38 results about "Single Molecule Imaging" patented technology

Methods, compositions, and kits are provided for imaging a polypeptide of interest. Methods, compositions, and kits are also provided for site-specific transcriptional regulation of one or more genetic elements.

Various embodiments of the invention describe the synthesis of upconverting nanoparticles (UCNPs), lanthanide-doped hexagonal β-phase sodium yttrium fluoride NaYF4:Er3+ / Yb3 nanocrystals, less than 10 nanometers in diameter that are over an order of magnitude brighter under single-particle imaging conditions than existing compositions, allowing visualization of single UCNPs as small (d=4.8 nm) as fluorescent proteins. We use Advanced single-particle characterization and theoretical modeling is demonstrated to find that surface effects become critical at diameters under 20 nm, and that the fluences used in single-molecule imaging change the dominant determinants of nanocrystal brightness. These results demonstrate that factors known to increase brightness in bulk experiments lose importance at higher excitation powers, and that, paradoxically, the brightest probes under single-molecule excitation are barely luminescent at the ensemble level.

The invention discloses a single-cell / single-molecule imaging light / electricity comprehensive tester based on a multifunctional probe. The tester comprises the multifunctional nano-probe, a sample pool, an atomic force microscope system, a light source unit, an electricity testing unit, a cell sample locating system and a photon testing unit. The light source unit, the electricity testing unit, the cell sample locating system and the photon testing unit are respectively connected with a data collection and analyzing unit. The multifunctional nano-probe comprises an optical fiber layer, a nano electrode layer and an insulation layer. The nano electrode layer and the insulation layer are wrapped on the outer wall of the optical fiber layer in sequence. The synchronization comprehensive tester comprises a manufacturing method of the nano-probe which can carry out atomic force scanning imaging on single-cells / single-molecules and carry out light / electricity testing at the same time, a manufacturing method of the sample pool and relevant optics, electricity and mechanics testing and analyzing instruments. Temporal-spatial resolution and the range of target objects which can be tested are greatly improved, the tester can be widely used in biology and medicine and be used for fundamental research and disease testing under the level of sub-cells and molecules, and the tester has great development significance on tumor early diagnosis, medicine development and agricultural science.

Laminar flow of a carrier liquid and polymeric molecules through micro-channels is used to straighten the polymeric molecules and attach the straightened molecules to a wall of the micro-channel for subsequent treatment and analysis. Micro-channels can be manufactured using an elastic molding material. One micro-channel embodiment provides fluid flow using a standard laboratory centrifuge.

The invention provides a fluorescent chemical sensor and detection method for detecting oxidative damage in telomeres and an application of the fluorescent chemical sensor and the detection method. Specificity of a repair enzyme and polymerization mediated by terminal deoxynucleotidyl transferase are combined so as to start a cycle cutting signal probe induced by endonuclease IV, induce an AP sitein an Endo IV splitting signal probe, cause release of AF488 fluorophore on the signal probe, and besides, generate a signal probe having free 3'-OH end. Each signal probe having free 3'-OH can be effectively extended through TdT to generate a long poly-A sequence. The released poly-A sequence and a newly-generated poly-A sequence can induce the repeated cycle cutting of the signal probe so as torelease more AF488 fluorophore. Under magnetic separation, the fluorophore in supernatant can be imaged and quantified by unimolecule. The fluorescent chemical sensor and detection method have favorable actual application value.

RNA molecules are an important member of an intracellular genetic information delivery system. Conventional RNA labeling methods cannot simultaneously have the characteristics of living cells, no invasion, high temporal and spatial resolution, single molecules, etc., so a novel method for labeling and tracking RNA need to be developed. The invention discloses a novel labeling method for messengerRNA and circular RNA based on self-ligation label bimolecular fluorescence complementation. A RNA aptamer subsystem is cooperatively used, aptamers are inserted at proper positions in the RNA, and twoparts of a self-ligation label are respectively fused with the binding proteins of the RNA aptamers. When the fusion proteins do not bind to RNA, RNA molecules do not fluoresce; when and only when the fusion proteins bind to RNA, the RNA molecules fluoresce, so the fluorescence background of an intracellular RNA marker is reduced. The novel RNA labeling method based on self-ligation label bimolecular fluorescence complementation can be used for long-term interference-free single-molecule imaging observation and tracking of messenger RNA and circular RNA of living cells and fixed cells, and has good application prospects in cell biology.

