Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.     

Noncontact measurement of the local mechanical properties of living cells using pressure applied via a pipette

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.     

Vertical dimension of hydrated biological samples in tapping mode scanning force microscopy.

The vertical dimensions of the well-characterized test samples tobacco mosaic virus, T4 bacteriophage polyhead, purple membrane, and hexagonally packed intermediate (HPI) layer were investigated by tapping mode scanning force microscopy (SFM) in solution. Purple membrane and HPI layer were imaged in both contact mode and tapping mode SFM for direct comparison. All vertical dimensions match the known heights. The practical implications of the absence of frictional forces in tapping mode are discussed.     

The topography of soft,adhesive diatom ‘trails’ as observed by Atomic Force Microscopy

Gliding diatoms foul surfaces by leaving behind ‘trails’ of secreted mucilage. Atomic force microscopy (AFM) used in ‘fluid tapping’ mode enabled the topography of the soft, adhesive trails in the natural hydrated state to be imaged, and without the artefacts resulting from fixation and/or dehydration. Diatom trails consist of a continuous, swollen ridge of material that dominates the trail, as well as a diffuse hydrated mucilage coating observed on either side of the main trail. The main trail material is evenly attached to the coverslip along its entire length, and appears to cure, or become less soft/adhesive, over time. Diatom trails observed with the scanning electron microscope were severely damaged by dehydration, while trails imaged by the AFM in ‘contact’ mode were damaged and/or removed by the action of the cantilever. The AFM used in ‘fluid tapping’ mode is an excellent tool for topographical studies of soft/adhesive biological molecules in the hydrated state, and will have great value for measuring their physical and mechanical properties when operated in ‘force modulation’ mode.     

Manipulation and Molecular Resolution of a Phosphatidylcholine-Supported Planar Bilayer by Atomic Force Microscopy

The morphology of supported planar bilayers has been investigated below phase transition temperature by atomic force microscopy in contact and tapping mode. The bilayers were formed by the vesicle-spreading technique. In contact mode at low scanning forces of about 1 nN true molecular resolution could be achieved for supported phosphatidylcholine bilayers. The resolution was confirmed by experiments that captured the location, average area of individual lipid headgroups and the manipulation of the bilayer surface. Repeated scanning in contact mode shifted the random topology of the surface consecutively to a striped pattern. Height profiles of defect-containing bilayers were analyzed. The shape of the defects became smooth by repeated scanning. The height profiles allowed the estimation of the indentation of the tip into the surface-adsorbed membrane. In tapping mode a disordered pattern of headgroups became visible. Our morphological data at molecular resolution suggest that the native arrangement of the choline headgroups is disordered, free of large packing defects and becomes ordered in Schallamach waves by scanning in contact mode. Received: 12 March 1997/Revised: 3 October 1997     

Scanning ion conductance microscopy of living cells.

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.     

Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy

Contact and tapping mode atomic force microscopy have been used to visualize the surface of cultured CV-1 kidney cells in aqueous medium. The height images obtained from living cells were comparable when using contact and tapping modes. In contrast, the corresponding, and simultaneously acquired, deflection images differed markedly. Whereas, as expected, deflection images enhanced the surface features in the contact mode, they revealed the presence of a filamentous network when using the tapping mode. This network became disorganized upon addition of cytochalasin, which strongly suggests that it corresponded to the submembraneous cytoskeleton. Examination of fixed cells further supported this assumption. These data show that, in addition to the structural information on the cell surface, the use of the tapping mode in liquid can also provide a good visualization of the membrane cytoskeleton. Tapping mode atomic force microscopy appears to he a promising technique for studying interactions between cell surface and subsurface structures, a critical step in many biological processes.     

Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.     

Patch clamp recording of the responses to three bitter stimuli in mouse taste cells.

Although several pathways of bitter taste signal transduction have been proposed in taste cells, these mechanisms have not been elucidated in detail. To investigate the diversity of responses to bitter stimuli, we recorded the electrophysiological responses to quinine, denatonium and naringin using whole-cell patch clamp technique in isolated taste cells of C57BL/6J mice. Ten mM quinine induced depolarizing response under the current clamp mode, and inward current response under the voltage-clamp mode (holding potential -80 mV) using both K+ (with pseudo intracellular solution) and Cs+ (K+ was substituted by Cs+ in the pseudo intracellular solution) pipettes. However, when the K+ pipette was used, the membrane conductance was suppressed and activated in succession. On the other hand, the membrane conductance was only activated when the Cs+ pipette was used. Half to one mM denatonium induced depolarizing response under the current clamp mode, and outward current response under the voltage clamp mode with both pipettes. Using these pipettes, the membrane conductance was activated or suppressed in the individual case. Naringin-induced responses were not detected in these measurements. These electrophysiological recordings suggest that multiple transduction mechanisms are involved in bitter taste perception in mouse taste cells.     

