134 results about "Two photon fluorescence" patented technology

A method for quantitatively measuring nucleic acid amplification reactions, especially the polymerase chain reaction, employing microparticles as hybridization solid phase, a probe sequence labeled with a fluorescent label and a fluorescence detection system which is based on two-photon fluorescence excitation, contacting all the amplification reaction components and the solid phase simultaneously in a closed cuvette, performing the amplification reactions in the same cuvette, focusing a two-photon exciting laser beam into the cuvette during the amplification cycles and measuring the fluorescence signal emitted by the microparticles from one particle at a time when they randomly float through the focal volume of the laser beam. The features of this invention allow a method and device for performing a fast quantitative nucleic acid amplification assay of single or multiple target sequences in a very small closed sample volume.

The invention discloses a method for two-photon fluorescence stimulated emission differential super-resolution microscopy. The method includes the steps that (1) after being collimated, pulsed laser beams are converted into linear polarized light and polarization modulation is conducted on the linear polarized light, so that radial polarized light is obtained; (2) the radial polarized light is converted into circular polarized light and projected onto a sample to be tested, two-photon stimulated emission is conducted, fluorescence is collected, and therefore first signal light intensity I1 is obtained; (3) polarization modulation is conducted on the linear polarized light obtained in the step (1) and the linear polarized light is converted into tangential polarized light; (4) the tangential polarized light is converted into circular polarized light and projected onto the sample to be tested, two-photon stimulated emission is conducted, fluorescence is collected, and therefore second signal light intensity I2 is obtained; (5) effective signal light intensity I is calculated according to a formula I=I1-gammaI2 , so that super-resolution imaging is achieved. The invention further discloses a device for two-photon fluorescence stimulated emission differential super-resolution microscopy. The device is simple, free of light division, low in light power, capable of weakening the photobleaching effect, higher in resolution and larger in imaging depth.

The invention provides a liquid-phase suspension biochip based on multi-optical trap encoding bead array and two-photon fluorescence detection. The liquid-phase suspension biochip has a configuration as follows: a near-infrared laser beam emitted by a near-infrared laser is expanded by a beam expanding system, is sequentially reflected by a telescope system and a dichroscope by using a holographic technology or a time-share scanning technology, and is focused into a sample pool through a high numerical aperture objective lens, to form multi-optical-trap optical tweezers; the multi-optical-trap optical tweezers capture a plurality of encoding beads enriched with objects to be detected, to form a bead array in the solution; after an infrared laser signal is filtered by a band-pass filter, a two-photon fluorescence signal from each bead is focused to an image detector by a lens and is subjected to imaging detection. The liquid-phase suspension biochip can perform real-time quantitative analysis on nucleic acids, proteins, virus particles and a plurality of objects to be detected, and has the advantages of high sensitivity, strong anti-interference ability, simultaneous determination of a plurality of components and the like.

The invention discloses a nitrogen-doped graphene quantum dot, a preparation method thereof and application thereof as a two-photon fluorescence probe to imaging of living cells. The nitrogen-doped graphene quantum dot is simple in preparation method, is good in imaging effect when applied to two-photon fluorescence imaging of living cells, and has extremely strong two-photon fluorescence in a relatively wide pH scope, and the two-photon absorption cross section can reach 48000 GM; the nitrogen-doped graphene quantum dot is extremely easy to dissolve in water, free of toxicity and good in light stability; and a solution of the nitrogen-doped graphene quantum dot has extremely strong stability, is capable of keeping stable within 24 months, and has wide application prospect.

A method for measuring the end point and for monitoring the real time kinetics of a bioaffinity reaction in biological fluids and suspensions, employing microparticles as bioaffinity binding solid phase, biospecific reagent labelled with a fluorescent label and a fluorescence detection system which is based on two-photon fluorescence excitation, contacting the analyte, the labelled reagent and the solid phase simultaneously, focusing a two-photon exciting laser beam into the reaction suspension and measuring the fluorescence signal emitted by the microparticles from one particle at a time when they randomly float through the focal volume of the laser beam. In this method the signal is monitored kinetically to obtain information about the analyte concentration before the reaction approaches the highest point of the response. Since the growth rate of the signal intensity is directly proportional to the analyte concentration, the analyte concentration can be predicted in the initial phase of the reaction. The growth rate monitoring also predicts whether the analyte concentration is higher than the binding capacity of the reagent and whether the reaction will be continued over the highest point of the response curve.

