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Michael AC, Borland LM, editors. Electrochemical Methods for Neuroscience. Boca Raton (FL): CRC Press/Taylor & Francis; 2007.

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Electrochemical Methods for Neuroscience.

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Chapter 17Scanning Electrochemical Microscopy as a Tool in Neuroscience

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Introduction

During evolution, humans and other vertebrates developed a dense network of highly specialized secretory cells, which were eventually fine-tuned to effectively synthesize, store, and release small, diffusible chemical messenger molecules. These transmitters work for intercellular communication and the associated control of physical and cognitive activities such as fine motor coordination, body movement and balance, sensing, thinking, learning, and establishing memory.

In fact, chemical signal transduction all through the body is governed by a vast number of neurotransmitter-releasing nerve cells or neurons from the central and peripheral nervous system including the brain and spinal cord and many hormone-releasing secretory cells inside the glands of the endocrine system, all acting together in a well-synchronized fashion. With some exceptions such as the gaseous messenger nitric oxide (NO), neurotransmitters from nerve cell terminals at synapses and hormones from neuroendocrine cells are released via the exocytosis of bubble-like membranous storage vesicles that are formed inside the cytoplasm by the Golgi apparatus.

As part of their life cycle, the initially empty vesicles fill with a transmitter that is characteristic for a given type of cells and later move and dock via firm membrane protein interactions to the inner side of the plasma membrane. The resulting complex between the two membranes travels through a sequence of maturation steps before, in rapid response to a sudden raise in the intracellular Ca2+ concentration as the stimulus, full-membrane fusion and exocytosis occur. Initially, only a narrow aqueous channel or “fusion pore” forms that connects the interior of a fused vesicle with the outside of the cell. However, only fractions of seconds later the fusion pore usually expands and the vesicle collapses, resulting in the complete release of its transmitter contents into the extracellular fluid. Once released, neurotransmitters move through the narrow synaptic cleft to reach and interact rapidly with receptors on the nearby postsynaptic membrane, whereas hormones usually are brought to more distant target cells using the blood stream as a carrier.

Knowledge about the mechanisms and molecular dynamics of chemical cell-to-cell signaling in particular, about the process of vesicular transmitter release and the modulation of exocytosis by endogenous and exogenous factors, not only is fundamental to unveiling the complexity of brain function and motivated behavior, but also to understanding the onset and progression of neuro-degenerative diseases such as Alzheimer’s disease and Parkinson’s syndrome.

Fortunately, many isolated and cultured neuronal and endocrine cells keep their ability to transduce and convey the relevant biochemical and -physical signals and to release their secretory products in response to proper stimulation. Accordingly, individual secretory cells became an excellent scaled-down model system for exploring the process of neurotransmitter and hormone release in controlled manner and steering clear of the difficulties of in-vivo measurements with sensors implanted e.g., directly in brain tissue. Living cells, on the other hand, are tiny objects and their responses to signal transduction and metabolism are diminutive and often occur rapidly. Sophisticated methodologies with an adequate sensitivity, selectivity, and an excellent spatio-temporal resolution are needed to observe neurochemical events on the level of single cells.

Scientists have developed innovative optical assays combining confocal, multi-photon or evanescent-wave microscopy with the application of fluorescence-labeled cellular constituents for looking inside cells and imaging the function of their molecular machinery, in particular, the kinetics and directions of protein dynamics [1–3]. Structural information against which fluorescent images can be interpreted typically comes from high-resolution scanning electron microscopy [4]. The patch-clamp technique Invented by Neher and Sakmann for detecting tiny currents flowing through single ion channels in cell membranes, matured into the most prominent and powerful tool in single-cell electrophysiology [5]. The capability to look at single ion channel activity led to major advancements in the conception of cell excitability and knowledge about the dependency of the function of ion channels on specific metabolic or pharmacological factors. Moreover, patch-clamp capacitance measurements proved to be an excellent approach for monitoring in real time the fusion of intracellular secretory vesicles with the plasma membrane, which is important for instance when studying exocytosis [6–8].

Common secretory products of nerve and endocrine cells are catecholamine and indolamine derivatives (e.g. dopamine, epinephrine, norepinephrine, and serotonin), histamine, insulin, and NO. These compounds can be oxidized at a suitable electrode surface at an appropriate applied potential and thus they are electrochemically detectable. As early as in the 1970s, R. Adams and coworkers established through their pioneering work, voltammetry at implanted microelectrodes. The result was a powerful electroanalytical approach for in vivo measurements of bulk catechol-amine release in animal brains [9–11]. Carbon microelectrodes showed improved characteristics over noble metal electrodes for neurochemical studies due to their lower residual background currents and their better resistance against electrode fouling causing improved long-term sensitivity.

