Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging

We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.     

Measuring the ionic flux of an electrochemically actuated conducting polymer using modified scanning ion conductance microscopy

We propose a modification of a scanning ion conductance microscope suitable for probing an electrode in an operating electrochemical cell. We demonstrate its use by measuring salt concentration variations near a conducting polymer electrode as the polymer is electrochemically oxidized and reduced. The electrochemical control circuit is opened to isolate the working electrode, at a frequency sufficiently high that the electrode capacitance maintains the electrode potential. The local solution conductivity variations are detected through the probe current during the open-circuit time. We demonstrate two-stage ion exchange during oxidation and reduction of poly(3,4-ethylenedioxythiophene) films that develops strongly with repeated cycling and is correlated with actuation changes. Spatial composition variations of the film, caused by redox current distribution over the surface, and electromigration to the probe tip, causing local solution composition changes, have clear and characteristic effects on the measured transients.     

Transport of redox probes through single pores measured by scanning electrochemical-scanning ion conductance microscopy (SECM-SICM)

We report scanning electrochemical microscopy-scanning ion conductance microscopy (SECM-SICM) experiments that describe transport of redox active molecules which emanate from single pores of a track-etch membrane. Experiments are performed with electrodes which consist of a thin gold layer deposited on one side of a nanopipet. Subsequent insulation of the electrode with parylene results in a hybrid electrode for SECM-SICM measurements. Electrode fabrication is straightforward and highly parallel. For image collection, ionic current measured at the nanopipet both controls the position of the electrode with respect to the membrane surface and reports the local conductance in the vicinity of the nanopipet, while faradaic current measured at the Au electrode reports the presence of redox-active molecules. Application of a transmembrane potential difference affords additional control over migration of charged species across the membrane.     

Effects of pipette modulation and imaging distances on ion currents measured with scanning ion conductance microscopy (SICM)

Local conductance variations can be estimated by measuring ion current magnitudes with scanning ion conductance microscopy (SICM). Factors which influence image quality and quantitation of ion currents measured with SICM have been evaluated. Specifically, effects of probe-sample separation and pipette modulation have been systematically studied for the case of imaging conductance variations at pores in a polymer membrane under transmembrane concentration gradients. The influence of probe-sample separation on ion current images was evaluated using distance-modulated (ac) feedback. Approach curves obtained using non-modulated (dc) feedback were also recorded to determine the relative influence of pipette-generated convection by comparison of ion currents measured with both ac and dc feedback modes. To better interpret results obtained, comparison to a model based on a disk-shaped geometry for nanopores in the membrane, as well as relevant position-dependent parameters of the experiment is described. These results advance our current understanding of conductance measurements with SICM.     

Electrochemical concept of scanning tunnel microscopy and scanning tunnel spectroscopy

The history of investigation, development and application of electrochemical scanning tunneling microscopy (ESTM) are reviewed.     

High-speed multifunctional scanning ion conductance microscopy: Innovative strategies to study dynamic cellular processes

The latest contributions of scanning ion conductance microscopy (SICM) to current research in life sciences are reviewed. The continued efforts toward technical improvements in SICM hardware and software resulted in development of new scanning strategies and instrumental designs. Enhanced high-speed mapping supported the capture of very fast cellular processes at the nanoscale, such as secretory events or cilia movements or the contraction cycle of a cardiomyocyte. The noninvasive scanning capability allowed researchers to monitor the response of individual live cells to chemical stimulations in real time. Access to new kinds of information that can be extracted from experimental data with the support of numerical simulations was demonstrated, thus extending further SICM multifunction capability.     

Correlating surface microstructures with reactivity on commercially pure zirconium using scanning electrochemical microscopy and scanning electron microscopy

Scanning electrochemical microscopy and scanning electron microscopy were employed to correlate the surface microstructures with surface reactivity of commercially pure zirconium. It was found that heightened reactivity was associated with iron impurities lying beneath the oxide surface. This could result in failure of nuclear reactor components fabricated using zirconium alloys due to hydrogen ingress and corrosion. COMSOL multiphysics software was used to quantify the electrochemical kinetic constants associated with the differences in surface reactivity.     

In situ scanning tunneling microscopy: New insight for electrochemical electrode-surface investigations

The successful expansion which the scanning tunneling microscopy (STM) has had is dependent on its ability to examine surfaces on a sub-nanometric scale and on providing in situ (i.e. in the presence of bulk electrolyte) sample examination. In addition to the ability to study metals and semiconductors in vacuo, the application of the technique to surfaces in contact with an electrolytic solution has prompted increased interest amongst electrochemists. We discuss herein the technique, with particular reference to advances in electrochemical applications. A new scanning tunneling microscope for operation in electrolytic environments is described. Atomic force microscopy, scanning electrochemical microscopy and scanning ion-conducting microscopy are compared with the STM.     

Nanoscale surface modification via scanning electrochemical probe microscopy

Micro- and nanoscale surface modification using scanning probe microscopy techniques in combination with electrochemically induced surface structuring provides a maskless in situ fabrication strategy enabling deposition or etching of three-dimensional nanostructures. This current opinion article focuses on scanning electrochemical probe microscopy techniques highlighting recent progress in nanoscale 3D surface modification along with a spotlight on approaches of practical relevance.     

Simultaneous detection of uric acid and glucose on a dual-enzyme chip using scanning electrochemical microscopy/scanning chemiluminescence microscopy

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1∼2 μm) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s⁻¹ while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable.     

