CN108548798A - With structure and the application of the relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure and its related drugs screening technique - Google Patents
With structure and the application of the relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure and its related drugs screening technique Download PDFInfo
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
- G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
- G01Q60/18—SNOM [Scanning Near-Field Optical Microscopy] or apparatus therefor, e.g. SNOM probes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The present invention relates to a kind of structure and applications with the relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure and its related drugs screening technique; by the size of the large biological molecules particles such as optics or image checking intracellular level abundant myosin, tubulin or Agglomerate, shape, position, quantity or its protein marker density and intensity, to evaluate variation and the zone location of intracellular colloid osmotic pressure.The of the present invention and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure can analyze the change of intracellular colloid osmotic pressure, and combine osmometer, parse crystal osmotic pressure and colloid osmotic is pressed in the composition and function of intracellular osmotic pressure.
Description
Technical field
The present invention relates to a kind of large biological molecule optical detecting methods, and in particular to a kind of with intracellular colloid osmotic pressure phase
Structure and the application of the large biological molecule optical detecting method and its related drugs screening technique of pass.
Background technology
Chemistry, electricity and mechanics activity are to maintain the citation form of cell activities, although the above two are furtherd investigate,
It is that intracellular mechanics activity is little affected by concern, is the blind spot in current cell research field.The a variety of physiology of cell and pathological activity according to
Rely its internal mechanics activity, such as cell growth and division, contraction, oedema, differentiation, the polarization of nerve, malignant tumour invasion transfer
Deng the cellular morphology change result that necessarily its interior mechanics pulls.
Although microfilament and micro-pipe traction force based on power molecular (myosin, dynein and kinesin) and with
Crystalline percolation pressure based on ion channel is identified very early, and as the movable important driving of regulating cell interior mechanics because
Element.But power is physics vector characteristic index, different and scalar, not only has size, also direction to distinguish, intracellular mechanical function
It is how to realize that vector (direction) changes not knowing.
The depolymerization of microfilament and micro-pipe can not only eliminate microfilament and micro-pipe relies on the drawing tension that power molecular is implemented, because of its solution
Content is extremely abundant in the cell for polycondensation monomer (β-myosin, α, 'beta '-tubulin), and each monomer accounts for about intracellular total protein concentration
1-10% is formed by large biological molecule particle and its condensate size between 4-100nm, can effectively form colloid and ooze
Pressure thoroughly, inducing cell expands outward, reverses microfilament and the inside drawing tension of micro-pipe, realizes that the movable vector of intracellular mechanics turns
Become, constitutes the movable new model of cell interior mechanics and new regulatory mechanism.
Invention content
Present invention aim to address the deficiency that the prior art identifies living cells and intracellular colloid osmotic pressure, provide it is a kind of with
The intracellular relevant large biological molecule optical detecting method of colloid osmotic pressure passes through inspection using the optical property of protein colloid
Intracellular level abundant protein particulate and its condensate, i.e. the main composition source of intracellular colloid osmotic pressure are surveyed, born of the same parents are measured
The variation of interior colloid osmotic pressure and the variation of eucaryotic cell structure tension vector.
To achieve the above object, technical solution provided by the invention is：
A kind of and the relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, optics or image checking cell
The size of the large biological molecules such as myosin, tubulin of interior rich content particle or Agglomerate, shape, position, number
The density and intensity of amount or its protein marker, to evaluate variation and the zone location of intracellular colloid osmotic pressure, method includes
Following steps：
A) smudge cells extract cytoplasm, carry out luminous intensity detection using the imaging with enough resolution ratio, determine life
The size and number of object macromolecular particle or Agglomerate establishes colloid in conjunction with the osmotic pressure numerical value that osmotic pressure instrument detects
Osmotic pressure changes the correspondence with colloid optics difference；
B) imaging is carried out to cell or tissue cell using the imaging with enough depth resolutions and optical strength is examined
It surveys, determines the position of large biological molecule particle or Agglomerate, the density and intensity of quantity or its protein marker；By
The relationship of the osmotic pressure and optics difference of foundation, thus it is speculated that the regional change of colloid osmotic pressure.
The present invention observes cell or thin according to protein adhesive volume property --- Tyndall effect using details in a play not acted out on stage, but told through dialogues microtechnic
Cytoplasm extracting solution detects its scattering (backward, vertical and forward direction) or transmitted light, to measure its particle position, size and number ratio
Example.Specifically used dark field microscope such as comes card, Olympus brand dark-field microscope, realizes the survey of protein colloid particle
It is fixed.
According to Tyndall effect, cytoplasm extracting solution is detected using the fluorescence detector with enough depth resolutions, is commented
Valence and the relevant protein particulate of colloid osmotic pressure or its protein marker, quantify intracellular protein particle or it combines fluorescence
The brightness or fluorescence volume that matter is sent out.And its characteristic and quantity is associated with colloid osmotic pressure numerical value, as evaluation colloid osmotic
The function of pressure.The mode specifically taken includes receiving using nanometer particle size analyzer Zetasizer Nano ZS90 and same type
Grain of rice diameter analyzer, by scattering or transmiting its absorbance of light detection or turbidity, granular size and quantity.It is sensitive to increase detection
Degree, can be used fluorescent label protein body, in conjunction with fluorescence excitation and corresponding fluoroscopic examination, improves and is measured to nano particle
Accuracy.
It is combined, and is had with the protein particulate of intracellular rich content using the fluorescent molecular that can be combined with large biological molecule
Nano-particle characteristic only just sends out characteristic fluorescence signal when being combined with large biological molecule.Part fluorescent material can penetrate cell
Film is combined with the protein particulate of cell.Or nano-metal particle may pass through capilary barrier, realize that animal and tissue are horizontal special
Determine the label of intracellular biological macromolecular, and the macromolecular of fluorescent marker is determined in conjunction with super-resolution fluorescence micro-imaging technique
Position and counting.Fluorescent dye or nano-metal particle are chosen according to specific research contents first.The wherein described fluorescent material is
Fluorescent molecular selected from the following：Oxazine dye AOI987, curcumin derivate matter, thioflavin S, thioflavin T, it is Congo red, its
Meaning combination and its arbitrary physiological compatibility derivative；The fluorescence protein gene that above-mentioned protein body coupling can also be used, is transferred to
Cell marks unimolecule protein body；Or selection nano-metal particle fluorescent material, such as the silicon ball body core of polyethylene glycol involucrum.
