Imaging Proteins in Membranes of Living Cells by High‐Resolution Scanning Ion Conductance Microscopy

Shevchuk, Andrew; Frolenkov, Gregory I.; Sánchez, Daniel; James, P S; Freedman, Noah; Lab, Max J.; Jones, Roy; Klenerman, David; Korchev, Yuri E.

doi:10.1002/anie.200503915

Cited by 205 publications

(193 citation statements)

References 22 publications

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“…Evidently, the hopping-mode scanning algorithm (defined briefly above; also see Materials and Methods) is able to visualize the structures clearly. Indeed, the topographical features of boar spermatozoon and PC12 revealed by SECM are very similar to those previously obtained with SICM (15,34), which is regarded highly as an ultrahigh-resolution in situ imaging technique. Further evidence for the powerful nature of the SECM technique comes from the fact that the neurite and varicosity, which administrate neuron transduction and axon formation, of PC12 cells were visualized much more clearly compared with a previous SECM report using more conventional imaging protocols (14).…”

Section: Resultssupporting

confidence: 82%

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.

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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: Resultssupporting

confidence: 82%

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.

Self Cite

206

224

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“…The work discussed herein [40][41][42]44,87], and studies performed in other groups [47,101] has demonstrated that SICM can be used to provide information about the polarity and magnitude of substrate charge. In fact, SICM is sensitive to substrate surface charge for similar tip-substrate distances to which the standard SICM response is observed, approximately one tip diameter away from the surface [55,99]. The response of the ionic current upon approach to a charged interface differs significantly from that to an uncharged surface, with the response depending on the magnitude and sign of the applied bias, the nanopipette characteristics (geometry, charge) and ionic strength of the bulk solution [40,41,47].…”

Section: (Iii) Surface-induced Rectificationmentioning

confidence: 93%

Multifunctional scanning ion conductance microscopy

2017

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Scanning ion conductance microscopy (SICM) is a nanopipette-based technique that has traditionally been used to image topography or to deliver species to an interface, particularly in a biological setting. This article highlights the recent blossoming of SICM into a technique with a much greater diversity of applications and capability that can be used either standalone, with advanced control (potential-time) functions, or in tandem with other methods. SICM can be used to elucidate functional information about interfaces, such as surface charge density or electrochemical activity (ion fluxes). Using a multi-barrel probe format, SICM-related techniques can be employed to deposit nanoscale three-dimensional structures and further functionality is realized when SICM is combined with scanning electrochemical microscopy (SECM), with simultaneous measurements from a single probe opening up considerable prospects for multifunctional imaging. SICM studies are greatly enhanced by finite-element method modelling for quantitative treatment of issues such as resolution, surface charge and (tip) geometry effects. SICM is particularly applicable to the study of living systems, notably single cells, although applications extend to materials characterization and to new methods of printing and nanofabrication. A more thorough understanding of the electrochemical principles and properties of SICM provides a foundation for significant applications of SICM in electrochemistry and interfacial science.

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“…Therefore, SICM can capture topographic images without any tip-sample contact. Although high spatial resolution (~6 nm) has been reported for SICM (Shevchuk et al 2006), it cannot be attained routinely. The spatial resolution is approximately equal to the diameter of the pipette opening, when the tip end-sample separation is similar to the diameter of the pipette opening.…”

Section: Non-contact Imagingmentioning

confidence: 99%

High-speed atomic force microscopy and its future prospects

2017

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Various techniques have been developed and used to investigate how proteins produce complex biological architectures and phenomena. Among these techniques, high-speed atomic force microscopy (HS-AFM) holds a unique position. It is only HS-AFM that allows the simultaneous assessment of structure and dynamics of single protein molecules in action. This new microscopy tool has been successfully applied to a variety of proteins, from motor proteins to membrane proteins, antibodies, enzymes, and even to intrinsically disordered proteins. And yet there still remain many biomolecular phenomena that cannot be addressed by HS-AFM in its current form. Here, I present a brief history of HS-AFM development, describe the current state of HS-AFM, and then discuss which new biological scanning probe microscopy techniques will be coming up next.

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Imaging Proteins in Membranes of Living Cells by High‐Resolution Scanning Ion Conductance Microscopy

Cited by 205 publications

References 22 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

Multifunctional scanning ion conductance microscopy

High-speed atomic force microscopy and its future prospects

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