Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.     

Immobilization method of yeast cells for intermittent contact mode imaging using the atomic force microscope

The atomic force microscope (AFM) is widely used for studying the surface morphology and growth of live cells. There are relatively fewer reports on the AFM imaging of yeast cells [1] (Kasas and Ikai, 1995), [2] (Gad and Ikai, 1995). Yeasts have thick and mechanically strong cell walls and are therefore difficult to attach to a solid substrate. In this report, a new immobilization technique for the height mode imaging of living yeast cells in solid media using AFM is presented. The proposed technique allows the cell surface to be almost completely exposed to the environment and studied using AFM. Apart from the new immobilization protocol, for the first time, height mode imaging of live yeast cell surface in intermittent contact mode is presented in this report. Stable and reproducible imaging over a 10-h time span is observed. A significant improvement in operational stability will facilitate the investigation of growth patterns and surface patterns of yeast cells.     

A torsional resonance mode AFM for in-plane tip surface interactions

Changing the method of tip/sample interaction leads to contact, tapping and other dynamic imaging modes in atomic force microscopy (AFM) feedback controls. A common characteristic of these feedback controls is that the primary control signals are based on flexural deflection of the cantilever probes, statically or dynamically. We introduce a new AFM mode using the torsional resonance amplitude (or phase) to control the feedback loop and maintain the tip/surface relative position through lateral interaction. The torsional resonance mode (TRmode™) provides complementary information to tapping mode for surface imaging and studies. The nature of tip/surface interaction of the TRmode facilitates phase measurements to resolve the in-plane anisotropy of materials as well as measurements of dynamic friction at nanometer scale. TRmode can image surfaces interleaved with TappingMode™ with the same probe and in the same area. In this way we are able to probe samples dynamically in both vertical and lateral dimensions with high sensitivity to local mechanical and tribological properties. The benefit of TRmode has been proven in studies of water adsorption on HOPG surface steps. TR phase data yields approximately 20 times stronger contrast than tapping phase at step edges, revealing detailed structures that cannot be resolved in tapping mode imaging. The effect of sample rotation relative to the torsional oscillation axis of the cantilever on TR phase contrast has been observed. Tip wear studies of TRmode demonstrated that the interaction forces between tip and sample could be controlled for minimum tip damage by the feedback loop.     

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.     

Controlling tip–sample interaction forces during a single tap for improved topography and mechanical property imaging of soft materials by AFM

We introduce a method that exploits the “active” nature of the force-sensing integrated readout and active tip (FIRAT), a recently introduced atomic force microscopy (AFM) probe, to control the interaction forces during individual tapping events in tapping mode (TM) AFM. In this method the probe tip is actively retracted if the tip–sample interaction force exceeds a user-specified force threshold during a single tap while the tip is still in contact with the surface. The active tip control (ATC) circuitry designed for this method makes it possible to control the repulsive forces and indentation into soft samples, limiting the repulsive forces during the scan while avoiding instability due to attractive forces. We demonstrate the accurate topographical imaging capability of this method on suitable samples that possess both soft and stiff features.     

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.     

An integrated approach to the study of living cells by atomic force microscopy

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long-term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.
The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.     

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.     

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).     

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.     

Dynamic analysis of tapping mode atomic force microscope (AFM) for critical dimension measurement

Transient dynamics of tapping mode atomic force microscope (AFM) for critical dimension measurement are analyzed. A simplified nonlinear model of AFM is presented to describe the forced vibration of the micro cantilever-tip system with consideration of both contact and non-contact transient behavior for critical dimension measurement. The governing motion equations of the AFM cantilever system are derived from the developed model. Based on the established dynamic model, motion state of the AFM cantilever system is calculated utilizing the method of averaging with the form of slow flow equations. Further analytical solutions are obtained to reveal the effects of critical parameters on the system dynamic performance. In addition, features of dynamic response of tapping mode AFM in critical dimension measurement are studied, where the effects of equivalent contact stiffness, quality factor and resonance frequency of cantilever on the system dynamic behavior are investigated. Contact behavior between the tip and sample is also analyzed and the frequency drift in contact phase is further explored. Influence of the interaction between the tip and sample on the subsequent non-contact phase is studied with regard to different parameters. The dependence of the minimum amplitude of tip displacement and maximum phase difference on the equivalent contact stiffness, quality factor and resonance frequency are investigated. This study brings further insights into the dynamic characteristics of tapping mode AFM for critical dimension measurement, and thus provides guidelines for the high fidelity tapping mode AFM scanning.     