Second step selects the super-resolution fluorescence micro-imaging technique with nm class resolution ratios, has：Random optical reconstructs microtechnic
(STORM/dSTORM), photoactivation positioning microtechnic (PALM), the photosensitive position finding microscope of fluorescence (FPALM), scattering formula near field
Scanning optical microscope (s-SNOM), stimulated emission depletion microtechnic (STED), interference photoactivation position microtechnic
(iPALM), rotating disk Confocal laser endomicroscopy (SDCM), super-resolution optical fluctuation imaging technique (SOFI), saturated structures illumination
Microtechnic (SSIM), reversible saturation optical transition technology (RESOLFT), scan angle interference microtechnic (SAIM) etc., implement light
Learn detection.
Protein particulate concentration is measured according to the special light absorptive that specific protein has.Such as it is a large amount of existing in cell
Actin, tubulin.Tyrosine and tryptophan are rich in two kinds of albumen, and tyrosine and tryptophan have at 280nm
There is absorption maximum, therefore actin and tubulin are measured by measuring absorbance value of the protein solution at 280nm, claims
For ultraviolet absorption method.
The deficiency for solving prior art identification living cells and intracellular colloid osmotic pressure of the present invention provides a kind of optics inspection
Survey and optical imaging method.As the of the present invention and relevant large biological molecule optical detection side of intracellular colloid osmotic pressure
Method analyzes the change of intracellular colloid osmotic pressure, and combines osmometer, parses crystal osmotic pressure and colloid osmotic is pressed in intracellular and oozes
The composition and function pressed thoroughly.
It is another object of the present invention to build related drugs according to the research of optical detection of the present invention and screen cell platform,
Screening adjusts relevant drug with colloid osmotic pressure.
Compared with prior art, the beneficial effects of the invention are as follows：
The of the present invention and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, can analyze intracellular glue
The change of body osmotic pressure, and combine osmometer, parse crystal osmotic pressure and colloid osmotic be pressed in intracellular osmotic pressure composition and
Function, with important research significance.Its related drugs screening cell platform, screening and colloid osmotic pressure tune can be built accordingly
Save relevant drug.
Description of the drawings
Fig. 1：Using nanometer particle size analyzer ZetasizerNanoZS90 oblique fire or scattering light detection its absorbance, particle
Grain size and quantity.
Fig. 2：Myosin or tubulin particle or its polymeric osmotic pressure are corresponding with Count rate (kcps)
Curve.
Fig. 3：The eucaryotic cell structure correction map of Image J softwares processing.
Fig. 4：The standard curve that actin or tubulin concentration are measured by using ultraviolet absorption method.
Specific implementation mode
Various embodiments of the present invention and aspect can illustrate in detail below.
Following description and diagram explain the present invention but are not construed as limitation of the present invention.Described a large amount of spies
Determine particulars and provides comprehensive understanding for various embodiments of the present invention.However, in some cases, in order to the present invention's
Embodiment offer is concisely discussed, and does not describe some it is known that or conventional particulars.
Including terms used herein "comprises/comprising" is interpreted as including and be it is open, it is inexclusive.Especially
Include in claims used in this specification, "comprises/comprising" and its variant indicate special characteristic, step or ingredient by including
Wherein.These terms cannot be construed to exclude the presence of other features, step or ingredient.
Terms used herein " exemplary " mean " can be used as example, example or illustration ", are not construed as than herein
Disclosed other configurations are preferably or advantageous.
Terms used herein " about/about " and " general ", when with particle size range, mix ingredients or other physics
When property and feature are used in conjunction, mean and cover in size range minor change that may be present in bound, it is averagely big to be not excluded for
The embodiment that portion size meets but the size except the range statistically may be present.The application is not intended to exclude such as
These embodiment.
The protein bodies such as myosin, tubulin can generate from cytoskeletal structure depolymerization, also itself can synthesize increase,
It polymerize and regional change becomes the important mechanisms of the outside tension regulation and control of cell, constitutes the outside stretching force of cell, driving is thin
The outside mechanics activity of born of the same parents' form.
Intracellular protein amounts of particles and its size can be quantified using Tyndall phenomenon.Use dark field imaging technique
To determine the subcellular localization variation of protein particulate in the cell.Meanwhile polarization is had studied using coupling fluorescin
Forward position, such as the growth cone of neuron, the distribution of large biological molecule particle and its variation of colloid osmotic pressure.In addition, utilizing high score
Resolution imaging technique is studied being located at the distribution of nerve cell large biological molecule.This form is with dark-field microscopy to intracellular
It is almost the same that protein particulate carries out cellular localization.Therefore, the development and application of high-resolution imaging technology are conducive to intracellular life
The observation and detection of object macromolecular.
Analyze intracellular protein particulate labels.Alternate labels object includes fluorescent material, spectral signal, differential polarization letter
Number, optical path difference or scattering the equation of light it is different.These signals may be from intracellular protein particle.Then higher magnifying power can be utilized
There is the region of marker in display in amplification expected areas, and the shape and ruler of marker are then assessed in the region differentiated before
Very little feature and intensity and spatially distributed signal.
The first possible marker includes the fluorescent molecular nontoxic to body cell comprising but be not limited to, intelligent light
Learn probe (only just emitting characteristic fluorescence signal when being combined with protein particulate) and other fluorescent dyes, such as near-infrared fluorescent
Oxazine dye μ OI987, curcumin derivate CRANAD-2, thioflavin T, thioflavin S or derivatives thereof, rhodamine or the Congo
It is red.If using fluorescence, the method for tissue and cell imaging includes that can excite selected point with single or double photon excitation
The wavelength of son detaches the filtration system of incident light and fluorescence, and the detector to sensitive fluorescent.Select detector optical filter and
Detector protrudes the wavelength of fluorescence of the marker combined with protein molecular, so that background tissues fluorescence unobvious.
