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The Evolution of Scanning Ion Conductance Microscopy
Abstract
This chapter recollects the historical evolution of the techniques that set the stage for the development of scanning ion conductance microscopy (SICM). We elaborate how techniques evolved that finally resulted in instruments that now allow researchers to obtain contact-free, three-dimensional images of the surface of living cells with a resolution in the range of a hundredth of a micrometer. The starting point for this as well as for other bioelectric techniques was the discovery of bioelectricity a little more than 200 years ago. After the introduction of the first galvanometers to detect bioelectrical signals in the nineteenth century, in the early decades of the twentieth century, extracellular techniques were developed to record electrocardiographic and electroencephalographic potential changes using chlorinated silver electrodes. These were miniaturized to allow for the detection of signals from individual cells in the middle of the twentieth century by the development of glass microelectrodes with sub-micrometer tip openings as well as appropriate current and voltage clamp amplifiers. The development of operational amplifiers based on transistors finally led to the detection of the tiny currents flowing through single transmembrane proteins. In the 1980s of the last century, scanning techniques were developed taking advantage of computer-guided piezo actuators to control the fine positioning of microelectrode tips on a scale smaller than the resolution of light microscopy. By combining amplifiers originally developed for electrophysiological recordings with nanoscale scanning techniques, it is now possible to quantitatively analyze the topography of the membranes of living cells with a resolution of down to 10 nm. Combinations with other microanalytical techniques, such as fluorescence microscopy, set the stage for future analysis of protein dynamics in moving membranes at unprecedented accuracy.
Scanning ion conductance microscopy (SICM) has been developing rapidly and has been a versatile tool for nanoscale imaging. However, the imaging quality and scanning speed of SICM have been significant restriction factors for its various applications. One of the main reasons is the mutual restriction of bandwidth and noise of the ion current feedback. In this paper, a low-noise two-stage transimpedance amplifier (TIA) with 500 kHz bandwidth was proposed and a low-capacitance pipette was combined to depress the high-frequency ion current noise of SICM's feedback circuit. The effect of pipette capacitance, which is a major cuase of high-frequency noise, was significantly reduced by covering a thin polydimethylsiloxane (PDMS) film on the outer wall of the pipette. Finite element method (FEM) was employed for the first time to simulate the pipette capacitance model. Impedance spectroscopy experiments verified that this method can greatly reduce the pipette capacitance (about 0.7 times). Through the comparative analysis of ion current noise, as well as the scanning experiments of standard silicon material sample and PMDS sample, it was demonstrated that the proposed method has a significant effect in reducing the high frequency noise of ion current measurement. Therefore, our method can provide an avenue to realize high-speed imaging with high positioning accuracy.
Platelets are small blood cells involved in hemostasis, wound healing, and immune response. After adhesion and spreading, platelets can migrate at sites of injury inducing an early immune response to inflammation or infection. Platelet migration requires fibrinogen-integrin binding and fibrinogen depletion from the substrate inducing a self-generated ligand gradient guiding the direction of migration. This type of cellular motion is referred to as haptotactic migration. The underlying mechanisms of haptotactic platelet migration have just recently been discovered, but the connection to platelet mechanics has remained unknown yet. Using scanning ion conductance microscopy (SICM), we investigated the three-dimensional morphology and mechanics of platelets during haptotactic migration for the first time. Migrating platelets showed a polarized, anisotropic shape oriented in the direction of migration. This polarization goes hand in hand with a characteristic subcellular stiffness distribution showing a region of increased stiffness at the leading edge. Moreover, the mechanical properties of the leading edge revealed a highly dynamic stiffening and softening process with rapid changes of the elastic modulus by a factor of up to 5× per minute. Inhibition of actin polymerization stopped the dynamic stiffening and softening process and halted the migration. By combining SICM with confocal fluorescence microscopy, we found that the increased stiffness and mechanical dynamics at the leading edge coincided with an increased volumetric F-actin density. Our data provide a connection between platelet mechanics and the cytoskeletal contribution to the migration process of platelets.
High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution. Methods for imaging the 3D cell surface often require physical interaction. Here the authors report the combination of scanning ion conductance microscopy (SICM) and live-cell super-resolution optical fluctuation imaging (SOFI) for the non-invasive topographical imaging of soft biological samples.