Characterization and theory of electrocatalysts based on scanning electrochemical microscopy screening methods

A strategy for finding new electrocatalysts for the oxygen reduction reaction (ORR) in acidic solutions is outlined and illustrated with results for Pd-Co catalysts. This is based on establishing guidelines for selecting test systems, rapid preparation of arrays, and rapid screening by scanning electrochemical microscopy. Promising candidates are further tested as supported electrocatalysts by larger scale electrochemical methods and in fuel cells, with optimization of the composition and structure. Those that emerge are characterized by a variety of methods, including X-ray diffraction, scanning electron microscopy, and X-ray photoemission spectroscopy. Finally, density functional theory is used for detailed calculations of oxygen adsorption and dissociation on the material and provides better guidelines for further testing.     

Imaging and detection of morphological changes of single cells before and after secretion using scanning electrochemical microscopy

Images of Human umbilical vein endothelial cells (HUVECs) have been obtained and the regulation of cell morphology changes after nitric oxide release has been recorded and discerned quantitatively for the first time using scanning electrochemical microscopy.     

Visualization of DNA microarrays by scanning electrochemical microscopy (SECM)

DNA duplex regions of the spots on a DNA microarray were successfully visualized by scanning electrochemical microscopy (SECM) in the electrolyte containing ferrocenyl naphthalene diimide as a hybridization indicator.     

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.     

Biological applications of scanning electrochemical microscopy: chemical imaging of single living cells and beyond

Recent applications of scanning electrochemical microscopy (SECM) to studies of single biological cells are reviewed. This scanning probe microscopic technique allows the imaging of an individual cell on the basis of not only its surface topography but also such cellular activities as photosynthesis, respiration, electron transfer, single vesicular exocytosis and membrane transport. The operational principles of SECM are also introduced in the context of these biological applications. Recent progress in techniques for high-resolution SECM imaging are also reviewed. Future directions, such as single-channel detection by SECM, high-resolution imaging with nanometer-sized probes, and combined SECM techniques for multidimensional imaging are also discussed.     

扫描电化学显微镜在生物医学领域的应用研究进展

研究病变细胞和组织的异常表现可为理解重大疾病发生发展的病理机理和新型药物筛选提供重要参考.扫描电化学显微镜(Scanning electrochemical microscopy, SECM)是一种基于电化学原理的扫描探针显微镜,通过记录探针在样品表面扫描时的电流或电位等信息,对活细胞的形态和多种化学信息进行原位、实时...     

Electrochemical detection of receptor-mediated endocytosis by scanning electrochemical microscopy

We report a scanning electrochemical microscopy (SECM)-based receptor-mediated endocytosis detection method. Epidermal growth factor receptor (EGFR), which is one of the key membrane proteins associated with cancer, was used as a model for receptor-mediated endocytosis. EGFR molecules on the outer cell membrane were detected by SECM by using alkaline phosphatase (ALP) as a labeling enzyme. Since SECM detected the ALP activity on the outer membrane, the procedure helped discriminate the EGFR on the outer membrane from the intracellular EGFR involved in endocytosis. SECM showed a marked decrease in the current responses generated due to ALP activity by 93% on addition of the epidermal growth factor, indicating clearly that EGF triggered the endocytosis, which led to the withdrawal of most EGFRs from the outer membrane.     

Scanning electrochemical microscopy under illumination: an elegant tool to directly determine the mobility of charge carriers within dye-sensitized nanostructured semiconductors

The diffusion constant of the ferrocenium ion in dye-sensitized nanostructured materials has been determined by time-of-flight experiments under working solar cell conditions with scanning electrochemical microscopy.     

Application of scanning electrochemical microscopy and scanning electron microscopy for the characterization of carbon-spray modified electrodes

Different gold surfaces modified by carbon-spray have been investigated by scanning electron microscopy (SEM) and scanning electrochemical microscopy (SECM). A transformation of the SECM image to a distance-location profile is proposed which assists the correlation of both images. The structures found in the transformed SECM images of carbon-spray layers on gold substrates can be explained by the topographic features visible in the SEM pictures. Tempering the carbon spray results in an increased density of electrochemically reactive carbon particles which could be confirmed by cyclic voltammetric investigations. Gold minigrids modified with carbon spray expose some areas of especially large currents which could not be predicted from their SEM images. This effect may result from particles located at the edge of a wire intersection having relatively large active surfaces per particle. They contribute significantly to the total current of the minigrid.     

Quantitative imaging of ion transport through single nanopores by high-resolution scanning electrochemical microscopy

Here we report on the unprecedentedly high resolution imaging of ion transport through single nanopores by scanning electrochemical microscopy (SECM). The quantitative SECM image of single nanopores allows for the determination of their structural properties, including their density, shape, and size, which are essential for understanding the permeability of the entire nanoporous membrane. Nanoscale spatial resolution was achieved by scanning a 17 nm radius pipet tip at a distance as low as 1.3 nm from a highly porous nanocrystalline silicon membrane in order to obtain the peak current response controlled by the nanopore-mediated diffusional transport of tetrabutylammonium ions to the nanopipet-supported liquid-liquid interface. A 280 nm × 500 nm image resolved 13 nanopores, which corresponds to a high density of 93 nanopores/μm(2). A finite element simulation of the SECM image was performed to assess quantitatively the spatial resolution limited by the tip diameter in resolving two adjacent pores and to determine the actual size of a nanopore, which was approximated as an elliptical cylinder with a depth of 30 nm and major and minor axes of 53 and 41 nm, respectively. These structural parameters were consistent with those determined by transmission electron microscopy, thereby confirming the reliability of quantitative SECM imaging at the nanoscale level.