Some workable fluorescent markers are the markers that can pass through blood-brain barrier.This allows to detect in animal level
It can implement to be injected intravenously in the process.
The fluorescent dye delivered alternatively by liposome methods.After this dyestuff discharges in similar cranial nerve cell
It can be positioned in axoneure.In the case of the single or double photon excitation of fluorescence, excitation beam can penetrate multi-layer cellular, swash
Fluorescing dye simultaneously returns to fluorescence.Above-mentioned fluorescence can be additionally used in the structure and shape that determine protein particulate.Equally, protein
Grain can use fluorochrome label.
Second of possible marker of protein particulate is spectrum.This include but not limited to Raman spectrum, absorption spectrum,
Fluorescence correlation spectroscopy, NMR spectra, quasi-elastic light scattering spectrum, circular dichroism spectra and Fourier trasform spectroscopy, spectral signal can be by
The protein particulate being rich in individually generates or the protein particulate by being combined with dyestuff generates.
As the third possible marker, intracellular protein particle is inhaled by the difference of polarised light (i.e. optical activity)
It receives, transmission, scatter or reflect, or by polarization spectrum or pass through the difference of the polarised light from intracellular protein particle
It reflects to show.In addition, optically active dyestuff can be used.Pass through known comparison of the enhancing with difference polarization characteristic structure
Polarization imaging method (such as the confocal laser scanning microscopy improved using Muller matrix polarimetry) or detection structure, reason
It is different to be that they have the function of the polarization characteristic of light.
4th kind of possible marker is to include protein particulate below with scattering signatures and difference endogenous characteristic
Light.Show using dark-field imaging or using super-resolution microscope.
Other than using above-mentioned marker, other optical technologies also can be used to assess the close of intracellular protein particle
Degree.It can be used by scattering light measurement about 4nm to the other methods of the particle of hundreds of nm sizes, because of each intracellular protein
Particle can form difference scattering light.These methods include aforementioned proteins particle scattering characteristics or difference endogenous it is optical
Matter, to determine protein particulate.
According to the principle for measuring after protein is marked used by aforementioned present invention people or directly assessing, the present invention
People is imaged protein particulate, positions and is measured using following technology or instrument：Dark-field microscope, super-resolution fluorescence are aobvious
Micro- imaging technique, using dynamic light scattering as the nanometer particle size analyzer of cardinal principle, it is micro than turbid instrument, according to specific protein
Special light absorptive measures protein particulate concentration method.
The first instrument is the nanometer particle size analyzer using dynamic light scattering as cardinal principle, such as Zetasizer Nano
The nanometer particle size analyzer product of ZS90 ex hoc genus anne types.
Proteins In Aqueous Solutions particle can make scattered light intensity fluctuating fluctuation and the dynamic that is formed at any time because of Brownian movement.When
By cytoplasm, the actin and tubulin particle that encounter in solution can scatter a branch of interference light to all directions.If
Grain does random Brownian movement, and scattered light intensity the random fluctuation near average intensity, scattering light frequency can also be sent out at any time
Raw minor change can obtain light intensity auto-correlation function at any time according to the fluctuation situation of this light intensity under certain angle
Variation, and then extrapolate actin and tubulin granular size and distribution.
In order to realize the purpose of detection intracellular colloid osmotic pressure numerical value change, the present invention provides one kind by using nanometer
Particle size determination instrument, draws myosin or tubulin particle or its polymeric particle ratio is corresponding with colloid osmotic pressure bent
The method of line speculates the variation of intracellular colloid osmotic pressure and for the diagnosing and treating medicine with colloid osmotic pressure relevant disease with this
The screening of object.
Wherein, the detection method includes：
1, cell 15min is stimulated using (1,1/2,1/4,1/8,1/16,0 times) of microfilament or microtubule depolymerization agent various dose,
Induce intracellular myosin or tubulin particle and its different degrees of formation of Agglomerate.Wherein, latrunculin-A is
Cytochalasin D (Cyto D), initial concentration are 5 μM；Microtubule depolymerization agent is Nocodazole (Noc), and initial concentration is 2 μ
M。
2, the culture solution in Tissue Culture Flask is outwelled, is cleaned twice with appropriate 4 DEG C of PBS.
3, (12000rpm, 30min, 4 DEG C) is centrifuged, sedimentation cell abandons supernatant culture medium, retains cell precipitation.
4,15-30 seconds broken using hypervelocity.
5,40000rpm centrifuges 15min, draws supernatant, i.e. cytoplasm (20-40 μ l).
6, cytoplasm osmotic pressure value is measured using osmometer (such as U.S. Advanced freezing point osmotic pressure gauges).
7, using nanometer particle size analyzer ZetasizerNanoZS90 oblique fire or scattering light detection its absorbance, particle
Diameter and quantity (as shown in Figure 1).
The data obtained when in order to enable detecting solution to be measured are more accurate, and ZetasizerNanoZS90 is used for preparing
When the sample of detection, the concentration of solution to be measured can be further qualified, Cmin 0.5g/l, maximum concentration should be with not
Subject to so that protein body is mutually assembled, being gelled.
8, pair of myosin or tubulin particle or its polymeric osmotic pressure and Count rate (kcps) is drawn
Curve is answered, the composition of cytoplasm crystal (or ion) osmotic pressure is parsed.Nerve is stimulated using drug (microfilament or microtubule depolymerization agent)
Spongiocyte 15min.It repeats the above steps (in addition to the first step).Using cytoplasmic penetration pressure as Y-axis, the actin of depolymerization generation
Or tubulin quantity is X-axis, is handled by SPSS17.0 softwares, obtains linear Y=0.3149X+152.92.As X=0, Y
=152.9, indicate that ionic osmotic pressure is 152.9Osm/Kg in cytoplasm.Thus cytoplasm colloid osmotic pressure under normal circumstances is
147.1Osm/Kg left and right.(see Fig. 2)
9, the numerical value correspondence of colloid osmotic pressure and Count rate can be directly obtained.