The scanning tunneling microscope is proposed as a method to measure forces as small as 10-18 N. As one application for this concept, we introduce a new type of microscope capable of investigating surfaces of insulators on an atomic scale. The atomic force microscope is a combination of the principles of the scanning tunneling microscope and the stylus profilometer. It incorporates a probe that does not damage the surface. Our preliminary results in air demonstrate a lateral resolution of 30 ÅA and a vertical resolution less than 1 Å.
Mapping the mechanical properties of living cells with high spatial and temporal resolution is important for the exploration of cell function. Widely used imaging techniques such as the atomic force microscope are generally based on direct mechanical contact between the probe and the cell, thereby involving the risk of damaging the cell. Here, we present a noncontact method for fast and quantitative stiffness mapping of living cells with sub-micrometer lateral resolution. This was achieved by repeatedly moving a pressurized nanopipette toward and away from the sample in a scanning ion conductance microscope (SICM). The pressure-induced microfluidic flow through the nanopipette produced a time-varying force on the sample surface, thereby locally indenting it without direct mechanical contact. Maps of sample stiffness (quantified by the Young's modulus) were then determined from ion current approach curves using a finite element model. To demonstrate the capability of the method we visualized the dynamics of individual cytoskeleton fibers in living cells over several hours. Additionally, we found that spreading extensions of migrating fibroblast cells tend to be softer than their lamellum, which is consistent with a mechanism of cell migration by osmotic swelling.
The migration of oligodendrocyte progenitor cells (OPCs) to the white matter is an indispensable requirement for an intact brain function. The mechanism of cell migration in general is not yet completely understood. Nevertheless, evidence is accumulating that besides the coordinated rearrangement of the cytoskeleton, a finetuned interplay of ion and water fluxes across the cell membrane is essential for cell migration. One part of a general hypothesis is that a local volume increase towards the direction of movement triggers a mechano-activated calcium influx that regulates various procedures at the rear end of a migrating cell. Here, we investigated cell volume changes of migrating OPCs using scanning ion conductance microscopy. We found that during accelerated migration OPCs undergo an increase in the frontal cell body volume. These findings are supplemented with time lapse calcium imaging data that hint an increase in calcium content the frontal part of the cell soma.
During the 1790s, Alexander von Humboldt (1769-1859), who showed an early interest in many facets of natural philosophy and natural history, delved into the controversial subject of galvanism and animal electricity, hoping to shed light on the basic nature of the nerve force. He was motivated by his broad worldview, the experiments of Luigi Galvani, who favored animal electricity in more than a few specialized fishes, and the thinking of Alessandro Volta, who accepted specialized fish electricity but was not willing to generalize to other animals, thinking Galvani's frog experiments flawed by his use of metals. Differing from many German Naturphilosophen, who shunned "violent" experiments, the newest instruments, and detailed measurement, Humboldt conducted thousands of galvanic experiments on animals and animal parts, as well as many on his own body, some of which caused him great pain. He interpreted his results as supporting some but not all of the claims made by both Galvani and Volta. Notably, because of certain negative findings and phenomenological differences, he remained skeptical about the intrinsic animal force being qualitatively identical to true electricity. Hence, he referred to a "galvanic force," not animal electricity, in his letters and publications, a theoretical position he would abandon with Volta's help early in the new century.
Scanning ion conductance microscopy (SICM) is an emerging tool for the noncontact investigation of biological samples such as live cells. It uses an ion current through the opening of a tapered nanopipette filled with an electrolyte for topography measurements. Despite its successful application to numerous systems no systematic investigation of the image formation process has yet been performed. Here, we use finite element modeling to investigate how the scanning ion conductance microscope images small particles on a planar surface, providing a fundamental characterization of the imaging process. We find that a small particle appears with a height that is only a fraction of its actual height. This has significant consequences for the quantitative interpretation of SICM images. Furthermore, small and low particles are imaged as rings in certain cases. This can cause small, closely spaced particles to appear with a lateral orientation that is rotated by 90°. Considering both real space and spatial frequency space we find that a reasonable and useful definition of lateral resolution of SICM is the smallest distance at which two small particles can clearly be resolved from each other in an image. We find that this resolution is approximately equal to three times the inner radius of the pipette tip opening.
Surface microscopy using vacuum tunneling is demonstrated for the
first time. Topographic pictures of surfaces on an atomic scale have
been obtained. Examples of resolved monoatomic steps and surface
reconstructions are shown for (110) surfaces of CaIrSn4 and Au.
The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits
create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter
is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled
cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center
of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to
overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.