The invention discloses a novel dimolecular fluorescence probe of which the chemical name is 6-((4-nitrobenzyl)oxy)-2,3,4,4a-tetrahydro-xanthene-1-one. The invention also discloses application of the two-photon fluorescence probe in detecting nitroreductase in cells. The fluorescence intensity of the probe obviously increases as the nitroreductase content increases. The probe disclosed by the invention can be used for detecting the nitroreductase content in the tumor anoxic zone by a fluorescent imaging technique, thereby evaluating and researching the anoxic level of the tumor anoxic zone.

The invention relates to a two-photon fluorescence biological silk material and a preparation method thereof. The material is formed by evenly compounding two-photon inorganic fluorescent substances and biological silk protein materials at a mass ratio of (0.001-50):100. The excitation wavelength of the material is positioned near infrared, and the fluorescence wavelength is shorter than the excitation wavelength. The preparation method comprises the following steps: adding a fibroin solution into a solution containing the two-photon inorganic fluorescent substances; stirring to obtain a mixed solution; and drying, standing or spinning to obtain the two-photon fluorescence biological silk material. Alternatively, the preparation method comprises the following steps: at room temperature, adding pre-processed silk into a fibroin solution containing the two-photon inorganic fluorescent substances, and dipping;, and stirring, washing and drying to obtain the two-photon fluorescence biological silk material. The material provided by the invention has strong two-photon adsorption and adjustable two-photon fluorescence performance under the irradiation of near infrared laser; the wavelength of an excitation light source is bio-optically transparent and does not damage biological tissues; and the material has the advantages of good biocompatibility, strong mechanical property and good economic benefit, is easy to prepare and is suitable for industrial production.

The invention disclose a two photon absorption material capable of imaging alive cells, which is a terpyridine derivative Zn(II)S2 complex. The complex is a two photon absorption material having a property of two-photon fluorescence imaging of alive tissue cells, has the characteristics of large absorption area, low excitation energy, large wavelength, high penetrability, less light injuries, non-toxicity and the like and is suitable to be used for detecting alive cells.

The invention belongs to the field of chemistry and relates to an organic near-infrared two-photon fluorescent dye with a structure in a formula I in the specification. The organic near-infrared two-photon fluorescent dye has the beneficial effects that after being excited, the organic near-infrared two-photon fluorescent dye simultaneously shows stronger luminous efficiency of single-photon near-infrared fluorescence and two-photon fluorescence; the efficiency of two-photon fluorescence is obviously improved compared with that of other carbocyanine dyes; living cell fluorescence microscope experiments show that probes prepared from the fluorescent dye can enter tumor cell lysosome through endocytosis and can be imaged and simultaneously monitored by single-photon fluorescence and two-photon fluorescence microscopes; the dye can achieve synchronous single-photon near-infrared fluorescence and two-photon fluorescence imaging in the in-vivo and in-vitro states; and the fluorescent dye has the characteristics o good water solubility and stable chemical properties, can be used or bio-macromolecular markers and in the field of medical image diagnosis and especially has strong application value in optical image guided tumor excision.

Image formation via photoinduced fluorescence changes in a polymeric medium with two-photon fluorescence readout of a multi-layer structure. Fluorophore-containing polymers, possessing one or more basic functional groups, underwent protonation in the presence of a photoinduced acid generator upon exposure to a broad-band UV light source or fast-pulsed red to near-IR laser irradiation. Solution studies demonstrated formation of monoprotonated and diprotonated species upon irradiation, each resulting in distinctly different absorption and fluorescence properties. The fluorescence of the original, neutral, fluorophore was reduced upon monoprotonation, leading to a concomitant increase in fluorescence at longer wavelengths due to the monoprotonated form, the basis for multichannel data readout. Experiments in polymer films demonstrate the changes in fluorescence properties of the photosensitive polymer compositions and polymers can be employed for a high storage density, write-once read-many (WORM) data storage medium with two-photon fluorescence readout. Two-channel, two-photon fluorescence imaging provided both “positive” and “negative” image readout capability.