Imaging soft samples with the atomic force microscope: gelatin in water and propanol.

We have imaged mica coated with thin gelatin films in water, propanol, and mixtures of these two liquids by atomic force microscopy (AFM). The elastic modulus (Young's modulus) can be tuned from 20 kPa to more than 0.1 GPa depending on the ratio of propanol to water. The resolution is best in pure propanol, on the order of 20 nm, and becomes worse for the softer samples. The degradation in resolution can be understood by considering the elastic indentation of the gelatin caused by the AFM tip. This indentation becomes larger and thus the contact area becomes larger the softer the sample is. Therefore this study may be used to estimate the resolution to be expected with an AFM on other soft samples, such as cells. Nondestructive imaging was possible only by imaging at forces < 1 nN. This was difficult to achieve in contact mode because of drift in the zero load deflection of the cantilever, supposedly caused by temperature drift, but straightforward in tapping mode.     

Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope.

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.     

Atomic force microscopy of insulin single crystals: direct visualization of molecules and crystal growth.

Atomic force microscopy performed on single crystals of three different polymorphs of bovine insulin revealed molecularly smooth (001) layers separated by steps whose heights reflect the dimensions of a single insulin hexamer. Whereas contact mode imaging caused etching that prevented molecular-scale resolution, tapping mode imaging in solution provided molecular-scale contrast that enabled determination of lattice parameters and polymorph identification while simultaneously enabling real-time examination of growth modes and assessment of crystal quality. Crystallization proceeds layer by layer, a process in which the protein molecules assemble homoepitaxially with nearly perfect orientational and translational commensurism. Tapping mode imaging also revealed insulin aggregates attached to the (001) faces, their incorporation into growing terraces, and their role in defect formation. These observations demonstrate that tapping mode imaging is ideal for real-time in situ investigation of the crystallization of soft protein crystals of relatively small proteins such as insulin, which cannot withstand the lateral shear forces exerted by the scanning probe in conventional imaging modes.     

Dynamic Model for Characterizing Contractile Behaviors and Mechanical Properties of a Cardiomyocyte

Studies on the contractile dynamics of heart cells have attracted broad attention for the development of both heart disease therapies and cardiomyocyte-actuated micro-robotics. In this study, a linear dynamic model of a single cardiomyocyte cell was proposed at the subcellular scale to characterize the contractile behaviors of heart cells, with system parameters representing the mechanical properties of the subcellular components of living cardiomyocytes. The system parameters of the dynamic model were identified with the cellular beating pattern measured by a scanning ion conductance microscope. The experiments were implemented with cardiomyocytes in one control group and two experimental groups with the drugs cytochalasin-D or nocodazole, to identify the system parameters of the model based on scanning ion conductance microscope measurements, measurement of the cellular Young’s modulus with atomic force microscopy indentation, measurement of cellular contraction forces using the micro-pillar technique, and immunofluorescence staining and imaging of the cytoskeleton. The proposed mathematical model was both indirectly and qualitatively verified by the variation in cytoskeleton, beating amplitude, and contractility of cardiomyocytes among the control and the experimental groups, as well as directly and quantitatively validated by the simulation and the significant consistency of 90.5% in the comparison between the ratios of the Young’s modulus and the equivalent comprehensive cellular elasticities of cells in the experimental groups to those in the control group. Apart from mechanical properties (mass, elasticity, and viscosity) of subcellular structures, other properties of cardiomyocytes have also been studied, such as the properties of the relative action potential pattern and cellular beating frequency. This work has potential implications for research on cytobiology, drug screening, mechanisms of the heart, and cardiomyocyte-based bio-syncretic robotics.     

Photodynamic effect on melanoma cells investigated by atomic force microscopy

Atomic force microscopy (AFM) is a modern experimental method for imaging of conducting or non-conducting samples. New trends in the application of scanning probe microscopy (SPM) give us the ability to scan live cells directly in their ingenuous surroundings or in air. Our apparatus was replenished with an inverse optical microscope, so we could observe the position of the scanning tip in every individual cell. The aim of the presented study is to picture the cell surface in air. A dry scanner in non-contact or tapping mode was used in the biological application of AFM. In our work the cell line G361 was used as a biological sample. We imaged the cell line before and after induction of a photodynamic effect (PDE) by irradiation of ZnTPPS4-loaded cells with a light dose of 15 J/cm(2). Individual cells before PDE induction had a smooth surface without protrusion on the entire surface. Cells after PDE induction did not have a smooth surface but their surface was rough with protrusion and in some places cleaved.     

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.     