We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions.
Luigi Galvani became one of the greatest scientists of the 18th century with his research and the development of his theory on animal electricity. His work was appreciated by many scientists. Nevertheless, it gave rise to one of the most passionate scientific debates in history when Alessandro Volta postulated that Galvani had confused intrinsic animal electricity with small currents produced by metals. This debate would result in the creation of electrophysiology, electromagnetism, electrochemistry and the electrical battery. Galvani responded to each of the postulated theories of Volta giving irrefutable proof of the involvement of electricity in the contraction of muscles. However, his work was subsequently abandoned and silenced for many years but his ideas and theories were finally confirmed by the creation of new instruments and the interest of new scientists who helped position Galvani as the father of electrophysiology.
Correlation microscopy combining fluorescence and scanning probe or electron microscopy is limited to fixed samples due to the sample preparation and non-physiological imaging conditions required by most probe or electron microscopy techniques. Among the few scanning probe techniques that allow imaging of living cells under physiological conditions, Scanning Ion Conductance Microscopy (SICM) has been shown to be the technique which minimizes the impact on the investigated sample. However, combinations of SICM and fluorescence microscopy suffered from the mismatch in resolution due to the limited resolution of conventional light microscopy. In the last years, the diffraction limit of light microscopy has been circumvented by various techniques, one of which is Stimulated Emission Depletion (STED) microscopy. Here, we aimed at demonstrating the combination of STED and a SICM. We show that both methods allow to record living cellular specimen and provide a SICM and STED image of the same sample, which allowed us to correlate the membrane surface topography and the distribution of the cytoskeletal protein actin. Our proof-of-concept study exemplifies the benefit of correlating SICM with a sub-diffraction fluorescence method and might form the basis for the development of a combined instrument which would allow the simultaneous recording of sub-diffraction fluorescence and topography information.
The scanning ion conductance microscope (SICM) is a powerful tool for imaging the topography of soft samples in an aqueous environment. Despite the rising popularity of the SICM, the image formation process and the fundamental limit of the lateral resolution are still a matter of debate. Using microfabricated samples, we investigated the imaging of small cylindrical particles, elongated objects, and topography steps and present the first direct comparison of numerical and experimental data. For the lateral resolution we considered two alternative definitions: the distance, at which two small particles can clearly be resolved from each other in an image, and the apparent full width at half maximum of small particles. For both definitions, we found a lateral resolution of about three times the inner opening radius of the pipet. We further validated this resolution limit in measurements on supported lipid bilayers and a polycarbonate sample using pipets with opening radii down to 8 nm.
Nanopipettes are emerging as simple but powerful tools for probing chemistry at the nanoscale. In this contribution the use of nanopipettes for simultaneous surface charge mapping and topographical imaging is demonstrated, using a scanning ion conductance microscopy (SICM) format. When a nanopipette is positioned close to a surface in electrolyte solution the direct ion current (DC), driven by an applied bias between a quasi-reference counter electrode (QRCE) in the nanopipette and a second QRCE in the bulk solution is sensitive to surface charge. The charge sensitivity arises because the diffuse double layers at the nanopipette and the surface interact, creating a perm-selective region which becomes increasingly significant at low ionic strengths (10 mM 1:1 aqueous electrolyte herein). This leads to a polarity-dependent ion current and surface-induced rectification as the bias is varied. Using distance-modulated SICM, which induces an alternating ion current component (AC) by periodically modulating the distance between the nanopipette and the surface, the effect of surface charge on the DC and AC is explored and rationalized. The impact of surface charge on the AC phase (with respect to the driving sinusoidal signal) is highlighted in particular; this quantity shows a shift that is highly sensitive to interfacial charge and provides the basis for visualizing charge simultaneously with topography. The studies herein highlight the use of nanopipettes for functional imaging with applications from cell biology to materials characterization where understanding surface charge is of key importance. They also provide a framework for the design of SICM experiments, which may be convoluted by topographical and surface charge effects, especially for small nanopipettes.
The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.
This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant nerve fibre (Hodgkinet al., 1952,J. Physiol.116, 424–448; Hodgkin and Huxley, 1952,J. Physiol.116, 449–566). Its general object is to discuss the results of the preceding papers (Section 1), to put them into mathematical form (Section 2) and to show that they will account for conduction and excitation in quantitative terms (Sections 3–6).
Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes.