The invention provides a method for preparing two-waveband single-photon and two-photon fluorescent carbon quantum dots and application, and the method comprises the following steps: preparing an o-phenylenediamine solution or a mixed solution of the o-phenylenediamine and an acid; putting the o-phenylenediamine solution or the o-phenylenediamine mixed solution into a reaction kettle, heating, reacting to obtain a product, and carrying out centrifugal separation on the product to obtain the two-waveband fluorescent carbon quantum dots with single-photon and two-photon fluorescence emission characteristics at the same time. The preparation method disclosed by the invention is simple, economical and easy to operate, can be used for preparing two-waveband long-waveband fluorescence luminous carbon quantum dots at one time, has the characteristics of long-waveband single-photon and two-photon fluorescence excitation and emission, and has a good application prospect in the biomedical fieldsof fluorescence labeling of biological cells and tissues, living body imaging and the like.

The invention provides a dual-color two-photon fluorescence imaging method and a device, which uses six dual-color beams for emitting along plus and minus X, plus and minus Y, plus and minus Z six directions into the sample 3, thereby forming a three-dimensional lattice for exciting fluorescence, and uses a two-dimension small hole array 6 for filtering background fluorescence in the detecting light path, uses femtosecond pulsed laser with two different kinds of frequencies for composing a dual-color beam, wherein the two are coaxial, and the wavelengths are lambada 1 and lambada 2 respectively, moreover, lambada 1 is equal to lambada 2. Two kinds of light with different wavelength form two different three-dimensional lattice structures in the sample 3, and delay line 10 is modulated for causing the six beams to have the same optical path, only in the zone where two kinds of three-dimensional lattices coincide mutually, the dual-color two-photon fluorescence can be excitated. The device provided by the invention can effectively improve resolution response and imaging speed of three-dimensional imaging, and can be conveniently operated.

The invention belongs to the field of organic nonlinear optical materials, and particularly relates to a triphenylamine two-photon fluorescence probe compound and a preparation method and an application thereof. The preparation method of the triphenylamine two-photon fluorescence probe compound comprises the following steps of: injecting anhydrous benzene into 4-nitrapyrin-2, 6-dimethyl isophthalate and triphenylphosphine under the protection of nitrogen; increasing temperature, refluxing, and cooling; filtering and washing to obtain chlorination-4-methylene triphenylphosphine group-pyridine-2,6-dimethyl isophthalate, and dissolving into methanol; mixing and stirring tri(4-formylphenyl)amine and absolute methanol; adding the methanol solution of the chlorination-4-methylene triphenylphosphine group-pyridine-2,6-dimethyl isophthalate and the methanol solution of sodium methylate; stirring at 0-5 DEG C for 1-2 hours, stirring at normal temperature for 3-4 hours; separating through column chromatography. The triphenylamine two-photon fluorescence probe compound disclosed by the invention has the advantages of outstandingly enhanced two-photon absorption section, good water solubility, higher reaction activity and very high coordination capacity and can be applied to silver ion detection and pH detection.

This invention discloses a method for imaging live nucleus and cytoplasm, which comprises the following steps that: (a) live cells are irradiated by a near-infrared femtosecond laser which is focused by a microscope; (b) the live cells produce two-photon fluorescence and second harmonic under the action of the near-infrared femtosecond laser focused by the microscope; and (c) the two-photon fluorescence and the second harmonic are subjected to microscopic imaging at the same time, the two-photon fluorescence performs microscopic imaging on target molecules which are marked by fluorescence, and the second harmonic performs microscopic imaging on the nucleus. The invention also discloses application of the method for imaging the live nucleus and cytoplasm in monitoring a live nucleus and cytoplasm signal pathway, eliminates photoinduced toxicity and photobleaching, and can observe the live cells for a long time without affecting the activity of the cells and furthest maintain the functional environment of the cells. The method can be applied to the level layer research of cell molecules in a tumor generation mechanism and the research of a pharmacodynamical mechanism of traditional Chinese medicinal herbs.

The invention discloses a preparation method of a gold-graphene composite nano material. The preparation method is environment-friendly, efficient, simple, convenient and feasible, and the prepared gold-carbon composite material has a good application prospect in the fields of two-photon fluorescence, Raman enhancement, near-infrared absorption and the like.

