Biomedical subjects
Publications and source records attributed to Yuri E Korchev.
A fluorescent nanosensor based on reporter dye molecules trapped in the tip of a nanopipette has been developed. This 100 nm sized nanosensor has been shown to be capable of measuring local pH and mapping sodium concentration with a temporal resolution of a few milliseconds.
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We present a new methodology which provides for the miniaturization of one of the most common tools in use in chemistry and biology laboratories today-the micropipet. We have used glass-fabricated double-barrel nanopipets to controllably produce arrayed water droplets with volumes as small as a few attoliters under an organic layer. We have addressed individual droplets and added controlled amounts of either additional volume or reagents from one of the barrels of the pipet. We demonstrate that this method can be used for miniaturized cell-free protein expression.
We report here the novel use of scanning ion conductance microscopy (SICM) to produce surface images of embryonic stem cell-derived cardiomyocytes (ESCM) to identify individual contracting cardiomyocytes among different cell types. By measuring amplitude and rhythm we can quantitate contraction of ESCM. This method gives, within the same experiment, an assessment of the number and position of ESCM within the layer of mixed cell types, as well as an accurate measure of the response of individual ESCM. Using different modulators of contraction as examples we showed how SICM could be used for recording their responses. We subsequently demonstrated that this model can be used to investigate the protective effect of antiarrhythmogenic drugs.
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The ability to address specific nanoscale features is required to produce diverse biological nanoarrays or perform local assembly using biological building blocks and is an important unsolved problem in nanotechnology. In this work, we describe the use of a novel nanofabricated gold surface, with regions of distinct topography and chemical functionalities, to solve this problem. First, nanoarrays of IgG antibodies were produced by selective immobilization in nanoholes on the surface. The smallest feature size was determined by the hole size (fwhm 90 nm) and not surface diffusion. Using holes of 300 nm diameter, we selectively addressed specific features in the array by nanopipet delivery of a functional antibody, anti-IgG. To our knowledge, this is the first example of addressing specific biologically functional features on a surface at the nanoscale.
BACKGROUND: Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. METHOD: Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. RESULTS: This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 microm(-2) for extended cells to 2.5 microm(2) for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. CONCLUSIONS: Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells.
The study of the properties of DNA under high electric fields is of both fundamental and practical interest. We have exploited the high electric fields produced locally in the tip of a nanopipette to probe the motion of double- and single-stranded 40-mer DNA, a 1-kb single-stranded DNA, and a single-nucleotide triphosphate (dCTP) just inside and outside the pipette tip at different frequencies and amplitudes of applied voltages. We used dual laser excitation and dual color detection to simultaneously follow two fluorophore-labeled DNA sequences with millisecond time resolution, significantly faster than studies to date. A strong trapping effect was observed during the negative half cycle for all DNA samples and also the dCTP. This effect was maximum below 1 Hz and decreased with higher frequency. We assign this trapping to strong dielectrophoresis due to the high electric field and electric field gradient in the pipette tip. Dielectrophoresis in electrodeless tapered nanostructures has potential applications for controlled mixing and manipulation of short lengths of DNA and other biomolecules, opening new possibilities in miniaturized biological analysis.
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We have used a nanopipet as a nanopen to locally and controllably deposit complex biomolecules, including antibodies and DNA, onto a surface to create multicomponent and functional submicron features. Key advantages of this method are that the biomolecules are always in solution and the applied voltage provides fine control of the delivery down to the single molecule level. Two consecutive cycles of deposition, to produce spatially varying features with different biological properties, were demonstrated with fluorescently labeled antibodies or biotin. This approach combines "top-down" fabrication, using the nanopen for local application, and "bottom-up" fabrication, using molecular recognition for self-assembly at defined positions, and opens up new possibilities in nanotechnology.
Cardiac toxicity is an uncommon but potentially serious complication of cancer therapy, especially with anthracyclines. One of the most effective anticancer drugs is doxorubicin, but its value is limited by the risk of developing cardiomyopathy and ventricular arrhythmia. When applied to a network of periodically contracting cardiomyocytes in culture, doxorubicin induces rhythm disturbances. Using a novel rapid assay based on non-invasive ion-conductance microscopy we show that the beta-antagonist esmolol can restore rhythm in doxorubicin-treated cultures of cardiomyocytes. Moreover, esmolol pre-treatment can protect the culture from doxorubicin-induced arrhythmia.
Although the dynamics of cell membranes and associated structures is vital for cell function, little is known due to lack of suitable methods. We found, using scanning ion conductance microscopy, that microvilli, membrane projections supported by internal actin bundles, undergo a life cycle: fast height-dependent growth, relatively short steady state, and slow height-independent retraction. The microvilli can aggregate into relatively stable structures where the steady state is extended. We suggest that the intrinsic dynamics of microvilli, combined with their ability to make stable structures, allows them to act as elementary "building blocks" for the assembly of specialized structures on the cell surface.
We present a new, general method for the controlled deposition of biological molecules on surfaces, based on a nanopipet operating in ionic solution. The potential applied to the pipet tip controls the flux of biological molecules from the pipet, allowing fine control of the delivery rate. We used the ion current to control the distance of the pipet from the surface of a glass slide and deposited the fluorescently labeled DNA or protein G at a defined location onto the surface. Features of 830 nm size were obtained by depositing the biotinylated DNA onto a streptavidin surface; 1.3 mum size spots were obtained by depositing protein G onto a positively charged glass surface.
We have developed a novel light source suitable for imaging of biological samples. The method is based on the use of a micropipet filled with fluo-3. A fluorogenic complex is formed when fluo-3 meets calcium in the bath solution. The complex is excited by focusing a laser beam at the pipet tip to produce a submicrometer light source. This source is continually renewed at the tip, eliminating problems with photobleaching, and can be controlled by varying the applied potential. We first characterized the light source using fluorescence correlation measurements in order to optimize its properties. We then recorded an image of a model sample under buffer with submicrometer resolution using ion conductance distance control to demonstrate the feasibility of this approach.
Cell specialization is often governed by the spatial distribution of ion channels and receptors on the cell surface. So far, little is known about functional ion channel localization. This is due to a lack of satisfactory methods for investigating ion channels in an intact cell and simultaneously determining the channels' positions accurately. We have developed a novel high-resolution scanning patch-clamp technique that enables the study of ion channels, not only in small cells, such as sperm, but in submicrometer cellular structures, such as epithelial microvilli, fine neuronal dendrites, and, particularly, T-tubule openings of cardiac myocytes. In cardiac myocytes, as in most excitable cells, action potential propagation depends essentially on the properties of ion channels that are functionally and spatially coupled. We found that the L-type calcium and chloride channels are distributed and colocalized in the region of T-tubule openings, but not in other regions of the myocyte. In addition, chloride channels were found in narrowly defined regions of Z-grooves. This finding suggests a new synergism between these types of channels that may be relevant for action potential propagation along the T-tubule system and excitation-contraction coupling.
We report the pulsed delivery of single-stranded DNA molecules through a nanopipet. The conical geometry of the pipet leads to a localized electric field, since all of the potential drop occurs in the tip region. Pulsatile delivery of DNA molecules can be achieved in an experimentally simple way with high precision by controlling the applied voltage. Single-molecule detection and fluorescence correlation spectroscopy in the nanopipet enable us to determine the number of molecules delivered. Anomalous slow diffusion of the DNA molecules in the pipet has also been observed. This nanopumping technique may have potential applications in local drug delivery and nanofabrication of biomolecules on surfaces in aqueous environments.
We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.