Second is micro than turbid instrument.It may be used including photoelectric turbidimetry, Immunity transmission turbidity, immunization powder than turbid instrument
Penetrate a variety of methods including turbidimetry, resonance light-scattering turbidimetry method.Using exciting light irradiation by cytoplasm extracting solution, flesh moves egg
White or tubulin or other antigen antibody complexs scatterings and absorption make the transit dose of light reduce.Using than turbid instrument
Accurately measure optical density or light transmittance.In a certain range, protein or antigen antibody complex granule density and light transmittance
It is inversely proportional, it is directly proportional to optical density (or scattering light).Optical density is measured with a series of suspension of known amounts of particles, it is close to make light extraction
Degree-protein particulate number standard curve.Then, with the optical density measured by sample liquid, actin is found from standard curve
Or tubulin quantity.
In order to realize the purpose of detection intracellular colloid osmotic pressure numerical value change, the present invention provides one kind by using immune
Scattered light urbidmetry, draws myosin or tubulin particle or its polymeric particle ratio is corresponding with colloid osmotic pressure bent
The method of line speculates the variation of intracellular colloid osmotic pressure and for the diagnosing and treating medicine with colloid osmotic pressure relevant disease with this
The screening of object.
Scattered light urbidmetry is using IMMAGE automatically than turbid instrument (BECKMAN COULTER.Inc.) and matched reagent and school
Quasi- product.Quality-control product is homemade cytoplasm extracting solution.
Wherein, the detection method includes：
1, determining instrument withinrun precision and betweenrun precision.It can be used when coefficient of variation CV≤5%.
2, the titer for selecting known actin or tubulin or its polymer concentration, by gradient dilution at 5.1%,
7.5%, 10.5%, 13.5%, 15.0% sample solution
3,6 parts of samples are repeatedly measured using scattered light urbidmetry, measurement result is handled by SPSS17.0 softwares, obtains line
Property standard curve.Wherein Y is scattering turbidimetry instrument measured value, and X is protein body concentration.
4, can sample to be tested be measured by using scattered light urbidmetry, protein solution concentration is calculated using standard curve,
The variation of intracellular colloid osmotic pressure is speculated with this.
The third detecting instrument is dark-field microscope, also known as dark field microscope, is designed according to Tyndall effect principle
It is a kind of under the conditions of black background observe be detected object microscope, it is observed that the extremely small object that light field can't see
Body can differentiate presence and movement of the diameter in the particle of 4-200nm ranges, therefore dark field microscope can be used for measuring including flesh
Intracellular protein including filamentous actin, tubulin and polymer.Dark field microscope uses central barn door or dark field optically focused
Device (the most commonly used is parabolic condensers), makes the central beam of light source be blocked, and from bottom to top cannot enter object by sample
Mirror is obliquely radiated on the cell to be observed to make light change approach, and cell meets light and reflection or scattering, scattering occurs
Light input object lens in, thus whole visual field is dark.It is intracellular protein particle what is observed in dark field
Diffracted light image (not protein particulate itself), can observe the presence and movement of protein particulate, so as to born of the same parents
Intracellular protein and polymer including interior actin, tubulin carry out relative quantification and positioning.
In order to realize the purpose of detection intracellular colloid osmotic pressure numerical value change, it is micro- using details in a play not acted out on stage, but told through dialogues that the present invention provides a kind of
Mirror collocation total internal reflection illumination, dynamic imaging and phase are carried out to myosin in living cells or tubulin particle or its condensate
To quantitative method, the variation of intracellular colloid osmotic pressure is speculated with this and is used for the diagnosis with colloid osmotic pressure relevant disease and controls
Treat the screening of drug.
Wherein, using dark field microscope arrange in pairs or groups total internal reflection illumination to myosin in living cells or tubulin particle or
Its condensate carries out dynamic imaging and the method for relative quantification includes：
It is cultivated under conditions of 1, all cells to be contained to 5% carbon dioxide at 37 DEG C.
2, in cell growth and experimentation, cell is suspended in the cell culture medium for the fetal calf serum for being added to 10%
In (the modification type culture medium (DMEM) of Dulbecco).
3, the Glass bottom culture dish of ibidi GmbH, Martinsried, Germany models, ibidi μ-Dish 1.5h are selected
The coverslip of (170 μm +/- 5 μm) model.Glass bottom culture dish and coverslip are thoroughly cleaned up.Before each experiment, will
Cell detachment is transferred in Tissue Culture Flask in Glass bottom culture dish, is covered with coverslip.
4, using a kind of parallel, linear polarization 488nm laser beams deflecting from two axis dip sweeping mirrors.488mm's
Laser is found sample excitation in suitable region and is imaged.
5, acousto-optic tunable filter is placed at beam channel beginning, ensures that laser power is constant in P=5.0mW.One
Beam diameter is expanded to 14 millimeters by 20x optical beam expanders, and then go directly scanning mirror.
6, specific diaphragm is placed in polypropylene, to constitute dark field detection, stops all total internal reflection light.Only dissipate
Light is penetrated by diaphragm and lens L4, and image is formed on sCMOS cameras.
7, Image Acquisition is carried out with the frame per second of 100Hz
8, caused perseverance is reflected in order to eliminate the interface being added on each original image by microscope optics
Fixed background interference, it is necessary to an individual background image be subtracted from all original images, obtain that one clear, reflection is repaiied
Positive eucaryotic cell structure clearly final image.
9, image real time transfer completes (see Fig. 3) in Image J softwares.
