Albert Schulte

Extrasynaptic Vesicular Transmitter Release from the Somata of Substantia Nigra Neurons in Rat Midbrain Slices

Substantia nigra neurons release dopamine from their somatodendritic regions. A long-unresolved question is whether this release occurs by exocytosis or by a nonvesicular mechanism. We used carbon fiber microelectrodes in a brainstem slice to assay secretion from single cell bodies that had been cleared of connective tissue. Amperometry at the carbon fiber microelectrodes revealed unitary events in ∼90% of cells in resting conditions. These events had charge integrals ranging from a few femtocoulombs to several hundred femtocoulombs (fC). Local glutamate application enhanced the event frequency by 3.5-fold on average and up to 10-fold in highly responsive cells, although the mean charge integral was not modified. Local application of a high K+-containing saline had effects similar to those of glutamate. The frequency of resting and stimulated amperometric events was much lower at 21–22°C than at 32–35°C. The addition of Cd2+ (50 μm), a blocker of voltage-dependent Ca2+ channels, to the bath solution blocked the stimulatory effects of glutamate. These results suggest that dopamine is released from the somata of substantia nigra neurons by exocytosis and that this mechanism is regulated by neuronal electrical activity. More generally, this study demonstrates the applicability of carbon fiber microelectrodes to the measurement of quantal monoamine secretion in brain slices.

Localised electrochemical impedance spectroscopy with high lateral resolution by means of alternating current scanning electrochemical microscopy

A new method for measuring local interfacial impedance properties with high lateral resolution was developed by combination of electrochemical impedance spectroscopy (EIS) with scanning electrochemical microscopy (SECM). Alternating current scanning electrochemical microscopy (AC-SECM) allowed to identify and visualise microscopic domains of different conductivity/electrochemical activities on solid/liquid interfaces immersed into an electrolyte. The performance of the method was illustrated by imaging an array of Pt-band microelectrodes in solutions of low conductivity in the absence of any redox mediator.

Effective Insulation of Scanning Tunneling Microscopy Tips for Electrochemical Studies Using an Electropainting Method

We report here on a new, straightforward, and effective method for insulating etched and cut Pt/Ir scanning tunneling microscopy (STM) tips for in situ electrochemical studies. The coating was formed by electrophoretic painting and subsequent heating. It covered all but the very end of the tip, which may be attributed to shrinking of the polymer during heating. We have characterized these tips by voltammetric methods, scanning electron microscopy, and by in situ STM imaging.

Scanning electrochemical microscopy in neuroscience.

This article reviews recent work involving the application of scanning electrochemical microscopy (SECM) to the study of individual cultured living cells, with an emphasis on topographical and functional imaging of neuronal and secretory cells of the nervous and endocrine system. The basic principles of biological SECM and associated negative amperometric-feedback and generator/collector-mode SECM imaging are discussed, and successful use of the methodology for screening soft and fragile membranous objects is outlined. The drawbacks of the constant-height mode of probe movement and the benefits of the constant-distance mode of SECM operation are described. Finally, representative examples of constant-height and constant-distance mode SECM on a variety of live cells are highlighted to demonstrate the current status of single-cell SECM in general and of SECM in neuroscience in particular.

Precursor sites for localised corrosion on lacquered tinplates visualised by means of alternating current scanning electrochemical microscopy

In solutions of low conductivity and at high frequencies the impedance of a SECM tip-auxiliary electrode cell is dominated by the solution resistance between the tip and counter electrode. Alternating current scanning electrochemical microscopy (AC-SECM) utilises the effect of an increasing (decreasing) solution resistance as the SECM tip approaches an insulator (conductor) for mapping domains of different conductivity/electrochemical activity on surfaces immersed into electrolytes. In the present study, we employed AC-SECM in aqueous solutions to evaluate the integrity of the solid/liquid interface of lacquered tinplates as commonly used in industry to manufacture, i.e. food cans. Significant differences were determined between the AC response and the phase shift measured with the SECM tip above the intact coating and above defects where the surface of the steel base is exposed. This allowed with high lateral resolution to detect and to visualise artificial micro cavities which we consider as an experimental model of microscopically small precursor sites for localised corrosion.

