Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: Application to single virus-like particle entry into a cell

Gorelik, Julia; Shevchuk, Andrew; Ramalho, Michal; Elliott, Martin; Chao, Lei; Higgins, Claire A.; Lab, Max J.; Klenerman, David; Krauzewicz, Nina; Korchev, Yuri E.

doi:10.1073/pnas.252458399

Cited by 97 publications

(87 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Next, we combined the sample height information obtained by constant-current mode SECM combined with fluorescence microscopy so that we could perform measurements at the specific fluorophore-labeled structures on a cell, in this case at the synapse of neuron. Previously, a hybrid SICM-confocal system was developed based on a similar concept (41,42), and we used a similar system in which the SECM scanner was integrated on an inverted confocal microscope. For the fluorescence measurement, the hippocampal neurons were stained with FM 1-43, which labeled synaptic vesicles (36).…”

Section: Resultsmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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 (

show abstract

Section: Resultsmentioning

confidence: 99%

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

View full text Add to dashboard Cite

show abstract

“…SICM is a scanning probe microscopy technique that uses a glass nanopipette as a sensitive probe that detects ion current and uses the current as an interaction signal to control the vertical (zaxis) position of the cell relative to the pipette tip (14). In SSCM the cell is moved up and down in the z direction while it is scanned in the x and y directions, so its surface is always the same distance from the nanopipette (typically, 25-75 nm).…”

Section: Scanning Ion Conductance Microscopy and Scanning Surface Conmentioning

confidence: 99%

Immortalization of Human Alveolar Epithelial Cells to Investigate Nanoparticle Uptake

Kemp

Thorley

Gorelik

et al. 2008

Am J Respir Cell Mol Biol

133

117

View full text Add to dashboard Cite

Primary human alveolar type 2 (AT2) cells were immortalized by transduction with the catalytic subunit of telomerase and simian virus 40 large-tumor antigen. Characterization by immunochemical and morphologic methods demonstrated an AT1-like cell phenotype. Unlike primary AT2 cells, immortalized cells no longer expressed alkaline phosphatase, pro-surfactant protein C, and thyroid transcription factor-1, but expressed increased caveolin-1 and receptor for advanced glycation end products (RAGE). Live cell imaging using scanning ion conductance microscopy showed that the cuboidal primary AT2 cells were approximately 15 mm and enriched with surface microvilli, while the immortal AT1 cells were attenuated more than 40 mm, resembling these cells in situ. Transmission electron microscopy highlighted the attenuated morphology and showed endosomal vesicles in some immortal AT1 cells (but not primary AT2 cells) as found in situ. Particulate air pollution exacerbates cardiopulmonary disease. Interaction of ultrafine, nano-sized particles with the alveolar epithelium and/or translocation into the cardiovasculature may be a contributory factor. We hypothesized differential uptake of nanoparticles by AT1 and AT2 cells, depending on particle size and surface charge. Uptake of 50-nm and 1-mm fluorescent latex particles was investigated using confocal microscopy and scanning surface confocal microscopy of live cells. Fewer than 10% of primary AT2 cells internalized particles. In contrast, 75% immortal AT1 cells internalized negatively charged particles, while less than 55% of these cells internalized positively charged particles; charge, rather than size, mattered. The process was rapid: one-third of the total cellassociated negatively charged 50-nm particle fluorescence measured at 24 hours was internalized during the first hour. AT1 cells could be important in translocation of particles from the lung into the circulation.

show abstract

“…This removes problems in photobleaching fluorophores before they are imaged, an issue with conventional confocal microscopy that uses z slices to build up an image of the surface. We have used surface confocal microscopy to identify viral particles [8,9] and clathrin-coated pits (CCPs) on the cell surface [10,11]. The pipette can also be used to perform a series of nanoscale local assays on the sample, including single-channel recording, mechanical measurements by local pressure application, local voltage delivery of reagents and local chemical mapping.…”

Section: Local Measurements Combined With Scanning Ion Conductance MImentioning

confidence: 99%

Imaging the cell surface and its organization down to the level of single molecules

Klenerman

Shevchuk

Novák

et al. 2013

Phil. Trans. R. Soc. B

View full text Add to dashboard Cite

Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.

show abstract

Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: Application to single virus-like particle entry into a cell

Cited by 97 publications

References 30 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

Immortalization of Human Alveolar Epithelial Cells to Investigate Nanoparticle Uptake

Imaging the cell surface and its organization down to the level of single molecules

Contact Info

Product

Resources

About