According to image-forming principle, the brightness of regional area is directly proportional to protein particulate concentration in image, with light transmittance at anti-
Than.The protein particulate in protein particulate concentration (light transmittance the is high) region low higher than brightness in brightness height (light transmittance is low) region is dense
Degree.By way of relative quantification, the large biological molecules such as intracellular myosin, tubulin particle can be detected or particle is poly-
The density and intensity of fit position, quantity or its protein marker, it is fixed with the variation and region of evaluating intracellular colloid osmotic pressure
Position.
4th kind of detection technique is super-resolution fluorescence micro-imaging technique, including random optical reconstruct microtechnic
(STORM/dSTORM), photoactivation positioning microtechnic (PALM), the photosensitive position finding microscope of fluorescence (FPALM), scattering formula near field
Scanning optical microscope (s-SNOM), stimulated emission depletion microtechnic (STED), interference photoactivation position microtechnic
(iPALM) etc..It is described in detail followed by enumerating：
1) photoactivation positioning microtechnic (Photoactivated Localization Microscope, PALM).Make
Mark myosin or tubulin with fluorescin, by adjust laser energy, low energy irradiating cell surface, one
Then the secondary several fluorescent moleculars for only activating sparse distribution under the visual field adjust laser irradiation and excite fluorescence again, quasi- by Gauss
It closes to be accurately positioned the position of myosin or tubulin.It is located correct to bleach these then to reuse laser irradiation
Fluorescent molecular, so that them is not come out by the laser reactivation of next round.And then secondary recycling laser is activated and is bleached
Other fluorescent moleculars.After continuing repeatedly to recycle, intracellular most of fluorescent moleculars are accurately positioned.By these molecules
Image is synthesized on a figure, is constantly repeated to excite and be detected, enough fluorescent moleculars may finally be precisely located out, profit
Myosin or the image of tubulin super-resolution are reconstructed with these several subgraphs, to realize quantifying for protein.This
Outside, PALM technologies are combined with the principle of interference of light, i.e., referred to as interferometry photoactivation positions microtechnic
(interferometric Photoactivated Localization Microscopy,iPALM).IPALM can be used for
Nanoscale protein microstructure is observed, the measurement of actin and tubulin can be equally realized according to above-mentioned steps.
2) random optical reconstruct microtechnic (Stochastic Optical Reconstruction Microscope,
STORM).Using light conversion fluorescence dyestuff as fluorescence probe.Using Alexa Fluor647, Alexa Fluor532 and
The cyanine dye labellings such as ATTO532 myosin or tubulin, this fluorescent dye have in the solution containing thiol compound
In can realize light and shade convert, can be directly realized by light conversion.Fluorescence point may be implemented in irradiation by controlling different wave length exciting light
Son is mutually converted between illuminated state and dark-state, realizes the mesh for being separated from each other to reach single molecular imaging of adjacent phosphor molecule
, to realize the nanoscale imaging of labelled protein particle.STORM technologies by reconditioning, positioning and quenching fluorescence molecule,
The super-resolution image of a width cell inner mark albumen can be reconstructed by so recycling after hundreds of times to thousands of times, to realize born of the same parents
The measurement of interior protein body and metering.
3) the photosensitive position finding microscope of fluorescence (Fluorescence Photoactivation Localization
Microscopy,FPALM).Using can photoactivation green fluorescent protein (PA-GFP) fluorescin come mark myosin or
Tubulin, the thousands of single fluorescent molecular of each collection analysis are glimmering with low excitation intensity one-time positioning small part therein
Optical molecule.For these can photoactivation molecule, active rate controls by activation illumination；Non-fluorescence inactive molecule is by high frequency
(405-nm) laser active, then sends out fluorescence when lower frequency excites.Digital image sensor (CCD) camera is to fluorescence
Be imaged, then by these molecules either reversibly inactivation or irreversibly photobleaching it to be removed from the visual field.Light
The speed of bleaching for exciting the intensity of the laser of fluorescence by controlling, usually using Ar+ ion lasers.Because when given
Between only a small amount of fluorescent molecular be visible, so their position can be accurately determined, positioning accuracy compares high resolution
10 times, reach the spatial resolution better than 10nm.Accordingly, it is determined that the quantity of the intracellular proteins such as myosin or tubulin and
Distribution.
4) stimulated emission depletion microtechnic (STED).Use fluorescent protein labeling myosin or tubulin.Fluorescence
Electronics in molecule is in ground state, is irradiated by exciting light, absorbs photon and jumps to unstable excitation state.Electronics is swashing
Hair state can transit to ground state, be simultaneously emitted by autofluorescence.When the electronics of excitation state encounter wavelength be equivalent to excitation state and ground state it
Between energy difference photon when, generate stimulated radiation.Sample exciting light is combined with loss light, and is focused on by object lens to be measured
On protein example.Then, sample is excited using the light of loss light and exciting light combination, two beam laser collective effects are in fluorescence point
On son, fluorescent molecular can spontaneous radiation fluorescence, and positions of those light irradiations that are depleted are excited loss effect and not will produce
Fluorescence, to realize the positioning and metering of the intracellulars labelled protein such as myosin or tubulin.
5) scattering formula near-field scanning optical microscope (scattering-type Scanning Near-field
Optical Microscopy,s-SNOM).Near field refers to the scale and probe and observing object for the probe for being used as physical observation object
Distance be respectively less than for observation radiation wavelength.Cytoplasm extracting solution sample to be measured is excited by far field excitation or evanescent wave, SNOM
Needle point collects optical signal as detection probe.Probe point by point scanning on cytoplasm extracting solution surface acquires intracellular protein particle
The variation of various scattered optical field information near surface near field range is imaged after record, obtains actin in cytoplasm point by point
With the pattern and optical information of the protein bodies such as tubulin, it is combined into optical imagery.
In order to realize detection intracellular colloid osmotic pressure numerical value change purpose, the present invention provides one kind by using
The method that STORM technologies are imaged actin or tubulin speculates that the variation of intracellular colloid osmotic pressure is used in combination with this
In the screening of the diagnosing and treating drug with colloid osmotic pressure relevant disease.