The invention discloses a nanopore-micro lens scanning based super-resolution microscopic imaging system, related to the technical field of microscopic particle super-resolution imaging and used for solving the existing problem of imaging of a living cell under single molecule scale. The nanopore-micro lens scanning based super-resolution microscopic imaging system comprises a scanning optical system, a Z-axis scanning detection system, an inverted fluorescent microscopic imaging system and an automatic focus fixing and focus locking system. A super-diffraction limit focusing spot generated bya nanopore-micro lens stimulates a fluorescence signal of a sample, distance between a nanopore-micro lens focusing spot and the sample is determined according to the Z-axis scanning detection systemof the nanopore-micro lens, a three-dimensional piezoelectric ceramic displacement platform is controlled to realize three-dimensional direction scanning for the sample, the automatic focus fixing and focus locking system determines a focal plane of a microscope and the sample and locks the focal plane, and finally, super-resolution imaging is executed for the sample through an inverted fluorescent microscope. A nanopore-micro lens scanning based super-resolution microscope provided by the invention can be applied to single molecule imaging at the inside of the living cell.

The invention discloses a modifying glass slide for fixing biomacromolecules and a method thereof. The modifying glass slide comprises a glass slide and a cover glass, wherein a modifying layer which is connected with the surface of the glass slide by using a covalent bond is arranged between the glass slide and the cover glass; a liquid adding hole is formed in the glass slide; the modifying layer is bovine serum albumin marked with biotin or a polyethylene glycol derivative marked with the biotin; the glass slide is a quartz glass slide; the bovine serum albumin marked with the biotin can be 6-8 biotin molecules per albumin molecule; and the mass ratio of polyethylene glycol derivative marked with the biotin to polyethylene glycol derivative without being marked is between 3 and 10 percent. An intermediate dielectric layer is added by virtue of the liquid adding hole to be incubated at room temperature, and sample solutions are added again so as to realize fixation of biomolecules. By using the modifying glass slide, nucleic acid and protein biomolecules can be conveniently fixed, and biological single-molecule imaging data acquisition is realized.

The present invention provides for single-molecule profiling of combinatorial protein modifications and single-molecule profiling of combinatorial protein modifications combined with single-molecule sequencing of protein / nucleic acids complexes. High-throughput single-molecule imaging was applied to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. Applicants identified bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. Applying genetic and chemical perturbations of chromatin enzymes show a preferential affect on nucleosomes harboring specific modification states. The present invention also combines this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes. This novel single-molecule technology can be used to address fundamental questions in chromatin biology and epigenetic regulation leading to novel therapeutics and diagnostics.

The present invention generally relates to imaging single molecules, or one or more collections of single molecules, and methods related to the imaging. In a method aspect, the present invention provides a method of imaging single molecules. The method comprises the steps of: a) exposing a test sample to a probe, wherein the probe comprises a first portion that specifically binds to a target molecule and a second portion that is detectable as the result of one or more chemical groups that interact with light at one or more wavelengths, wherein the probe binds to a target molecule to provide a complex; b) exposing the complex to one or more wavelengths of light that interact with the one or more chemical groups; c) detecting a result from the interacting of one or more wavelengths of light that interact with the one or more chemical groups to provide an image of one or more single molecules. The image possesses a resolution better than 450 nm over a view field area of at least 1×105 μm2, and wherein the image is obtained in a single detection step without variation of any detection settings.

This invention relates to a method to rapidly find crystallization conditions for a biomolecule in a desired conformation using single-molecule, fluorescent resonance energy transfer (smFRET) imaging techniques. The method provides significant cost and time advantages over the empricial exploration for crystallization conditions.

A system for characterizing and sorting individual molecules, for example chromatin or DNA molecules. In some configurations, the system can immobilize molecules of interest suspended in fluid in a sample stage for characterization, and selectively release the immobilized molecules to different output reservoirs, depending on the characterization result. For example, fluorescent markers may be hybridized onto molecules, which may then be attached to dielectric beads and immobilized using holographic optical tweezers or other means. The system may provide elongational flow to elongate molecules, and may detect the presence and spacing of multiple fluorescent markers hybridized to the molecules. Using advanced techniques, the system may be able to characterize features with a resolution better than the theoretical resolution of the system optics. The system may utilize a microfluidic cartridge.

Provided is an e-exponential type nonlinear nano-metal taper probe high in spatial resolution, high in sensitivity and capable of producing a strong longitudinal polarizing electric field and adjusting the intensity of the electric field. The probe is composed of an e-exponential type nonlinear nano-metal taper structure. When incident light (especially radially polarized light) is irradiated to the bottom surface of the e-exponential type nonlinear nano-metal taper probe, surface plasmons are excited from the metal surface and are propagated along the curved surface of a nonlinear nano-metal taper and compressed to the top end to form highly and locally reinforced electromagnetic field distribution, and accordingly strong nano focusing is obtained. The probe can be used for scanning near-field microscopes, atomic force microscopes and other scanning probe microscopes and needle-enhanced Raman spectrometers and has the important application value in the multiple fields such as single-molecule imaging, heat-assisted magnetic recording, nano sensing, nano imaging, nano photoetching and nano control.