An advanced biological scanning electrochemical microscope (Bio-SECM) for studying individual living cells

An inverted optical microscope was customized to incorporate a constant-distance mode scanning electrochemical microscope (SECM). The system worked well with an optical, shearforce-based feedback mechanism for maintaining a constant tip-to-sample separation throughout scanning. The highly accurate distance control of the established Bio-SECM allowed novel, flexible carbon-fibre microelectrodes with appropriate vibration characteristics and significantly reduced tip diameters to be used as vibrationable SECM tips for topographical and electrochemical measurements on soft biological samples such as adherently growing fibroblasts or adrenal chromaffin cells. Visual aid offered by the optical microscope helped identifying suitable cells and supported manual prepositioning of an electrode tip next to a selected cell. Precise, non-manual positioning of the tip of the microelectrode directly above a single living cell at a distance of a fraction of a micrometre was carried out by taking advantage of topography information available from constant-distance SECM line scans. In the case of catecholamine-releasing chromaffin cells, properly placed SECM tips succeeded to detect amperometrically the release of adrenaline and noradrenaline out of single secretory vesicles upon proper stimulation.

4D shearforce-based constant-distance mode scanning electrochemical microscopy.

4D shearforce-based constant-distance mode scanning electrochemical microscopy (4D SF/CD-SECM) is designed to assess SECM tip currents at several but constant distances to the sample topography at each point of the x,y-scanning grid. The distance dependent signal is achieved by a shearforce interaction between the in-resonance vibrating SECM tip and the sample surface. A 4D SF/CD-SECM measuring cycle at each grid point involves a shearforce controlled SECM tip z-approach to a point of closest distance and subsequent stepwise tip retractions. At the point of closest approach and during the retraction steps, pairs of tip current (I) and position are acquired for various distances above the sample surface. Such a sequence provides x,y,I maps, that can be compiled and displayed for each selected data acquisition distance. Thus, multiple SECM images are obtained at known and constant distances above the sample topography. 4D SF/CD-SECM supports distance-controlled tip operation while continuous scanning of the SECM tip in the shear-force distance is avoided. In this way, constant-distance mode SECM imaging can be performed at user-defined, large tip-to-sample distances. The feasibility and the potential of the proposed 4D SF/CD-SECM imaging is demonstrated using on the one hand amperometric feedback mode imaging of a Pt band electrode array and on the other hand the visualization of the diffusion zone of a redox active species above a microelectrode in a generator/collector arrangement.

Redox hydrogel-based bienzyme microelectrodes for amperometric monitoring of L-glutamate

Fabrication and characterization of amperometric bienzyme L-glutamate sensitive microelectrodes are the prerequisite for monitoring changes Of L-glutamate concentration at glutamate-secreting cell cultures. The design of the glutamate microelectrodes is based on incorporating L-glutamate oxidase and horseradish peroxidase into a redox-hydrogel containing PVI19-dmeOs as the redox mediator and immobilizing this system onto the surface of platinum microdisk electrodes using, a dip-coating procedure. For amperometric measurements Of L-glutamate, these redox hydrogel-based bienzyme microelectrodes can be operated at low working potentials (-50 mV vs. Ag/AgCl) decreasing the influence of electroactive interferants possibly present in biological samples. The L-glutamate microsensors are characterized by a good operation stability and sensitivity (0.038+/-0.005 mAM(-1)), a low detection limit (0.5 muM in a conventional amperometric set-up and 0.03 muM in a Faraday cage, defined as three times the signal-to-noise ratio), a linear range up to 50 muM and a response time of about 35 s. The glutamate biosensors have been applied for the direct measurement Of L-glutamate release (upon chemical stimulation) from a population of immortalized hippocampal neurons (HN10 cells) demonstrating the possibility to amperometrically monitor in-Situ L-glutamate secretion from these cells. (Less)

Pyrrole functionalised metalloporphyrins as electrocatalysts for the oxidation of nitric oxide

Pyrrole-functionalised tetracarboxyphenyl porphyrin and trimethoxyphenylcarboxy-phenyl porphyrin containing Ni, Mn and Pd as the central metal ion were used to modify Pt-disk microelectrodes (slashed circle 50 mum) (by repetitive cyclic voltammetry, dip-dry and pulse-amperometry methods) for the detection of nitric oxide (NO). Electrodes modified with Mn(II) trimethoxyphenylcarboxyphenyl porphyrin using the pulse amperomery approach, were found to be sensitive, stable and fast in response towards the oxidation of NO. Thus, they were used for the detection of NO release from a population of transformed human umbilical vein endothelial cells (T-HUVEC) into a droplet of electrolyte solution following stimulation with vascular endothelial growth factor (VEGF). The electrode surface was covered with an additional layer of Nafion(R) to prevent interference from anionic molecules such as nitrite.