Wherein, include using the method that STORM platforms carry out optical detection to intracellular actin or tubulin：
1, cell 15min is stimulated using microfilament or microtubule depolymerization agent, induce intracellular myosin or tubulin particle and
The different degrees of formation of its Agglomerate.Wherein, latrunculin-A be Cytochalasin D (Cyto D), a concentration of 5 μM；
Microtubule depolymerization agent be Nocodazole (Noc), a concentration of 2 μM.
2, #1.5 models are selected, the glass slide that specification is 22mm*22mm.Glass slide and coverslip are thoroughly cleaned up.
3, cell inoculation is cultivated in the orifice plate or culture dish handled well, cell density is unsuitable excessively high.
4, using containing 3% paraformaldehyde and 0.1% glutaraldehyde cytoskeleton buffer solution (Cytoskeletal Buffer,
CB, formula see the table below) actin filament of fixed cell；Micro-pipe egg is fixed using the phosphate buffer containing 4% paraformaldehyde
In vain.
5, using the fixed sample of paraformaldehyde, sample is rinsed so that its generation is quenched using the glycine solution containing 50mM
Autofluorescence；Using the fixed sample of paraformaldehyde, using the wash buffer containing 0.1% sodium borohydride of fresh configuration with
Eliminate autofluorescence.
6, permeable membrane is carried out using non-ionic detergents such as TritonX-100 or NP-40, is arrived for 0.05% using concentration range
0.5% (v/v).
7, fluorescent marker is carried out to actin or tubulin.
Actin staining procedure (by taking the ware of diameter 33mm as an example)：
(1) cell sample is taken out, culture solution is sucked.The CB wash buffers cell 2 times, every time 2 points preheated with 1mL37C
Clock.
(2) fixed：Fixers of the 1mL containing 3% paraformaldehyde and 0.1% glutaraldehyde is taken to fix cell 10 minutes, after fixed
It is rinsed 3 times, every time 5 minutes with 2mLPBS.
(3) permeable membrane：The 0.25%Triton X-100 room temperatures permeable membrane of the fresh configurations of 1mL is added 10 minutes, increases cell
Permeability.PBS is rinsed 2 times, every time 5 minutes.
(4) in order to reduce autofluorescence, 0.1% sodium borohydride solution of lmL Fresh is added, room temperature acts on 7 minutes.
PBS is rinsed, 10 minutes every time.
(5) it closes：Fresh configuration 3% bovine serum albumin(BSA) (BSA) the solution 37C closing cells of 1mL are added 1 hour, prevent
Antibody non-specific adsorption is rinsed 3 times with PBS later.
(6) it dyes：The phalloidine fluorescence antibody that Alexa Fluor647 are marked is diluted with the PBS solution containing 1%BSA
(7) it fixes afterwards：The paraformaldehyde solution that 1mL4% is added repeats to fix 10 minutes, and PBS is rinsed；With 50mM glycine
Solution rinses cell 2 times, every time 5 minutes.
Tubulin staining procedure：
(1) cell sample is taken out, culture solution is sucked, cell is rinsed with the PBS solution of 37C preheatings；
(2) phosphate buffer of 1mL4% paraformaldehydes is taken to fix cell 10 minutes, PBS is rinsed；
(3) permeable membrane and closing can refer to the experimental procedure of actin；
(4) primary antibody is incubated：2ugAnti-a-tublin antibody is diluted in 1mLPBS solution, cell sample, room is added
Temperature effect 1 hour, 2mLPBS is rinsed 3 times, every time 5 minutes.
(5) secondary antibody is incubated：2ug Alexa Fluor 647-labeled Goat Anti-Mouse antibody is diluted in
In PBS solutions of the 1mL containing 1%BSA, 37 DEG C are incubated 2 hours, and PBS is rinsed 3 times, every time 5 minutes.
(6) fixing step is same as above afterwards.
8, fluorescent microsphere benchmark particle is added in the sample, is fixed on slide, passes through calculating benchmark particle and mesh
The relative position of phosphor dot is marked to calibrate image.Stoste dilutes 1000 times with PBS solution, and 4C is preserved.In use, ultrasound can be used
Oscillation 10 minutes, to ensure that fluorescent microsphere is evenly distributed.
9, after benchmark particle is added in sample, imaging buffer solution is added in sample and is impregnated 1 minute, can be put into later aobvious
Micro- imaging system carries out super-resolution imaging.
10, the laser of 640mm is used to be imaged sample excitation to find suitable region.
11, after finding suitable imaging region (must assure that and contain benchmark particle in region), gradually increase exciting light
Intensity is until sample is largely converted to dark-state.
12, low intensive activation light (532nm) is added makes part fluorescent molecular be restored to illuminated state by dark-state, activates light
Intensity is generally located between 1.5%-3%.When single molecular fluorescence point is evenly distributed in image, i.e. available EMCCD carries out image
Acquisition, region acquires about 30,000 frame images.
13, image real time transfer is completed in Matlab softwares.
14, it estimates cytoplasm internal protein quantity, calculates cytoplasm colloid osmotic pressure.
5th kind of technology is to measure protein particulate concentration according to the special light absorptive that specific protein has.Such as cell
In a large amount of existing actin, tubulins.Tyrosine and tryptophan, and tyrosine and tryptophan are rich in two kinds of albumen
There is absorption maximum at 280nm, therefore it is to measure actin and micro-pipe egg to measure absorbance value of the cytoplasm at 280nm
White ultraviolet absorption method.When measurement, cytoplasmic droplet to be measured is added on micro optical detector, on ultraviolet spectrometry degree meter directly
The absorbance value for reading 280nm calculates protein particulate based on tyrosine and the special absorbance of tryptophan substantially
Concentration.
In order to realize the purpose of detection intracellular colloid osmotic pressure numerical value change, the present invention provides one kind by using ultraviolet
The method that absorption process is measured actin or tubulin concentration speculates that the variation of intracellular colloid osmotic pressure is used in combination with this
In the screening of the diagnosing and treating drug with colloid osmotic pressure relevant disease.