Advancements in instrumentation such as the development of low-noise electrochemical amplifiers with current sensitivities in the low pA range and improved micropositioning systems became available. The development of disk-shaped carbon-fiber microelectrodes (CFMEs) with overall tip diameters in the μm range and millisecond response times allowed then to proceed to localized voltammetry at single cells. Scientists obtained the first successful electrochemical single-cell secretion measurements on chromaffin cells from the adrenal medulla by carefully placing the tip of properly polarized disk-shaped CFMEs closest as possible to the cell membrane and monitoring the amperometric tip current as a function of time [12,13].

Stimulation of approached chromaffin cells resulted in the appearance of a series of anodic current spikes each of them related to the oxidation of catecholamine molecules leaking out of a single secretory vesicle that underwent exocytosis. Motivated by the capability to watch the rapid process of single-vesicle exocytosis with an exquisite sensitivity in real time, researchers have used carbon-fiber amperometry to study single-cell transmitter release from a number of different neuronal and neuroendocrine cells [14–24]. The analysis of individual current transients in ampero-metric recordings (e.g., the rise time, amplitude, charge, and half width) provides the number of transmitter molecules released per vesicle and features of the time course of release, both valuable information for elucidating the mechanism and kinetics of exocytosis. If not known, the chemical nature of the released transmitter and its concentration can be assessed by operating the CFME next to a secretory cell in the fast-scan cyclic voltammetry instead of the amperometry mode. A number of recently published comprehensive review articles [25–33] contain detailed information about the analysis, advantages, and limitations of constant-potential carbon-fiber amperometry and fast-scan cyclic voltammetry for the detection of chemical secretion from single cells as well as selected applications.

As expected, scientists have applied different variants of high-resolution scanning probe microscopes as biological imaging, sensing, or manipulation devices. In addition, they have opened new opportunities for probing the properties of individual cells under near-physiological conditions. Atomic force microscopy (AFM) found, for instance, not only widespread application for topographical imaging of cellular structures with up to nanometer lateral resolution [34,35] but also for evaluating adhesion forces between biological (macro) molecules and cell membranes [36] or the elasticity of cells [37]. The fine, nanometer-sized tip apertures of pulled fiber-optic wave guides operated as optical scanning probes and the special properties of near-field illumination made scanning near-field optical microscopy (SNOM) a promising technique for resolving cell structures and molecular dynamics well beyond the resolution of conventional light microscopes [38–40]. Even though offering a little lower spatial resolution than SNOM and AFM, scanning ion conductance microscopy (SICM) [41–43] became another attractive tool for imaging cell topography essentially because of its potential for functional mapping of cell volume changes and movements [44] and of ion fluxes through channels in the membrane surface of living cells.

In the late 1980s, the development of scanning electrochemical microscopy (SECM) brought the small tips of amperometric ultramicroelectrodes into play as electrochemical scanning probes (SECM tips) [45,46]. Scanning electrochemical microscopy imaging involves monitoring the current of SECM tips that are scanned close across a surface as a function of lateral tip position. In contrast to other scanning probe techniques, SECM is not only able to reveal the topography of samples in solution, but it may also detect and visualize local variations in the (electro) chemical reactivity of the investigated sample. Originally, researchers developed SECM merely for applications in surface science; however, the potential of the method for biological applications was recognized soon. Scanning electrochemical microscopy-based bioelectroanalysis developed into a powerful approach for imaging chemical gradients in the diffusion layer surrounding cells and tissue [47,48], the catalytic activity of immobilized enzymes [49,50] or antibodies [51]. Oxygen permeability of cartilage [52], photosynthetic electron transport in guard cells [53], cell respiration [54], calcium release during bone resorption [55,56], hybridization events on DNA microarrays [57,58] or differences in redox activities of cancer cells [59] are further biological phenomena that were investigated at high spatial resolution by SECM.