The invention discloses a ratio-type two-photon formaldehyde fluorescent probe, a preparation method therefor and use of the ratio-type two-photon formaldehyde fluorescent probe. The ratio-type two-photon formaldehyde fluorescent probe has a structure as follows shown in the description. Molecules of the fluorescent probe disclosed by the invention show relatively high selectivity and sensitivity to formaldehyde in a coexisting system of molecules of the formaldehyde and other aldehydes, amino acids and the like. Proven by cytotoxicity tests, the molecules of the fluorescent probe disclosed by the invention are almost free of toxic action on cells; and shown by two-photon fluorescence microscopy imaging experiments, the molecules of the fluorescent probe disclosed by the invention have good cell permeability to MCF-7 cells, so that the fluorescent probe is applicable to the detection of presence of formaldehyde molecules in the cells.

The invention relates to preparation of a probe capable of realizing two-photon fluorescence detection of nitroreductase (NTR) as shown in the figure (I) and belongs to the field of organic fluorescent probes. The structural formula of the probe capable of realizing two-photon fluorescence detection of the NTR is as shown in the description. The fluorescent probe provided by the invention can accurately detect the content of the NTR in cells and avoid the interference of other reducing agents in cells. In addition, the probe has the characteristics of better chemical stability, biological compatibility and selectively and the like. A laser confocal imaging experiment shows that the probe has better cell permeability, has no toxic and side effects on cells and organisms, can realize detection of content of cell level NTR and indicate the cell hypoxia condition, and can be further applied to research of precancerous detection.

The invention provides a two-photon lysosome pH fluorescence probe, a preparation method thereof and an application. The probe is a pH fluorescence probe based on an intramolecular charge transfer effect and structured by bridging benzimidazole with carbazole derivative. The preparation method includes steps of, dissolving benzimidazole and carbazole derivative in a few amount of N, N- dimethyl formamide; adding trimethylchlorosilane to catalyze and reacting it in a seal pipe; then adding saturated Na2CO3 solution and adjusting pH value to alkalescence; acquiring a pure product through vacuum concentration and silicagel column separation. The probe has pKa value approaching to pH value balance of lysosome. The probe has high sensitivity, good selectivity and big Stocks displacement to H+. After combining with H+, the probe has obvious color change, and can be identified by bared eyes. The probe takes the carbazole derivative as a two-photon fluorescence group; the two-photon fluorescence imaging technology is used for proving that the probe and the marketed green lysosome probe have good common polarization, and can be applied to detect pH value change in the HeLa cell lysosome.

The invention discloses a nitrogen-and-sulfur-codoping-based graphene quantum dot material as well as a preparation method and application thereof. The method provided by the invention adopts a bottom-up preparation method to synthesize nitrogen and sulfur codoped graphene quantum dots by one step in an alkali aqueous solution of ammonium sulfite from small organic molecular 1,3,6-trinitropyrene.The method provided by the invention is simple in step as well as high in repeatability and yield. As the surfaces of the nitrogen and sulfur codoped graphene quantum dots contain more hydroxyl functional groups, amino functional groups and sulfonic functional groups, and the nitrogen and sulfur codoped graphene quantum dots are very easily dissolved into water and stably exit in an aqueous solution for a long time. For the graphene quantum dots provided by the invention, the two-photon fluorescence performance is remarkably improved, the imaging effect is further improved, and the imaging depth in biological tissues is further increased. As a two-photon fluorescence probe, the nitrogen and sulfur codoped graphene quantum dots prepared by the method provided by the invention has a very good two-photon fluorescence characteristic, can emit stronger fluorescence under the excitation of near-infrared wavelengths and can show a good imaging effect to the biological tissues.

The invention discloses a two-photon fluorescence probe for detecting peroxynitrite as well as a preparation method and application of the two-photon fluorescence probe. The two-photon fluorescence probe has the structural formula as shown in the description. The two-photon fluorescence probe is named as TPNIR-FP. The two-photon fluorescence probe adopts a Nile red derivative as a fluorescent group and an alpha-keto-amide functional group as an ONOO-reaction group, can rapidly respond to ONOO- (within 10 seconds), and in addition is high in sensitivity and good in selectivity. The two-photon fluorescence probe can be applied to imaging research on ONOO- in antharcycline antibiotic induced cardiac muscle cell and cardiac muscle tissue damage.