Visualization of Oxygen Consumption of Single Living Cells by Scanning Electrochemical Microscopy: The Influence of the Faradaic Tip Reaction†

The respiration activity of an individual living cell is an indicator of its metabolic vitality. Closely positioned microelectrodes have been suggested for determination of the respiration activity by monitoring the local oxygen concentration. Although first attempts for visualization of the oxygen consumption rate of single living cells by means of scanning electrochemical microscopy (SECM) were already described in 1998, evaluation of the respiratory activity of individual cells remains challenging and the complexity is often underestimated. In particular, the dimensions of the cell itself lead to limitations of conventionally used constant-height mode SECM investigations. Apart from convolution of the oxygen reduction current at the SECM tip with topographic effects, constant-height mode experiments require working distances comparable or below the height of the cell body, thus increasing the risk of tip crash. Attempts to overcome these restrictions include among others positioning of the tip to distances outside the feedback range, embedding of the cells into cavities, or efforts to subtract topographic contributions after cell death. Moreover, as living cells are irregular in dimension, the tip-to-cell distance varies with the tip position. Therefore, constant-distance mode (cd-mode) SECM techniques are inherently advantageous for this purpose. In particular, coupling SECM with scanning probe techniques, such as atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) as well as shearforce and impedance-based techniques, led to efficient strategies to control the tip-to-sample separation. Recently, we described a shearforce-based cd method (4D SF/ CD-SECM) that is able to work at various tip-to-sample separations. It can hence detect complete diffusion profiles in the surroundings of sources or sinks of redox-active species. Although SECM distance control systems are available, the detection of the respiration activity of single living cells remains challenging. Owing to the small rate of oxygen consumption by a single cell, only small current variations ontop of a high background current are measured. Even more importantly, a biological cell acts as an immiscible liquid– liquid interface in a SECM experiment. Lipophilic redox mediators are known to undergo transmembrane diffusion processes and can be utilized to investigate intracellular redox activity. However, concentration changes in the vicinity of the cellular membrane, for example by the tip reaction, may induce local concentration gradients and cause a diffusional exchange of redox species over the lipid bilayer in a socalled SECM-induced transfer (SECM-IT) mode. The high solubility of oxygen in lipids promotes this transmembrane diffusion and oxygen can easily cross the cell membrane. This diffusion process superimposes the detection of cell respiration. As a result, in most reports addressing detection of cell metabolism based on the detection of variations in the local oxygen concentration, the positioned microelectrode does not act as a passive observer but actively influences the oxygen concentration inside the gap between tip and cell, resulting in imaging artifacts that have not previously been addressed. Even though mentioned occasionally, this effect was neglected in SECM investigations of respiration activity at living cells. Herein, we address the influence of the oxygen reduction rate at the SECM tip on imaging the respiration activity at living cells. We provide strategies to avoid limitations resulting from a strong tip reaction using a potential pulse profile at the tip with a time dependent data acquisition in the shearforce-based cd-mode of SECM. Commonly, the detection of the local oxygen concentration in close proximity to the cell body is performed by means of a variation of the generator-collector mode of SECM with the tip being continuously polarized at oxygen reduction potential. The tip competes with the respiring living cell for the available oxygen inside the gap between SECM tip and cell surface. Crossing the cell body during a SECM line scan should therefore lead to a decrease of the tip current owing to a locally lowered oxygen concentration caused by cell [*] Dr. M. Nebel, S. Gr tzke, Prof. Dr. W. Schuhmann Lehrstuhl f r Analytische Chemie, Elektroanalytik & Sensorik and Center for Electrochemical Sciences, CES Ruhr-Universit t Bochum Universit tsstrasse 150, 44780 Bochum (Germany) E-mail: wolfgang.schuhmann@rub.de