Scientists have proposed potentiometric SECM with ion-selective microelectrodes as scanning probes aimed at the determination of the local activity of a number of ions including protons, chloride, sodium, potassium, and calcium [60–65]. All of these ions are of physiological importance and, although not shown to date, assessing their local concentrations in vicinity of individual living cells with scanned H+ -, Na+ -, K+ -, Ca2+ - or Cl -selective tips could be attractive. However, this review focuses on the latest efforts in developing amperometric SECM into a high-resolution electrochemical imaging tool applicable for research and development in the field of neuroscience, with special emphasis given to the local amperometric detection of chemical secretion from single secretory cells. In order to help understand the method, this chapter will first introduce the relevant features of the amperometric microelectrodes and the basic operational principles and technical concept of SECM including the amperometric feedback and the generator collector mode of imaging.

Taking into account the fragile nature of soft living cells, this chapter will discuss the limitations of the ordinary constant-height mode of SECM and present approaches for establishing a constant-distance mode. Only by using SECM in constant-distance mode, the SECM tip is forced to carefully follow the contours of the cell throughout scanning to avoid damaging the tip and the investigated biological sample. Finally, this section will present selected examples of single-cell SECM studies, which highlight the potential impact of this modern electrochemical assay in neurochemistry.

Operational Principles of the Amperometric Feedback and the Generator/Collector Mode of SECM

The key component of SECM in the amperometric feedback and the generator/collector mode is the tip of a mobile disk-shaped carbon or noble metal microelectrode, which is connected to a low-noise potentiostat for sensitive current measurements and made accurately movable through the attachment to computer-controlled high-precision x -, y -, and z -micropositioning devices. Accompanied by fixed reference and counter electrodes, the SECM tip completes an electrochemical cell that is filled with supporting electrolyte containing as well a redox active compound (the “mediator”). Applying, for example, reducible redox mediators such as [Ru(NH3) [6]]3+, [Fe(CN)6] 3 − or even dissolved oxygen and tip potentials notably more negative than the standard potential of the chosen redox couple, a diffusion limited cathodic tip current is generated and used as specific signal for SECM imaging.

With the SECM tip located in the bulk of the electrolyte far above the sample surface, hemi-spherical diffusion of the mediator toward the electroactive disk of the microelectrode is controlling the tip current and a steady-state diffusion-limited current value is observed in cyclic voltammograms (Figure 17.1) or in constant-potential amperometry.

FIGURE 17.1. Voltammetric behavior of disk-shaped microelectrodes (scanning electrochemical microscopy (SECM) tips).

FIGURE 17.1

Voltammetric behavior of disk-shaped microelectrodes (scanning electrochemical microscopy (SECM) tips). Cyclic voltammetry at disk-shaped carbon or noble metal disk-shaped microelectrodes in solutions containing a supporting electrolyte and an e.g., reducible (more...)

However, when one maneuvers the SECM tip closer to a sample, the vicinity and nature of the approached surface will actually start to affect the tip response. For instance, electrochemical recycling of tip-consumed mediator molecules can take place at a neighboring electrochemically active surface. A considerable increase in tip current ( I > Ilim, positive feedback, Figure 17.2a) is observed if the kinetics of the electron-transfer reaction at the sample is faster than the diffusion of the redox mediator in the narrow gap between tip electrode and surface. In contrast, proximity to an electrochemically inactive surface physically obstructs diffusion of species toward the tip leading to a decrease in tip current ( I < Ilim, negative feedback, Figure 17.2b).

FIGURE 17.2. Z-approach curves showing the positive and negative feedback above electrochemically active and passive surfaces.

FIGURE 17.2

Z-approach curves showing the positive and negative feedback above electrochemically active and passive surfaces. In close proximity to a conducting surface, tip-consumed redox species can be recycled to its original oxidation state. As the tip-to-sample (more...)

Positive and negative feedback effects appear gradually with decreasing tip-to-sample distance, d, however, the associated up- or downward deflections in typical approach curves (I / Ilim vs. d / r ; r = radius of the active disk of the microelectrode) become steeper the closer the tip gets to the sample. For imaging in the feedback mode, approach curves have to be recorded and used to place the SECM tip at an appropriate working distance within the “electrochemical” near field, which starts at distances of about two times the tip electrode diameter. Image acquisition is then achieved by moving the SECM tip laterally at predetermined constant z -height along the sample surface and simultaneously monitoring the tip current as a function of x and y location.