Early detection of cytotoxic events between hepatic natural killer cells and colon carcinoma cells as probed with the atomic force microscope.

The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.     

Manipulation, dissection, and lithography using modified tapping mode atomic force microscope

A modified tapping mode of the atomic force microscope (AFM) was introduced for manipulation, dissection, and lithography. By sufficiently decreasing the amplitude of AFM tip in the normal tapping mode and adjusting the setpoint, the tip-sample interaction can be efficiently controlled. This modified tapping mode has some characteristics of the AFM contact mode and can be used to manipulate nanoparticles, dissect biomolecules, and make lithographs on various surfaces. This method did not need any additional equipment and it can be applied to any AFM system.     

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.     

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.     

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.     

Structural diversity of sphingomyelin microdomains

In cells plasma membrane, sphingomyelin (SM) plays a key role in the formation of a category of lipid microdomains enriched in cholesterol (Chl) often referred to as rafts. Atomic force microscopy (AFM) was used to analyze the mesoscopic topography of enriched SM microdomains in supported bilayers made of SM/dioleoylphosphatidylcholine (SM/DOPC) and SM/palmitoyl-oleoyl-phosphatidylcholine (SM/POPC) equimolar mixtures, in buffer, at room temperature. Gel–fluid phase separation occurs in both SM/DOPC and SM/POPC bilayers. The gel phase SM-enriched microdomains adopt a variety of size, shape and mesoscopic structure, from homogeneous flat domains of a few hundreds of nanometer in diameter to domains of several micrometers made of closely packed globular structures. Gel–gel phase separation in SM domains is also observed which gives rise to different structures for the diunsaturated and the mixed-saturated PC species. These differences could also extend to the interactions with Chl. This suggests that studies on rafts formation commonly performed using SM/DOPC mixture as a model should also include the physiologically more relevant POPC species.     

Time-lapse imaging of In Vitro myogenesis using atomic force microscopy

Myoblast therapy relies on the integration of skeletal muscle stem cells into distinct muscular compartments for the prevention of clinical conditions such as heart failure, or bladder dysfunction. Understanding the fundamentals of myogenesis is hence crucial for the success of these potential medical therapies. In this report, we followed the rearrangement of the surface membrane structure and the actin cytoskeletal organization in C2C12 myoblasts at different stages of myogenesis using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging of living myoblasts undergoing fusion unveiled that within minutes of making cell–cell contact, membrane tubules appear that unite the myoblasts and increase in girth as fusion proceeds. CLSM identified these membrane tubules as built on scaffolds of actin filaments that nucleate at points of contact between fusing myoblasts. In contrast, similarly behaving membrane tubules are absent during cytokinesis. The results from our study in combination with recent findings in literature further expand the understanding of the biochemical and membrane structural rearrangements involved in the two fundamental cellular processes of division and fusion.     

Tapping-mode atomic force microscopy on intact cells: optimal adjustment of tapping conditions by using the deflection signal

Difficulties in the proper adjustment of the scanning parameters are often encountered when using tapping-mode atomic force microscopy (TMAFM) for imaging thick and soft material, and particularly living cells, in aqueous buffer. A simple procedure that drastically enhances the successful imaging of the surface of intact cells by TMAFM is described. It is based on the observation, in liquid, of a deflection signal, concomitant with the damping of the amplitude that can be followed by amplitude-distance curves. For intact cells, the evolution of the deflection signal, steeper than the amplitude damping allows a precise adjustment of the feedback value. Besides its use in finding the appropriate tapping conditions, the deflection signal provides images of living cells that essentially reveal the organization of the membrane cytoskeleton. This allows to show that changes in the membrane surface topography are associated with a reorganization of the membrane skeleton. Studies on the relationships between the cell surface topography and membrane skeleton organization in living cells open a new field of applications for the atomic force microscope.     

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.