Wherein, the detection method includes the drafting of standard curve and calculates corresponding protein compression according to ultraviolet absorptivity
Degree：
1, draw 1.0 respectively with pipette, 1.5,2.0,2.5,3.0mL 3.00mg/mL standard proteins solution is in 5
In 10mL colorimetric cylinders, it is diluted to scale with 0.9%NaCl solution, is shaken up.This series containing protein is respectively：0.3、0.45、
0.6、0.75、0.9mg/mL。
2, with 1cm quartz colorimetric utensils each standard solution is measured respectively at 278nm using 0.9%NaCl solution as reference
Absorbance A 278 records data.
3, using standard protein solution concentration as abscissa, absorbance is ordinate, draws standard curve.(see Fig. 4)
4, it is handled by SPSS17.0 softwares, obtains linear Y=4.3298X+51.795.
5, the 50 μ L of testing protein solution of appropriate concentration are taken, measure the absorbance at 278nm, parallel survey as stated above
Determine three times.
6, establishing criteria curve measures the protein content of sample to be tested.
It is exemplified below several specific embodiments：
Embodiment 1
Nanometer particle size analyzer Zetasizer Nano ZS90 based on dynamic light scattering principle measure myosin or micro-
Tubulin particle and its Agglomerate
It should be noted that the present inventor be intended to by this embodiment illustrate nanometer particle size analyzer observation myosin or
The operating method and principle of tubulin particle and its Agglomerate, it is not limited to Zetasizer Nano ZS90 instruments,
Also it is not limited to actin or tubulin and its Agglomerate, but suitable for being all based on dynamic light scattering principle
Nanometer particle size analyzer and all protein and other macromoleculars, the above embodiments are merely illustrative of the technical solutions of the present invention and
It is unrestricted, although being described the invention in detail with reference to preferred embodiment, it will be understood by those of ordinary skill in the art that,
Technical scheme of the present invention can be modified or replaced equivalently, without departing from the spirit of the technical scheme of the invention and model
It encloses, is intended to be within the scope of the claims of the invention.
The drafting (Fig. 2) of standard curve
1, cell 15min is stimulated using (1,1/2,1/4,1/8,1/16,0 times) of microfilament or microtubule depolymerization agent various dose,
Induce intracellular myosin or tubulin particle and its different degrees of formation of Agglomerate
2, under the conditions of 4 DEG C, centrifugation cell abandons supernatant culture medium, retains cell precipitation, and 15-30 is crushed using hypervelocity
Second.
3,40000g centrifuges 15min, draws supernatant, i.e. cytoplasm (20-40 μ l).
4, cytoplasm osmotic pressure value is measured using osmometer
5, big using nanometer particle size analyzer ZetasizerNanoZS90 oblique fire or scattering light detection its absorbance, particle
Small and quantity.
6, pair of myosin or tubulin particle or its polymeric osmotic pressure and Count rate (kcps) is drawn
Answer curve.Composition and the colloid osmotic pressure for parsing cytoplasm crystal (or ion) osmotic pressure are corresponding with the numerical value of Count rate
Relationship.
As X=0, Y=152.9 indicates that ionic osmotic pressure is 152.9Osm/Kg in cytoplasm；Because in normal cell,
Osmotic pressure value is 300Osm/Kg, thus its colloid osmotic pressure is 147.1Osm/Kg or so.
It measures
1, using drug (glutamic acid) stimulation Deiter's cells 15min.
2,2,3,5 and 6 are repeated the above steps
7, according to above-mentioned formula, the composition and numerical value of cytoplasm crystal and ionic osmotic pressure are calculated
Note：Four groups of data are that normal cell, depolymerization of microfilaments cell, microtubule depolymerization cell, microfilament and microtubule depolymerization are thin respectively
The osmotic pressure value of born of the same parents and its corresponding protein body ratio.
Embodiment 2
Total internal reflection dark field microscope observes myosin or tubulin particle and its Agglomerate in the cell
Quantity and distribution
It should be noted that the present inventor is intended to illustrate that dark field microscope observes myosin or micro-pipe by this embodiment
The operating method and principle of protein body and its Agglomerate, it is not limited to which actin or tubulin and its particle are poly-
Zoarium, but be suitable for all protein and other macromoleculars, the above embodiments are merely illustrative of the technical solutions of the present invention and
It is unrestricted, although being described the invention in detail with reference to preferred embodiment, it will be understood by those of ordinary skill in the art that,
Technical scheme of the present invention can be modified or replaced equivalently, without departing from the spirit of the technical scheme of the invention and model
It encloses, is intended to be within the scope of the claims of the invention.
1. the cytoplasm of cell or extraction in culture dish/coverslip is placed under high-resolution dark field condenser.
2. can select to dye target protein, such as actin is coloured using conjugation rhodamine-phalloidine
About 1h makes actin particle convenient for observation.
3. condenser aperture is adjusted to 1.4.
4. the diaphragm hole of light source is adjusted to maximum.
5. putting a big drop cedar oil on concentrator, sample is set on objective table, screwing on concentrator makes oil be connect with glass slide
It touches (cannot have bubble).
6. being carried out matching optical registration object with low power objective and 7 × eyepiece.The height for adjusting concentrator, first on glass slide
There is the aperture that there is a stain in a centre, is finally a bright luminous point, luminous point is smaller better, and thus putting will be above and below concentrator
Luminous point is made to increase when mobile.
7. changing required eyepiece and high power lens, slowly rise object lens and focus, until luminous sample occurs in central region.
8. dripping cedar oil on the cover slip, and oil mirror is gone to and should adjust light distribution in position, is observed.
9. carrying out the record of image using CCD camera.
10. often completing primary experiment, concentrator distilled water and 70% ethyl alcohol rinse 3 times, then use ultraviolet disinfection 30
Minute.
Embodiment 3
Special light absorptive based on specific protein, is measured actin or tubulin granule density.