As schematically shown in Figure 17.3, increases or decreases in the tip current obtained in line scans at homogenously insulating or conducting surfaces can be understand as up and down in surface topography while current variations gained at flat surfaces with neighboring conductive and insulating regions represent the local changes in electrochemical activity of the sample surface. In samples with variations in both interfacial electrochemical activity and changes in surface topography, the tip response reflects a convolution of contributions from alterations in electro-chemical activity and topography, and, in fact, additional information about the morphology of the specimen is obligatory for an interpretation of the constant-height SECM images.

FIGURE 17.3. Principles of constant-height scanning electrochemical microscopy (SECM) feedback imaging.

FIGURE 17.3

Principles of constant-height scanning electrochemical microscopy (SECM) feedback imaging. In the feedback mode of operation, SECM imaging takes advantage of an amperometric tip current that originates from the redox conversion of a redox mediator, which (more...)

One can effectively detect and visualize the release of redox-active species liberated from localized spots on the surface via a properly positioned SECM tip capable of oxidizing or reducing the molecules that are ejected from the active site. Clearly, this strategy, the so-called substrate generator/tip collector mode of SECM is not only appealing when aiming on the local detection of pitting corrosion, diffusion through micropores of semi permeable polymer membranes or the confined activity of enzyme microstructures, but is also ideal for spotting the dynamic chemical release of biologically important species from single secretory cells (see Figure 17.4).

FIGURE 17.4. Generation/collection mode of scanning electrochemical microscopy (SECM).

FIGURE 17.4

Generation/collection mode of scanning electrochemical microscopy (SECM). A tip current originates from an electrochemical-induced conversion of a redox species that is generated at locally confined small release sites at the sample surface. Typical examples (more...)

Traditionally, SECM imaging is carried out with the tip scanned at constant height above the sample surface. However, due to the convolution of topography and electrochemical activity pointed out before constant-height mode SECM has significant drawbacks when applied for the investigation of samples with three-dimensional objects on the surfaces or a high relief as compared to the radii of the applied SECM tips. Tip crash and tip/sample damage may occur in case the tip is positioned in too close proximity to the surface. The following sections will discuss constant-height mode SECM measurements as well as constant-distance mode measurements at individual cells.

Constant-Height Mode SECM Measurement at Individual Cells

For electrochemical single-cell measurements, the SECM or any positioning unit that allows for the positioning of a microelectrode in close proximity to a single cell has to be placed on the stage of an inverted optical microscope in order to combine an independent inspection of the biological object together and the microelectrode tip. In addition, by this pre-positioning the tip relative to the object becomes possible. For an efficient detection of chemical release from a secretory cell the tip of the voltammetric microsensor has to be close to the release sites in the cell membrane. This is especially true for vesicle exocytosis events with a very limited amount of neurotransmitter molecules being released to the extracellular medium within a very short time.

Early theoretical calculations revealed that a separation between the collecting electrode and the exocytosis site of larger than about 1 μm already lead to a significant diffusional loss of the released compounds due to their escape from the gap between the disk-shaped sensor and the cell membrane [66]. Also in the case of stimulated release of NO, which diffuses through the cell membrane for longer times after intracellular formation the exact knowledge about the distance between the detecting sensor tip and the releasing cell is indispensable for a quantitative treatment of the obtained results.

Interestingly, scientists introduced rather early the detection of single vesicle exocytosis using a manual positioning of a carbon fiber disk electrode in close proximity to the surface of an individual cell followed by concepts as developed for patch-clamp measurements. In these “conventional” amperometric [37,67–73] or fast-scan cyclic voltammetry [74–76] measurements of single-vesicle exocytosis, the tip of a disk-shaped CFME is carefully moved with manually operated micromanipulators to a cell of choice until a small deformation of the cell can be seen in the optical microscope invoked by gently pressing the microelectrode against the cell’s membrane. The microelectrode is then retracted a little to ensure proximity and thus good collection efficiency. This approach heavily stimulated research in brain physiology and understanding of cell-to-cell communication and made accurately positioned microelectrodes an important tool in brain research. However, one potential drawback of this approach was seen in the direct physical contact between the microelectrode tip and the cell membrane, which may lead to a (local) damage of the cell and/or a contamination of the carbon microdisk electrode with cell components. In addition, some compounds are released already upon application of a shear stress to the cell and hence the conventional manual positioning may already lead to shear stress stimulated exocytosis during the microelectrode positioning.