The invention discloses a two-channel two-photon fluorescent polar probe, a preparation method and uses thereof, wherein the structure of the two-channel two-photon fluorescent polar probe is definedin the specification. According to the present invention, the two-channel two-photon fluorescent polar probe has specific response to polarity in a system containing the probe and other interference factors; and the cytotoxicity test results show that the probe has almost no toxic side effects on cells, and the two-photon confocal fluorescence microscopy imaging experiment results show that the probe has good permeability to HeLa cells and can effectively locate the mitochondria in the cells (positioning coefficient of 0.95), such that the probe is suitable for the two-channel two-photon fluorescence imaging and quantitative detection of the polarity in cell mitochondrial.

The invention belongs to the field of quantum information, in particular to a method for generating two-photon radiation on the basis of a single quantum dot. Generation of two photons has great significance in the quantum information field such as quantum key distribution, quantum entanglement, quantum measurement and the like. The method for generating two-photon radiation on the basis of the single quantum dot is provided and comprises steps as follows: firstly, a near infrared single quantum dot is prepared on a slide with a indium tin oxide film formed through evaporation plating; then, indium tin oxide nanoparticles cover the single quantum dot; the indium tin oxide nanoparticles are spin-coated with polystyrene polymer films, and influence of the external environment on the quantum dot is effectively isolated; laser co-focusing is performed on the sample, and two-photon fluorescence radiation is generated for the single quantum dot. With the adoption of the indium tin oxide nanoparticles, nonradiative auger recombination of biexciton of the quantum dot is effectively inhibited, and two-photon radiation of the single quantum dot is realized.

The invention discloses a femtosecond laser device based on single cladding neodymium optical fibers and a ring cavity and a manufacturing method. The femtosecond laser device comprises a cavity part and a space optical path part, wherein the cavity part comprises 808 nm single mode semiconductor optical pumps (1), 808 nm optical fiber type single mode isolators (2), 808 / 920 nm wavelength division multiplexers (3), a single cladding neodymium-doped gain optical fiber (4) and 920 nm optical fiber collimators (5); the space optical path part comprises a low-pass dichroscope (6), 920 nm 1 / 4 wave plates (7), 920 nm polarization beam splitter prisms (8), a 920 nm Faraday optical rotation device (9), 920 nm 1 / 2 wave plates (10) and a birefringence filtering piece (11). Mode locking potential of the neodymium-doped optical fiber is explored between 900 nm and 920 nm, on the premise that single pulse energy is guaranteed, light-light conversion efficiency is improved, and meanwhile the laser device provides a better light source facilitating integration for an integrated two-photon fluorescence microscope.

The invention discloses a two-photon fluorescent probe for detecting polarity and viscosity through two channels as well as a preparation method and application thereof. The two-photon fluorescent probe can monitor the real-time change of polarity and viscosity in cell mitochondria in two different channels at the same time, and the structural formula of the two-photon fluorescent probe is shown in the specification. The two-photon fluorescent probe molecule can perform fluorescence response on polarity (short wave with the wave length of 410 nm) and viscosity (long wave with the wave length of 580 nm) at the same time by utilizing two different fluorescence bands. Furthermore, a cytotoxicity test shows that the probe has almost no toxic or side effect on cells; a two-photon confocal fluorescence microimaging experiment shows that the probe has good permeability to HeLa cells, can effectively locate mitochondria (the locating coefficient is 0.97) in the cells, and is suitable for two-channel two-photon fluorescence imaging and detection of polarity and viscosity in the cell mitochondria.

The invention discloses design and synthesis of a near-infrared aggregation-induced emission material with strong two-photon absorption as well as organelle imaging and photodynamic therapy application of the near-infrared aggregation-induced emission material. Through reasonable molecular structure design, four near-infrared aggregation-induced emission materials with large two-photon absorptionsections and high brightness are obtained. The materials have relatively large Stokes shift, good biocompatibility and excellent light stability. Three neutral materials can specifically image lipid droplets, and the other cationic material can specifically image mitochondria. In view of their excellent two-photon properties, the four materials can be used for two-photon fluorescence imaging of cells and living tissues. The four materials can effectively generate singlet oxygen under illumination, so that the singlet oxygen can be used for two-photon excited organelle targeted photodynamic therapy. The material synthesized by the method shows the property of aggregation-induced emission, and effectively overcomes the defects of the traditional fluorescent dye.