The invention provides a logarithmic type non-linear metal taper probe which is high in space resolution and flexibility and capable of generating strong longitudinal polarization electric field. The probe is formed by a logarithmic type metal nano non-linear taper structure. When incident light, especially radial polarized light, irradiates the bottom of the logarithmic type non-linear metal taper probe, the incident light energy is converted into surface plasmons. The plasmons are transmitted along the bending surface of the non-linear metal nano taper, and are compressed into the top end so as to form highly locally enhanced electromagnetic field distribution, thereby obtaining strong nano focusing. According to the invention, the probe can be used as a probe of a scanning probe microscope like a scanning near-field microscope and an atomic force microscope, and of a tip enhanced roman spectroscopy, and has high application value in fields like single-molecule imaging, heat auxiliary magnetic recording, nano-sensing, nano-imaging, nano-lithography and nano-control.

Single-molecule imaging techniques have been applied herein and have demonstrated the conformation states of HIV-1 Env. The introduction of small organic fluorophores at selected positions within HIV-1 Env that do not affect infectivity permits the detection of changes in inter-dye distances as a measure of conformational changes. Implementation of the smFRET-based technologies disclosed herein enable conformational screening for molecules that block, induce or trap HIV-1 Env in specific conformational states. Methods for identifying anti-HIV drugs in a smFRET-based screening, and various reagents useful for implementation of such methods, are provided.

The invention provides a low-cost high-efficiency machining method capable of machining symmetric multi-node micro-nano metal cone structures. According to the method, the core experiment principle is electricity chemistry corrosion. Under precise control of a precision displacement table, the corrosion time of each portion is controlled and is from long to short, corrosion is carried out from deep to shallow, and finally linear and non-linear metal cones with the tip end transverse radius within one micrometer are obtained. The electricity chemistry corrosion method is applied, in the aspect of micro-nano structure machining, the beneficial effects of extremely low cost and extremely high manufacturing efficiency are achieved, the method can be used for manufacturing probes in scanning near field microscopes and atomic force microscopes, and the manufactured low-cost metal robes has the very high utilization value in the aspects of single molecular imaging, thermally assisted magnetic recording, nano sensing, nano imaging, nano photoetching, nano manipulation and the like.

The invention relates to the field of biological vaccines, in particular to a porcine circovirus type 4 Cap protein monoclonal antibody as well as a preparation method and application thereof. Immunogen used for preparing the monoclonal antibody is virus-like particles formed by a porcine circovirus type 4 Cap protein; The porcine circovirus type 4 Cap protein comprises 3 core antigen epitopes, namely an epitope 1: RRRSRWRRKNGIFHARFMREVTLSVSSFST, an epitope 2: NAYYRIRKIKVEFLPLI and an epitope 3: SIQNSNFVQVWTVRFTL, which are as shown in SEQ ID NO: 3-5. The porcine circovirus type 4 Cap protein monoclonal antibody is prepared, a PCV4 Cap protein VLP single-molecular imaging enzyme-linked immunosorbent assay iELISA method taking a 6D5 monoclonal antibody as a capture antibody is established, and the antibody has the advantages of high sensitivity, strong specificity, good stability, high flux and the like.

The invention discloses an intra-cellular miRNA quantifying method. The method comprises the following steps of (1) fixing excised cells, and then performing cell nucleus dyeing; (2) adding a buffer solution containing fluorescent probes to the cells, and enabling the fluorescent probes and intra-cellular miRNA to be detected to generate reversible combination; (3) placing the cells on an objective table of a TIRF microscope, performing detection, recording the shape of the cells at a bright field, performing fluorescence recording of the position of a cell nuclei at a wide field, and recording unimolecule fluorescence intensity-time track in an HILO mode; and (4) performing signal filtration to obtain a unimolecule imaging graph in which the unimolecule fluorescence intensity-time track presenting ON-OFF alternate changes, namely an intra-cellular miRNA unimolecule imaging graph, and performing counting so as to obtain the quantity of intra-cellular miRNA. According to the method disclosed by the invention, an "in-situ quantifying imaging" manner is provided, high-identification degree unimolecule dynamics fingerprint signals are used, unimolecule positioning and pinpoint countingare performed on the intra-cellular miRNA, and the technical bottleneck that intra-cellular miRNA quantifying depends on RNA extraction is broken through.