Based on the above, a major question, which had to be solved, was the accurate positioning of a microdisk electrode in known and extremely close proximity to an individual cell without touching the cell. As shown above, with the feedback mode of SECM the positioning of the microelectrode tip relative to the sample surface is supported by the distance-dependent current response of the amperometric SECM tip in combination with a computer-controlled positioning. Because living cells as well as the glass cover slips carrying them are behaving as electrochemically inactive surface, only the negative feedback mode of SECM is relevant for cell measurements. The effective working distance for imaging in the negative feedback mode is about the diameter of the electro-active disk of the SECM tip [77,78] with some dependence from the ratio between the diameter of the active electrode disk and the insulating glass sheath (Figure 17.5). As a consequence, the smaller the electroactive disk electrode of the SECM tip the smaller is the working distance and the more difficult are constant height scans over surfaces with pronounced three-dimensional topography.

FIGURE 17.5. Negative feedback approach curves in dependence from the active diameter of the scanning electrochemical microscopy (SECM) tip.

FIGURE 17.5

Negative feedback approach curves in dependence from the active diameter of the scanning electrochemical microscopy (SECM) tip. The working distance (i) is significantly decreased with decreasing electrode size. If the changes in topography are in the (more...)

Topographical imaging of adherently growing living cells can be achieved using feedback mode SECM using a free-diffusing redox-active compound which is converted at the SECM tip in a diffusion controlled manner at an appropriate electrode potential. In fact, if the SECM approach curve using negative feedback mode is performed at a x, y -location without a cell, the tip has to be retracted to a distance, which is at least as big as the expected three-dimensional topography of the sample. If the electrode is very small, one may even loose the feedback related nearfield distance established previously over the glass cover slip. Two-dimensional scans at this predetermined constant height will lead to enhanced negative feedback at areas where the tip is moving across cells and thus cells become visualized as areas of lower currents in plots of the electrochemical tip response vs. x, y tip position (Figure 17.6).

FIGURE 17.6. Tip movements from the bulk (a) toward the glass cover slip (b) allows to record the feedback approach curve which represents a measure for the absolute distance once the electrode diameter and the ratio between the diameter of the electrode and the insulating glass sheath is known.

FIGURE 17.6

Tip movements from the bulk (a) toward the glass cover slip (b) allows to record the feedback approach curve which represents a measure for the absolute distance once the electrode diameter and the ratio between the diameter of the electrode and the insulating (more...)

This procedure is useful for imaging the morphology of individual cells, however, potentially overlaid with information about cell metabolism. Researchers have successfully used a set of membrane-impermeable, hydrophilic, and membrane-permeable, hydrophobic redox mediators along with specific scanning protocols to probe and image the redox activity of individual metastatic and non-metastatic human breast cells [79]. By means of biocompatibility tests, a collection of redox active compounds was selected which should be most suitable for biological SECM experiments on cultured cells [80]. The best mediators were employed in SECM experiments on cultured PC12 pheochromocytoma cells, a model cell type for studying neuronal development and function. One could visualize the cell bodies and branching neurites of differentiated PC12 cells by means of constant height feedback SECM and changes in cell height induced by an exposure of PC12 cells to hypotonic or hypertonic solutions were detectable in real time.

Scientist exploited SECM in the constant-height mode for the local detection of variations in the O2 partial pressure at single living cells in the late 1990s of the last century [54,81–83]. A disk-shaped Pt microelectrode was kept at a constant potential sufficiently cathodic to drive diffusion-limited reduction of dissolved O2 and scanned in a horizontal plane over an individual cell or a population of adherently growing cells while simultaneously recording the amperometric O2 reduction current as a function of the SECM tip location. As a representative example, Figure 17.7 illustrates the constant height feedback SECM image of adherent Retzius cells that were dissected from a leech ganglion and maintained in cell culture before SECM imaging. In order to visualize the isolated neuron, dissolved oxygen as usually present in physiological buffer was used as redox mediator and a glass-insulated Pt disk microelectrode of 25 μm diameter as SECM tips. At a constant potential of −600 mV vs. Ag/AgCl the leech neuron clearly became visible in the x / y plot as area of reduced tip (oxygen) current. In fact, the measured current at the SECM tip is a convolution of potentially decreased O2 concentrations due to the respiratory activity of the cell and the topography modulating the diffusional access of molecular oxygen to the SECM tip surface.

FIGURE 17.7. ( See color insert following page 272.