The low conductance K(+) channel in human colonic crypt cells has a voltage-dependent permeability not affected by Mg(++)

The low conductance K(+) channel found in human colonocytes was investigated using the patch-clamp technique. The channel is Ca(++)-dependent and is blocked by Ba(++) (5 mM) with a decrease in open probability from 0.42 to 0.19. At -40 mV the slope conductance was 29 pS (using intracellular solution in the pipette). In inside-out patches, inward rectification was seen both with KCl (pipette)/NaCl (bath) solutions as well as KCl/KCl solutions. The rectification could not be affected by omitting Mg(++) from the pipette or the bath solution, neither by exposing the patches to the polyamine spermine (1 mM). Using the Goldman-Hodgkin-Katz equation we show that the permeability decreased in a linear fashion from approximately 5.2 x 10(-14) cm(3)/s to 1.8 x 10(-14) cm(3)/s (-100 to +100 mV), both with and without Mg(++) in the solutions. There was no significant difference in the nominal values of permeability. This property of the K(+) channel may facilitate the hyperpolarization needed to sustain a chloride secretion.     

Imaging mixed lipid monolayers by dynamic atomic force microscopy.

Phase imaging with tapping mode atomic force microscopy (AFM) and force modulation microscopy were used to probe the mechanical properties of phase-separated lipid monolayers made of a mixture (0.25:0.75) of the surface-active lipopeptide surfactin and of dipalmitoylphosphatidylcholine (DPPC). The pi-A isotherms and the result of a molecular modeling study revealed a loose, 2-D liquid-like organization for the surfactin molecules and a closely packed, 2-D solid-like organization for DPPC molecules. This difference in molecular organization was responsible for a significant contrast in height, tapping mode phase and force modulation amplitude images. Phase imaging at light tapping, i.e., with a ratio of the set-point tapping amplitude with respect to the free amplitude A(sp)/A(0) approximately 0.9, showed larger phase shifts on the solid-like DPPC domains attributed to larger Young's modulus. However, contrast inversion was observed for A(sp)/A(0)<0.7, suggesting that at moderate and hard tapping the image contrast was dominated by the probe-sample contact area. Surprisingly, force modulation amplitude images showed larger stiffness for the liquid-like surfactin domains, suggesting that the contrast was dominated by contact area effects rather than by Young's modulus. These data emphasize the complex nature of the contrast mechanisms of dynamic AFM images recorded on mixed lipid monolayers.     

Molecular dynamics - potential of mean force calculations as a tool for understanding ion permeation and selectivity in narrow channels

Ion channels catalyze the permeation of charged molecules across cell membranes and are essential for many vital physiological functions, including nerve and muscle activity. To understand better the mechanisms underlying ion conduction and valence selectivity of narrow ion channels, we have employed free energy techniques to calculate the potential of mean force (PMF) for ion movement through the prototypical gramicidin A channel. Employing modern all-atom molecular dynamics (MD) force fields with umbrella sampling methods that incorporate one hundred 1-2 ns trajectories, we find that it is possible to achieve semi-quantitative agreement with experimental binding and conductance measurements. We also examine the sensitivity of the MD-PMF results to the choice of MD force field and compare PMFs for potassium, calcium and chloride ions to explore the basis for the valence selectivity of this narrow and uncharged ion channel. A large central barrier is observed for both anions and divalent ions, consistent with lack of experimental conductance. Neither anion or divalent cation is seen to be stabilized inside the channel relative to the bulk electrolyte and each leads to large disruptions to the protein and membrane structure when held deep inside the channel. Weak binding of calcium ions outside the channel corresponds to a free energy well that is too shallow to demonstrate channel blocking. Our findings emphasize the success of the MD-PMF approach and the sensitivity of ion energetics to the choice of biomolecular force field.     

Dynamic response of HPV16/anti-HPV16 pairs with unbinding events studied by atomic force microscopy

This paper proposes an effective approach to distinguish whether samples include Human Papilloma virus type-16 (HPV16) by Atomic force microscopy (AFM). AFM is an important instrument in nanobiotechnology field. At first we identified the HPV16 by Polymerase chain reaction (PCR) analysis and Western blotting from specimen of the HPV patient (E12) and the normal (C2), and then we used an AFM to observe the surface ultrastructure by tapping mode and to measure the unbinding force between HPV16 coupled to an AFM tip and anti-HPV16 L1 coated on the substrate surface by contact mode. The experimental results by tapping mode show that the size of a single HPV viron was similar to its SEM image from the previous literatures; moreover, based on the purposed methods and the analysis, two obvious findings that we can determine whether or not the subject is a HPV patient can be derived from the results; one is based on the distribution of unbinding forces, and the other is based on the distribution of the stiffness. Furthermore, the proposed method could be a useful technique for further investigating the potential role among subtypes of HPVs in the oncogenesis of human cervical cancer.