Scanning Electrochemical Microscopy of Model Neurons:  Imaging and Real-Time Detection of Morphological Changes

Liebetrau, Johanna M.; Miller, Heather M.; Baur, John E.; Takacs, Sara A.; Anupunpisit, Vipavee; Garris, Paul A.; Wipf, David O.

doi:10.1021/ac026166v

Cited by 102 publications

(81 citation statements)

References 31 publications

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“…Hydrophilic redox mediators are useful for living cell topography imaging because they cannot cross the cell membrane, and so the diffusion-limited faradaic current for such a species in the bulk solution decreases with the SECM tip electrode to (cell membrane) surface in a defined manner (2). This is well illustrated, for example, by the studies of Baur and coworkers who imaged cell topography by both constant-height mode and constant-distance mode SECM using hydrophilic mediators (14,33). A variety of electrochemical mediators were assessed for toxicity to living cell and RuðNH 3 Þ 6 3þ was selected as a suitable mediator.…”

Section: Resultsmentioning

confidence: 95%

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Takahashi

Shevchuk

Novák

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

206

224

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We describe voltage-switching mode scanning electrochemical microscopy (VSM-SECM), in which a single SECM tip electrode was used to acquire high-quality topographical and electrochemical images of living cells simultaneously. This was achieved by switching the applied voltage so as to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest. This imaging method is robust, and a single nanoscale SECM electrode, which is simple to produce, is used for both topography and activity measurements. In order to minimize the delay at voltage switching, we used pyrolytic carbon nanoelectrodes with 6.5-100 nm radii that rapidly reached a steady-state current, typically in less than 20 ms for the largest electrodes and faster for smaller electrodes. In addition, these carbon nanoelectrodes are suitable for convoluted cell topography imaging because the RG value (ratio of overall probe diameter to active electrode diameter) is typically in the range of 1.5-3.0. We first evaluated the resolution of constant-current mode topography imaging using carbon nanoelectrodes. Next, we performed VSM-SECM measurements to visualize membrane proteins on A431 cells and to detect neurotransmitters from a PC12 cells. We also combined VSM-SECM with surface confocal microscopy to allow simultaneous fluorescence and topographical imaging. VSM-SECM opens up new opportunities in nanoscale chemical mapping at interfaces, and should find wide application in the physical and biological sciences.high-resolution imaging | living cell imaging | noninvasive | constant-distance mode S canning electrochemical microscopy (SECM) uses an electrode tip for detecting electroactive chemical species and is an effective tool for the investigation of the localized chemical properties of sample surfaces and interfaces (1). Because SECM has high temporal resolution and can be used under physiological conditions, it is particularly well suited for quantitative measurements of (short-lived) chemicals like neurotransmitters, nitric oxide, reactive oxygen species, and oxygen, which are released/ consumed by living cells. In conventional SECM, the probe is often micrometer scale and the probe vertical position is kept at a constant height, a plane, during probe scanning. If the sample topography is not flat, the electrode-sample separation changes during scanning, complicating the SECM measurement and its analysis.Various methods for SECM electrode miniaturization and control of the electrode-sample separation have been advocated in order to improve the resolution of SECM imaging. The reliable fabrication of nanoelectrodes with a small ratio of electrodeinsulation to active electrode (

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Section: Resultsmentioning

confidence: 95%

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Takahashi

Shevchuk

Novák

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

206

224

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“…Although SECM has been used over the years for investigating biological systems, 23,24 only recently has attention been given to SECM for single cell studies. [25][26][27][28][29][30][31][32][33][34][35][36][37][38] Mirkin et al 26,27,[32][33][34] have investigated single cells by SECM in order to determine various redox activities across different types of cells, examining the differences between nonmetastatic and metastatic human breast cells. They have identified series of redox mediators that can cross the cell membrane, investigated their distribution between cytosol and nucleus, and found them unable to penetrate the nuclear envelope.…”

Section: Introductionmentioning

confidence: 99%

Glucose and Lactate Biosensors for Scanning Electrochemical Microscopy Imaging of Single Live Cells

et al. 2008

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We have developed glucose and lactate ultramicroelectrode (UME) biosensors based on glucose oxidase and lactate oxidase (with enzymes immobilized onto Pt UMEs by either electropolymerization or casting) for scanning electrochemical microscopy (SECM), and have determined their sensitivity to glucose and lactate, respectively. The results of our evaluations reveal different advantages for sensors constructed by each method: improved sensitivity and shorter manufacturing time for hand-casting, and increased reproducibility for electropolymerization. We have acquired amperometric approach curves (ACs) for each type of manufactured biosensor UME, and these ACs can be used as a means of positioning the UME above a substrate at a known distance. We have used the glucose biosensor UMEs to record profiles of glucose uptake above individual fibroblasts. Likewise, we have employed the lactate biosensor UMEs for recording the lactate production above single cancer cells with the SECM. We also show that oxygen respiration profiles for single cancer cells do not mimic cell topography, but are rather more convoluted, with a higher respiration activity observed at the points where the cell touches the Petri dish. These UME biosensors, along with the application of others already described in the literature, could prove to be powerful tools for mapping metabolic analytes, such as glucose, lactate and oxygen, in single cancer cells.

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“…In general, the negative-feedback mode yields little information about the biological activity of the substrate and is mainly used to find the tip-to-substrate distance and image biological samples, as used recently with PC12 cells, dopamine-releasing immortal rat cells (11).…”

mentioning

confidence: 99%

Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells

Mauzeroll

Bard

2004

Proc. Natl. Acad. Sci. U.S.A.

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The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV-visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-m-diameter electrode situated 10 m from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster.

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Scanning Electrochemical Microscopy of Model Neurons: Imaging and Real-Time Detection of Morphological Changes

Cited by 102 publications

References 31 publications

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Topographical and electrochemical nanoscale imaging of living cells using voltage-switching mode scanning electrochemical microscopy

Glucose and Lactate Biosensors for Scanning Electrochemical Microscopy Imaging of Single Live Cells

Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells

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