FIGURE 17.7

( See color insert following page 272.) Constant-height scanning electrochemical microscopy (SECM) feedback image of a pair of Retzius cells from leech ganglion. The SECM image of the leech neuron was recorded in the negative feedback mode in physiological (more...)

One might partly overcome this problem by using very small electrodes (typically 3–5 μm diameter), which are retracted to a distance at which one could at least reduce the impact of the cell topography on the observed current variations. The approach was employed for local oxygen measurements at individual guard cells from plants [53], cells from cultivated cell lines [81,84], or single protoplasts from algae [85,86], and more recently for investigations at single bovine embryos [87] and individual neuronal cells [88]. Furthermore, SECM-based local detection of respiration- induced changes of O2 concentrations were performed in combination with specially designed micropatterns of cultivated cells on glass [89], or silicon [90] substrates to develop sensitive cell proliferation assays [91] or screening the mode of action of anticancer drugs [92,93].

As mentioned above, the interpretation of the results of single-cell oxygen measurements is often difficult taking into account the permeability of cells for molecular oxygen and the continuous consumption of molecular oxygen at the SECM tip leading to a hemispherical depletion of O2 in its vicinity. Thus, it seems to be indispensable to develop potential pulse detection schemes avoiding the competition between the positioned SECM tip and the cell for O2 or to follow the cell topography in a defined and constant distance. Alternatively, SECM negative feedback curves can be recorded with the SECM tip directly over a cell in absence or presence of a drug. The difference in the current-distance curve should then represent the changes in cell metabolism assuming constant cell morphology.

A further gaseous compound that plays an important role in the nervous system, is involved in vasodilatation, the control of cerebral blood flow, modulation of synaptic plasticity, memory formation, and the initiation of host defense against tumor cells and infectious pathogens is NO. Because of its diverse biological functions and the involvement of abnormalities in physiological NO levels in, e.g., hypertension, diabetes, ischemia, and atherosclerosis, the local detection of NO release from single cells or cell populations became a topic of increasing interest. Aiming at the elucidation of NO signaling pathways and the basic mechanisms underlying NO release, NO selective amperometric microsensors were developed [94–96] and applied in physiological systems [97]. Generally, electrodes chemically modified with NO oxidizing catalysts such as metalloporphyrins and phthalocyanines in combination with permselective and negatively charged membranes for exclusion of negatively charged potentially interfering compounds were proposed were proposed. Already in 1992, it was shown that porphyrin-modified CFMEs are suitable for NO determination on the level of a single cell [15]. However, although NO sensors became commercially available, quantification of NO released from a single cell or a cell population remains difficult due to the complex diffusion profile of NO in vicinity of the releasing cells. On one hand, a NO diffusion profile is established that depends on the rate of the enzymatic NO formation at the NO synthase by converting arginine to citrulline, reactions within the diffusion zone with molecular oxygen and other reactive oxygen species, its trapping by radical scavengers. On the other hand, the NO sensor locally decreases the NO concentration, which leads to a diffusional flux of NO toward the NO sensor surface. Due to the still-limited performance of available electrocatalysts, the sensitivity of the NO sensors requires often rather large sensor areas of several μm2. Therefore, the NO conversion at the electrode cannot be neglected with respect to the rate of NO formation inside a single cell and hence an exact knowledge about the distance of the NO sensor and the NO release site is indispensable.

In a recent attempt to improve the accuracy and reproducibility of the positioning of NO microsensors relative to NO releasing (endothelial) cells, scientists designed, and fabricated dual disk-shaped microelectrodes to support an SECM feedback mode z -approach and precise adjustment of the cell-to-sensor separation [98,99] One of the two electrodes, actually a bare 10 μm diameter Pt disk electrode, was operated as conventional amperometric SECM tip and thus could be used via feedback-mode z -approach curves as “travel guide” for the slightly larger second electrode, which was a porphyrin-modified 50 μm diameter Pt disk. With a set of stimulated NO release measurements made at different distances between the disk surface of NO microsensors and endothelial cells it could be clearly demonstrated that the magnitude of the acquired NO signals was as expected strongly dependent on the position of the NO sensor relative to the release sites. In view of that, the SECM-based strategy of employing dual disk, bifunctional probe tips for the reproducible and accurate positioning of NO-specific sensors at well-defined distances from NO releasing cells is a significant advancement for the local detection of NO release. This approach has been extended to the simultaneous detection of the release of NO and glutamate from adherently growing cell populations [100] and could even be integrated into a microtiter-plate compatible electro-chemical robotic system for cell assays with increased throughput [101]. However, this type of electrodes is not suitable for lateral scanning in the x, y plane and hence other techniques have to be combined with the conventional SECM modes for keeping the distance between SECM tip and sample constant.