It should be noted that the present inventor is intended to illustrate the special light absorptive of specific protein in target by this embodiment
Operating method in protein body assay and principle, it is not limited to actin or tubulin, but suitable for complete
Portion's protein and other macromoleculars, the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to compared with
Good embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the spirit of the technical scheme of the invention and range, should all be covered at this
In the right of invention.
The drafting (Fig. 4) of standard curve
1, cell 15min is stimulated using (1,1/2,1/4,1/8,1/16,0 times) of microfilament or microtubule depolymerization agent various dose,
Induce intracellular myosin or tubulin particle and its different degrees of formation of Agglomerate
2, under the conditions of 4 DEG C, centrifugation cell abandons supernatant culture medium, retains cell precipitation, and 15-30 is crushed using hypervelocity
Second.
3,40000g centrifuges 15min, draws supernatant, i.e. cytoplasm.
4, cytoplasm osmotic pressure value is measured using osmometer
5, using its absorbance of the ultraviolet light detections of 280nm or protein content.Because of β-myosin or alpha-tubulin β-
Tubulin richness tryptophan, high-content have more significant light absorption value.
6, myosin or tubulin particle are drawn or its polymeric osmotic pressure is corresponding with protein content (mg/ml)
Curve.Parse cytoplasm crystal and the composition and colloid osmotic pressure of ionic osmotic pressure and the numerical value correspondence of protein content.
It measures
1, using drug (glutamic acid) stimulation Deiter's cells 15min.
2,2,3,5 and 6 are repeated the above steps
7, according to above-mentioned formula, the composition and numerical value of cytoplasm crystal and ionic osmotic pressure are calculated.
Note：Four groups of data are that normal cell, depolymerization of microfilaments cell, microtubule depolymerization cell, microfilament and microtubule depolymerization are thin respectively
The osmotic pressure value of born of the same parents and its corresponding protein content ratio.
The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, any ripe
Professional and technical personnel is known, without departing from the scope of the present invention, according to the technical essence of the invention, to above real
Apply any simple modification, equivalent replacement and improvement etc. made by example, still fall within technical solution of the present invention protection domain it
It is interior.
Claims (10)
1. a kind of and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, it is characterised in that：Optics or at
As size, the shape of the large biological molecules particles such as detection intracellular level abundant myosin, tubulin or Agglomerate
Shape, position, quantity or its protein marker density and intensity, to evaluate variation and the zone location of intracellular colloid osmotic pressure,
Its method includes the following steps：
A) smudge cells extract cytoplasm, and luminous intensity detection is carried out using the imaging with enough resolution ratio, determine that biology is big
The size and number of molecule particles or Agglomerate establishes colloid osmotic in conjunction with the osmotic pressure numerical value that osmotic pressure instrument detects
Pressure changes the correspondence with colloid optics difference；
B) imaging is carried out to cell or tissue cell using the imaging with enough depth resolutions and optical strength detects, really
Determine the position of large biological molecule particle or Agglomerate, the density and intensity of quantity or its protein marker；By having been established
Osmotic pressure and optics difference relationship, thus it is speculated that the regional change of colloid osmotic pressure.
2. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：It is detected using cell or cytoplasm extracting solution, dyestuff and fluorochrome label, various fluorescins coupling mark can be used
Remember above-mentioned sample, improve detection sensitivity, and realizes living cells unimolecule or the observation of molecule aggregation fluorescence dynamic indicator and quantization
Evaluation.
3. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：The combination of fluorescence of natural light, polarised light and specific wavelength or both can be used in configuration excitation light source, with protrusion
The fluorescence volume that intracellular biological macromolecular particle or its fluorescent material combined are sent out, and make any background fluorescence unobvious.
4. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：It is detected using details in a play not acted out on stage, but told through dialogues micro-imaging or dark field optical, using Tyndall phenomenon, is observed and scattered by highly sensitive detector
Light, transmitted light come the sub- intracellular distribution of detection of particles, amounts of particles and dynamic change；Measure micelle optical signalling Assessment of Changes its
Quantity, distribution and its variation of related osmotic pressure.
5. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：Shown using ultrahigh resolution microscope, nm grades of particulate intracellular distributions can be observed；The optical signalling for measuring micelle becomes
Change the variation for evaluating its quantity and its related osmotic pressure.
6. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：Using with nanoparticle sub-feature oxazine dye AOI987, curcumin derivate matter, thioflavin S, thioflavin T and
Congo red arbitrary combination and its arbitrary physiological compatibility derivative, send out characteristic fluorescence signal when being combined with large biological molecule,
It is coupled the gene of fluorescin using above-mentioned protein body, is transferred to cell, marks unimolecule protein body.
7. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：Using nano-metal particle fluorescent material, capilary barrier is may pass through, combination dye label can realize animal and tissue
The measurement of the detection of large biological molecule particle and its related colloid osmotic pressure in horizontal specific cells.
8. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：Its marker is the interaction of protein particulate and imaging as a result, and being light according to protein particulate
Activity is learned, any one of Difference Absorption, scattering, transmission, projection and reflection of generation detect.
9. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：In conjunction with the cytoplasm osmotic pressure numerical value that freezing point, crystal and colloid isosmoticity instrument detect, ion and colloid osmotic are differentiated
The osmotic pressure effect of the composition and each self-generating that are pressed in cytoplasm, quantitative assessment large biological molecule particle optical signal and colloid
The relationship of osmotic pressure numerical value.
10. the according to claim 1 and relevant large biological molecule optical detecting method of intracellular colloid osmotic pressure, special
Sign is：Speculate regional imaging optical change according to colloid osmotic pressure and the relationship of large biological molecule particle optical signal numerical value,
Corresponding colloid osmotic pressure numerical value change.
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| CN113237807A (en) * | 2021-03-22 | 2021-08-10 | 南京中医药大学 | Quantitative analysis technology for transmembrane osmotic pressure of protein nanoparticles regulated by each component of biological body fluid |
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