be difficult to attain correct information regarding the bulk with such a surface-imaging technique. In this work, we present a way of imaging material phase separation in an improved manner by recording a series of AFM images at different tapping force. More specifically, we have employed the variable-force AFM method on organic mixtures, comprising a conjugated polymer (MEH-PPV) and an ion-conducting polymer electrolyte (PEO-XCF(3)SO(3), X=Li, K, Rb), and we demonstrate that it is capable of reversibly sampling such materials not only on the surface, but also (indirectly) in the topmost part of the bulk. The analysis of the evolution of AFM phase images allows us to (indirectly) gain information about the bulk-phase separation of materials. We find that the variable-force AFM results correlate well with the device performance of light-emitting electrochemical cells employing such organic mixtures as the active material.     

Note: ferrule-top atomic force microscope. II. Imaging in tapping mode and at low temperature

In a recent paper [D.Chavan et al., Rev. Sci. Instrum. 81, 123702 (2010)] we have demonstrated that ferrule-top cantilevers, obtained by carving the end of a ferruled fiber, can be used for contact mode atomic force microscopy in ambient conditions. Here we show that those probes can provide tapping mode images at both room and cryogenic temperatures (12 K).     

Prototype cantilevers for quantitative lateral force microscopy

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.     

Calibration of atomic force microscope cantilevers using piezolevers

The atomic force microscope (AFM) can provide qualitative information by numerous imaging modes, but it can also provide quantitative information when calibrated cantilevers are used. In this article a new technique is demonstrated to calibrate AFM cantilevers using a reference piezolever. Experiments are performed on 13 different commercially available cantilevers. The stiff cantilevers, whose stiffness is more than 0.4 N/m, are compared to the stiffness values measured using nanoindentation. The experimental data collected by the piezolever method is in good agreement with the nanoindentation data. Calibration with a piezolever is fast, easy, and nondestructive and a commercially available AFM is enough to perform the experiments. In addition, the AFM laser must not be calibrated. Calibration is reported here for cantilevers whose stiffness lies between 0.08 and 6.02 N/m.     

Mechanical properties of plasma membrane and nuclear envelope measured by scanning probe microscope

Atomic force microscopy has been used to visualize nano‐scale structures of various cellular components and to characterize mechanical properties of biomolecules. In spite of its ability to measure non‐fixed samples in liquid, the application of AFM for living cell manipulation has been hampered by the lack of knowledge of the mechanical properties of living cells. In this study, we successfully combine AFM imaging and force measurement to characterize the mechanical properties of the plasma membrane and the nuclear envelope of living HeLa cells in a culture medium. We examine cantilevers with different physical properties (spring constant, tip angle and length) to find out the one suitable for living cell imaging and manipulation. Our results of elasticity measurement revealed that both the plasma membrane and the nuclear envelope are soft enough to absorb a large deformation by the AFM probe. The penetrations of the plasma membrane and the nuclear envelope were possible when the probe indents the cell membranes far down close to a hard glass surface. These results provide useful information to the development of single‐cell manipulation techniques.