Constant-Distance Mode SECM at Individual Cells and Cell Populations

Monitoring simultaneously the morphology and the chemical activity of dynamic living cells in a non-invasive manner is the most challenging goal of biological SECM. For accomplishing proper tip positioning constant-distance mode SECM was developed, which enables to continuously control the tip-to-sample distance via a computer-controlled feedback circuitry and guides an SECM tip at constant spacing across the sample surface during scanning. Several strategies have been implemented for providing a distance-dependent input signal that is required for the closed-loop feedback of the distance control including shearforce-based feedback mechanisms with optical [102,103] piezoelectric [104], or tuning fork based [105,107] detection of shearforces between the vibrating tip and the sample surface. In addition, the maintenance of constant tip currents throughout line-scans [106–108] and a tip impedance-based feedback system have been proposed [108,109]. In any case, the established constant-distance mode forces the tip electrode to follow the specimens shape und thus provides visualization of the sample topography simultaneously with the local detection of electrochemical activity or released compounds. Tip-sample collisions can be avoided and precise positioning of SECM tips at preferred spots and adjusted distances is facilitated. The different schemes for establishing a distance-sensitive feedback signal have their specific assets and drawbacks. Tuning fork and piezoelectric shear force detection systems have been shown well feasible for SECM imaging of individual living cells allowing for positioning of the SECM tip in a constant distance of 100–300 nm (Figure 17.8).

FIGURE 17.8. ( See color insert following page 272.

FIGURE 17.8

( See color insert following page 272.) Constant-distance topography image of a PC12 cell. A vibrating carbon fiber tip was scanned over the surface using a shearforce-based constant distance mode with optical read-out for repositioning of the scanning (more...)

The constant-current mode, however, works with exogenous redox mediators in physiological buffer solution, compounds that may be harmful to cultured cells or change their behavior. Advantageous from this point of view are systems using the tip impedance or optically translated shearforces for the establishment of a distance control being applicable in absence of a possibly toxic mediator even in pure cell growth media. It is additionally useful that both positioning modes allow for synchronized detection of a faradaic and topographic signal due to the fact that the high-frequency tip impedance can be separated from the low-frequency voltammetric tip response and shear forces are current-independent in any case. Thus, efforts were put forward for establishing constant-distance mode biological SECM as a specialized tool for the synchronized topographical and chemical imaging of single cells. The setups that were realized are similar in their basic design and composed of the constant-distance SECM unit located on the stage of an inverted optical microscope (Figure 17.9).

FIGURE 17.9. Schematic representation of a biological scanning electrochemical microscopy (SECM) set-up designed for constant-distance mode topographical and chemical imaging on living cells.

FIGURE 17.9

Schematic representation of a biological scanning electrochemical microscopy (SECM) set-up designed for constant-distance mode topographical and chemical imaging on living cells. The main functional components of the BioSECM are a SECM tip, a micropositioning (more...)

As already mentioned earlier, researchers have used different strategies to provide the Bio-SECM with the input signal for the computer-controlled closed-loop feedback system that continuously detects the tip-to-sample separation and maintains it constant throughout lateral scanning. One possibility is to take advantage of optical and non-optical schemes for the detection of hydrodynamic shearforces occurring between liquid/solid interfaces and specially-designed, flexible SECM tips that are brought to vibration at resonance [102–104]. The distance control benefits from a shearforce-induced dampening of tip vibration in extreme proximity to surface. The integrated computer-controlled feedback loop of the device continually compares actual measured oscillation amplitudes and/or phase shifts with a user-defined set point by means of lock-in amplification and responds to alter damping caused by distance variations with tip repositioning. Thus, a constant level of damping is maintained and non-contact scanning at constant distance of about 100 nm to a few hundred nm is guaranteed. It has to be pointed out that vibration of the SECM tip in its resonance frequency as described in [102–104] has advantages over tuning-fork systems due to a softer interaction between tip and sample at an increased tip-to-sample distance.