Disclosed are several technical approaches of using low coherence interferometry techniques to create an autofocus apparatus for optical microscopy. These approaches allow automatic focusing on thin structures that are positioned closely to reflective surfaces and behind refractive material like a cover slip, and automated adjustment of focus position into the sample region without disturbance from reflection off adjacent surfaces. The measurement offset induced by refraction of material that covers the sample is compensated for. Proposed are techniques of an instrument that allows the automatic interchange of imaging objectives in a low coherence interferometry autofocus system, which is of major interest in combination with TDI (time delay integration) imaging, confocal and two-photon fluorescence microscopy.

The invention relates to a two-photon fluorescent bio-silk material and a preparation method thereof. The material is prepared by evenly compounding a two-photon organic fluorescent substance and a bio-silk protein material, wherein the mass ratio of the two-photon organic fluorescent substance to the bio-silk protein material is (0.001-50):100; the excitation wavelength of the two-photon fluorescent bio-silk material is in near-infrared, and the fluorescence wavelength of the same is shorter than the excitation wavelength. The preparation method of the two-photon fluorescent bio-silk material comprises the following steps of: adding pretreated silk into a dimethyl sulfoxide solution containing the two-photon organic fluorescent substance at 20-110 DEG C, stirring, washing and drying to obtain the finished product; or adding a silk protein solution to the dimethyl sulfoxide solution containing the two-photon organic fluorescent substance, stirring to obtain a mixed solution, and drying, standing or spinning to obtain the finished product. The two-photon fluorescent bio-silk material has stronger two-photon absorption performance and adjustable two-photon fluorescence performance under near-infrared laser irradiation; an excitation light source has photobiologically transparent wavelength and has no damages to biological tissues; and the two-photon fluorescent bio-silk material has favorable biocompatibility, strong mechanical property, easiness of preparation and good economic benefit and is suitable for industrial production.

A method and a device for increasing the chromatographic depth of two-color two-photon fluorescence imaging are provided. The method utilizes an outer light beam with an inner light beam inside thereof, and the two-color light coaxially passes through the same focusing objective lens (3) and focuses on a sample (4) to excite the sample to emit fluorescent light. Two femto-second pulsed lasers of different frequencies are used as excitation light sources to emit two laser beams, wherein one beam has a ring-shaped cross section and the other has a round cross section, the two beams are coaxial and complementary to each other, and the two beams are only superposed in a focal area. The aplanatic excitation two-photon fluorescence signals of the two beams in the focal area are regulated by means of delay lines, so as to eliminate background signals other than the focus. The device of the invention can efficiently increase the chromatographic depth of three-dimensional imaging, eliminate background fluorescence and increase speech-noise ratio, and be convenient for operation.

A multi-mode scanning device of a two-photon fluorescence microscope is disclosed. The scanning device comprises a first laser used for emitting a first laser source, a first incident light path comprising a first galvanometer scanner, a second incident light path comprising a resonance-type scanner, an emergent light path comprising a second galvanometer scanner and a micro objective, a first rotatable reflector used for communicating the first laser with the first incident light path or the second incident light path, and a second rotatable reflector used for communicating the emergent light path with the first incident light path or the second incident light path. The first laser source is adopted as incident light and switched and selected through the first rotatable reflector and the second rotatable reflector so as to allow the incident light to enter the emergent light path through the first incident light path or through the second incident light path. The scanning device is low in cost, high in efficiency and high in precision and facilitates real-time switching between global scanning and local scanning by a user.

The invention relates to a polyethylene glycol / stannic sulfide intercalation quantum dot and a hydrothermal synthesis method. The intercalation quantum dot takes stannic sulfide as the host and polyethylene glycol as the guest, and the polyethylene glycol enters stannic sulfide layers to obtain the quantum dot with a particle size of 3-5nm. Compared with the prior art, the preparation method is simple, the operation is easy, the obtained quantum dot is provided with superior single / two-photon fluorescence performance, and the quantum yield can reach up to 65%.