Knowledge of mechanical properties of living cells is essential to understand their physiological and pathological conditions. To measure local cellular elasticity, scanning probe techniques have been increasingly employed. In particular, non-contact scanning ion conductance microscopy (SICM) has been used for this purpose; thanks to the application of a hydrostatic pressure via the SICM pipette. However, the measurement of sample deformations induced by weak pressures at a short distance has not yet been carried out. A direct quantification of the applied pressure has not been also achieved up to now. These two issues are highly relevant, especially when one addresses the investigation of thin cell regions. In this paper, we present an approach to solve these problems based on the use of a setup integrating SICM, atomic force microscopy, and optical microscopy. In particular, we describe how we can directly image the pipette aperture in situ. Additionally, we can measure the force induced by a constant hydrostatic pressure applied via the pipette over the entire probe-sample distance range from a remote point to contact. Then, we demonstrate that the sample deformation induced by an external pressure applied to the pipette can be indirectly and reliably evaluated from the analysis of the current-displacement curves. This method allows us to measure the linear relationship between indentation and applied pressure on uniformly deformable elastomers of known Young's modulus. Finally, we apply the method to murine fibroblasts and we show that it is sensitive to local and temporally induced variations of the cell surface elasticity.
Surface microscopy using vacuum tunneling is demonstrated for the first time. Topographic pictures of surfaces on an atomic scale have been obtained. Examples of resolved monoatomic steps and surface reconstructions are shown for (110) surfaces of and Au.
In 1964, Giuseppe Moruzzi (1910–1986), a prominent neurophysiologist of worldwide fame with a scholarly interest in the history of science, published an extensive and insightful analysis of the work and personality of Carlo Matteucci, a 19th century Professor of Physics at the University of Pisa who had worked on “animal electricity” in the great tradition of Galvani. By discovering fundamental phenomena such as the demarcation potential and action current in muscle and nerve, Matteucci paved the way for the development of modern electrophysiology. Being written in Italian and having appeared in a not easily available journal of history of science, Moruzzi's essay has been lost to most members of the international neuroscience community. An English translation of it is published here with two aims: first, to make known to a large audience of neuroscientists a fascinating account of the origins of electrophysiology as seen through a perceptive interpretation of the successes and failures of a brilliant scientist and passionate man; and second, to pay tribute to the memory of Giuseppe Moruzzi and his everlasting contributions to learning and science on the occasion of the 10th anniversary of his death.
Do not touch! The surface of a living cell is soft and responsive and therefore high-resolution imaging of the cell membrane has not been possible to date. Now noncontact imaging of protein complexes in the plasma membrane of living cells has been demonstrated (see picture) and has been used to follow the cells' structural reorganization. This breakthrough opens up a wealth of new experiments in membrane and cell biology.
THE ionic channel associated with the acetylcholine (ACh) receptor at
the neuromuscular junction of skeletal muscle fibres is probably the
best described channel in biological membranes. Nevertheless, the
properties of individual channels are still unknown, as previous studies
were concerned with average population properties. Macroscopic
conductance fluctuations occurring in the presence of ACh were analysed
to provide estimates for single channel conductance and mean open
times1-3. The values obtained, however, depended on
assumptions about the shape of the elementary conductance
contribution-for example, that the elementary contribution is a square
pulse-like event2. Clearly, it would be of great interest to
refine techniques of conductance measurement in order to resolve
discrete changes in conductance which are expected to occur when single
channels open or close. This has not been possible so far because of
excessive extraneous background noise. We report on a more sensitive
method of conductance measurement, which, in appropriate conditions,
reveals discrete changes in conductance that show many of the features
that have been postulated for single ionic channels.
Scanning ion conductance microscopy (SICM) was used to interrogate ion currents emanating from nanometer-scale pores of a polymer membrane. The transport activity of individual pores was measured by examining ion current images and corresponding topographic images recorded simultaneously. Localized ion currents over individual nanopores were generated by introducing a concentration difference between the upper and lower chambers of a diffusion cell. To better estimate these localized ion currents, Goldman-Hodgkin-Katz (GHK) theory was used to model ion current through a permeable membrane under gradients of both concentration and applied potential. Experimental ion current profiles over a single pore fit well with theoretical plots calculated from the GHK model. On the basis of this analysis, nanoscale transport properties can be measured with SICM.
Observation of filamentous actin (F-actin) in living cells is currently limited to the resolution of the light microscope.