Alternatively, scientists applied pure electrochemical feedback signals for distance control, namely the tip impedance at high frequencies and the amperometric tip current that occurs at proper tip potentials in the presence of a suitable exogenous redox mediator [108]. With shearforce, impedance and current-based constant-distance mode, conventional-sized carbon fiber micro-electrodes (Ø 5–10 μm) and carbon microelectrodes of reduced diameter (Ø 1–2 μm) could be scanned in constant distance across cover slips carrying individual cells, thus revealing the topography image of the cell.

In order to detect single exocytosis events upon stimulation of a secretory cell, the cell is first visualized using non-contact constant-distance topography imaging (see Figure 17.8). The SECM tip is then brought right beside the cell, and using constant-distance scanning the tip is moved exactly above the cell. Scanning is stopped and by addition of a suitable stimulant by means of a positioned microcapillary, compound release is invoked (Figure 17.10).

FIGURE 17.10. Left: Shearforce-based constant-distance mode scanning electrochemical microscopy (SECM) scan for positioning the SECM tip exactly above a PC12 cell.

FIGURE 17.10

Left: Shearforce-based constant-distance mode scanning electrochemical microscopy (SECM) scan for positioning the SECM tip exactly above a PC12 cell. Right: Current trace after depolarization of the cell membrane by means of local application of high (more...)

Obviously, the knowledge about the exact topography of the cells made a precise positioning of SECM tips in close proximity of the membrane of secretory cells possible and allowed the local detection of transmitter release. In addition to the detection of neurotransmitter release from PC12 [108] cells (Figure 17.10), catecholamine release from bovine chromaffin cells [110,111] could be successfully demonstrated using unetched and etched carbon fiber electrodes (Figure 17.11).

FIGURE 17.11. Local amperometric detection of single-vesicle adrenaline release from an individual bovine chromaffin cell using constant-distance mode Bio-SECM.

FIGURE 17.11

Local amperometric detection of single-vesicle adrenaline release from an individual bovine chromaffin cell using constant-distance mode Bio-SECM. A 5 μm diameter carbon-fiber disk electrode was polarized to + 800 mV vs. Ag/AgCl and positioned (more...)

Recently, constant-distance positioning over single cells could be extended to catalyst-modified carbon fiber electrodes suitable for the selective determination of NO [112]. A Ni-phthalocyanine film was electrochemically deposited on the surface of a platinized carbon fiber and positioned using shearforce-based constant-distance positioning under simultaneous optical control through the inverted microscope. The NO microsensor was positioned exactly above an adherently growing human umbilical vein endothelial cell (HUVEC) using a constant-distance SECM line scan. NO release was then stimulated by local application of bradykinin leading to a current increase at a constant applied potential of 750 mV vs. Ag/AgCl (Figure 17.12).

FIGURE 17.12. Left: Shearforce-based constant-distance mode scanning electrochemical microscopy (SECM) scan for positioning the Ni-phthalocyanine-modified SECM tip exactly above a single human umbilical vein endothelial cell (HUVEC) cell.

FIGURE 17.12

Left: Shearforce-based constant-distance mode scanning electrochemical microscopy (SECM) scan for positioning the Ni-phthalocyanine-modified SECM tip exactly above a single human umbilical vein endothelial cell (HUVEC) cell. The insert shows the situation (more...)

Conclusion and Future Aspects

Due to significant technical advancements, SECM matured into a powerful and valuable high-resolution imaging instrument for applications at the cutting-edge of life science. Biological SECM, especially in the constant distance mode of scanning, has been proven well suitable for visualizing simultaneously the topography and activity of single living cells. Imaging the exact contours of neuronal and secretory cells concomitantly with the local detection of single-vesicle exocytosis, visualization of cell metabolism and release of NO at accurately positioned SECM tips have been successfully demonstrated.

The ability of Bio-SECM to monitor at cellular level dynamic morphological and functional processes is of special importance for the elucidation of underlying mechanisms of cell-to-cell communication. Taking Bio-SECM to the next level of sophistication clearly requires the development, implementation and routine operation of SECM tips with sizes down to the 100 nm range, an improvement of the sensitivity and selectivity of the scanned tips with respect to the target analytes, and an advancement of the software controlling the action of the instrument including the tip-to-sample distance control, data acquisition and data analysis. Achieving these goals in principle should allow approaching more complex cellular systems with Bio-SECM and investigate in detail the influence of the chemical environment of neuronal cells and morphology changes during neurodevelopment, synaptogenesis and neurodegeneration.

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