The invention discloses a single-cell / single-molecule imaging light / electricity comprehensive tester based on a multifunctional probe. The tester comprises the multifunctional nano-probe, a sample pool, an atomic force microscope system, a light source unit, an electricity testing unit, a cell sample locating system and a photon testing unit. The light source unit, the electricity testing unit, the cell sample locating system and the photon testing unit are respectively connected with a data collection and analyzing unit. The multifunctional nano-probe comprises an optical fiber layer, a nano electrode layer and an insulation layer. The nano electrode layer and the insulation layer are wrapped on the outer wall of the optical fiber layer in sequence. The synchronization comprehensive tester comprises a manufacturing method of the nano-probe which can carry out atomic force scanning imaging on single-cells / single-molecules and carry out light / electricity testing at the same time, a manufacturing method of the sample pool and relevant optics, electricity and mechanics testing and analyzing instruments. Temporal-spatial resolution and the range of target objects which can be tested are greatly improved, the tester can be widely used in biology and medicine and be used for fundamental research and disease testing under the level of sub-cells and molecules, and the tester has great development significance on tumor early diagnosis, medicine development and agricultural science.

The invention discloses a single molecule dynamic detection method based on external force regulation and control. According to the single-molecule dynamic detection method, an external force regulation and control module is used for regulating combination and dissociation between a target molecule and a detection probe, a capture probe and nanoparticles, and a single-molecule imaging system is used for obtaining single-molecule imaging information and analyzing the dynamic state of the target molecule. The invention also discloses a system for detecting the dynamic state of the single molecule and application of the system. According to the invention, rapid, ultra-sensitive and high-throughput single-molecule immunodetection can be realized, and the detection limit can reach fM level or even lower; and the problem that the probe does not have universality is solved, the device cost is lower, the use threshold is lower, and the flux is larger.

The invention discloses an imaging method of RNA tailing and structures of in-situ cells, and belongs to the technical field of cellular imaging. The imaging method of the RNA tailing and the structures of the in-situ cells takes intracellular RNA tailing and various structures as research targets; and then, click-encoded rolling FISH (ClickerFISH) is adopted for realizing RNA tailing as well as single-strand and double-strand structures while realizing single-cell single-molecule imaging. The reaction system utilized by the imaging method of the RNA tailing and the structures of the in-situ cells disclosed by the invention is simple, high in reaction efficiency, as well as capable of realizing rapid and accurate imaging of intracellular RNA. Being used for studying intracellular RNA tailing, it has been found that the occurrence location and the length of RNA tailing are different in different cell lines and cell cycles; and moreover, being used for studying intracellular RNA single-strand and double-strand structures, it has been found that the distribution of the single-strand and double-strand structures are different in different cell lines and cell cycles.

The invention discloses a modifying glass slide for fixing biomacromolecules and a method thereof. The modifying glass slide comprises a glass slide and a cover glass, wherein a modifying layer which is connected with the surface of the glass slide by using a covalent bond is arranged between the glass slide and the cover glass; a liquid adding hole is formed in the glass slide; the modifying layer is bovine serum albumin marked with biotin or a polyethylene glycol derivative marked with the biotin; the glass slide is a quartz glass slide; the bovine serum albumin marked with the biotin can be 6-8 biotin molecules per albumin molecule; and the mass ratio of polyethylene glycol derivative marked with the biotin to polyethylene glycol derivative without being marked is between 3 and 10 percent. An intermediate dielectric layer is added by virtue of the liquid adding hole to be incubated at room temperature, and sample solutions are added again so as to realize fixation of biomolecules. By using the modifying glass slide, nucleic acid and protein biomolecules can be conveniently fixed, and biological single-molecule imaging data acquisition is realized.

The invention belongs to the technical field of single molecule imaging and particularly relates to a sub-millisecond real-time three-dimensional super-resolution microscopic imaging system. The system comprises a first objective lens, a second objective lens, a first plane, a second plane, a first plane mirror, a concave mirror, a second plane mirror and an EMCCD. According to the system in the invention, on the basis of a basic inverted fluorescence microscope high-speed two-dimensional detection system, the second objective lens is built, a fluorescence signal of the upper hemisphere surface is collected to the second plane, and then the fluorescence signal is returned to a sample plane by the concave mirror on the second plane; after being amplified by the second objective lens, the distance between the single-molecule signal and the central axis of the concave mirror is close to the magnitude of the focal length of the concave mirror, so as to reach the optimal measurement precision in the vertical direction.