Higher resolution procedures require sample fixation and preclude dynamic studies. The atomic force microscope (AFM) can image
and manipulate samples at very high, sometimes atomic resolution by scanning a fine tip over the surface of interest and detecting
physical interactions between the tip and sample. This study demonstrates that F-actin can be readily resolved in living cells
with the AFM and that the dynamic properties of F-actin are easily observed.
A scanning ion-conductance microscope (SICM) has been developed that can image the topography of nonconducting surfaces that
are covered with electrolytes. The probe of the SICM is an electrolyte-filled micropipette. The flow of ions through the opening
of the pipette is blocked at short distances between the probe and the surface, thus, limiting the ion conductance. A feedback
mechanism can be used to maintain a given conductance and in turn determine the distance to the surface. The SICM can also
sample and image the local ion currents above the surfaces. To illustrate its potential for imaging ion currents through channels
in membranes, a topographic image of a membrane filter with 0.80-micrometer pores and an image of the ion currents flowing
through such pores are presented.
The atomic force microscope (AFM) can be used to image the surface of both conductors and nonconductors even if they are covered
with water or aqueous solutions. An AFM was used that combines microfabricated cantilevers with a previously described optical
lever system to monitor deflection. Images of mica demonstrate that atomic resolution is possible on rigid materials, thus
opening the possibility of atomic-scale corrosion experiments on nonconductors. Images of polyalanine, an amino acid polymer,
show the potential of the AFM for revealing the structure of molecules important in biology and medicine. Finally, a series
of ten images of the polymerization of fibrin, the basic component of blood clots, illustrate the potential of the AFM for
revealing subtle details of biological processes as they occur in real time.
We have constructed a combined TappingMode atomic force microscope and scanning ion conductance microscope. The design is based on a bent glass pipette that acts as both the force sensor and conductance probe. Measuring the pipette deflection allows more stable feedback than possible with previous versions of the scanning ion conductance microscope. Using this microscope, we have imaged synthetic membranes in both contact and tapping modes under fluid. Although contact mode operation is possible, we found that our microscope provided higher contrast and less apparent sample damage in the topographic and ionic conductance images in the tapping mode.
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.
Luigi Galvani and his famous experiments on frogs carried out in the second half of the 18th century belong more to legend than to the history of science. Galvani not only laid the foundations of a new science, electrophysiology, but also opened the way for the invention of the electric battery, and thus for the development of the physical investigations of electricity. However, in spite of the widespread celebration of his work, Galvani's scientific endeavours have been largely misrepresented in the history of science. The scholar of Bologna has a stereotyped image as an 'occasional' scientist, who started his studies by chance, largely ignored the scientific theories of his time and wandered aimlessly in mental elaborations until the physicist of Pavia, Alessandro Volta, entered the field, correctly interpreted Galvani's results and eventually developed the electric battery. With the present understanding of electrical phenomena in excitable membranes, it is now time to reconsider the real matter raised by Galvani's discoveries and by his hypothesis of an intrinsic 'animal electricity', and to make a clearer evaluation of a revolutionary phase of scientific progress.
A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.
Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried‐coated, wet‐fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde‐fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented.
AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip–sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high‐resolution real‐time studies of dynamic changes in submembranous structures.
The spatial distribution of ion channels in the cell plasma membrane has an important role in governing regional specialization, providing a precise and localized control over cell function. We report here a novel technique based on scanning ion conductance microscopy that allows, for the first time, mapping of single active ion channels in intact cell plasma membranes. We have mapped the distribution of ATP-regulated K+ channels (KATP channels) in cardiac myocytes. The channels are organized in small groups and anchored in the Z-grooves of the sarcolemma. The distinct pattern of distribution of these channels may have important functional implications.
We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.
Scanning ion conductance microscopy (SICM) takes advantage of the increase in the resistance which occurs if a glass microelectrode is closely approached to a poorly conducting membrane (Science 243 (1989) 641) and has been shown to be a promising technique to study membranes of living cells (Biophys J 73 (1997a) 653; J Microsc 188 (1997b) 17). Based on a newly designed set-up on top of an inverted light microscope in combination with a speed optimized low noise intracellular amplifier, a novel mode for control of the distance between the probe and surface has been developed. By application of current pulses, the change in the resistance is monitored independently from electrode drift and parasitic DC currents. We demonstrate the applicability by showing first high-resolution images of neural cells produced with the pulse-mode operated SICM.