CN108548798B - Biomacromolecule optical detection method related to intracellular colloid osmotic pressure and construction and application of related drug screening method - Google Patents
Biomacromolecule optical detection method related to intracellular colloid osmotic pressure and construction and application of related drug screening method Download PDFInfo
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- 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 invention relates to a biomacromolecule optical detection method related to intracellular colloid osmotic pressure and construction and application of a related drug screening method thereof. The optical detection method of biomacromolecules related to the intracellular colloid osmotic pressure can analyze the change of the intracellular colloid osmotic pressure and analyze the composition and the function of the crystal osmotic pressure and the colloid osmotic pressure in the intracellular osmotic pressure by combining an osmometer.
Description
Technical Field
The invention relates to an optical detection method for biomacromolecules, in particular to an optical detection method for biomacromolecules related to intracellular colloid osmotic pressure and construction and application of a related drug screening method.
Background
Chemical, electrical and mechanical activities are the basic forms of life activities of the maintenance cells, and although the former two are studied intensively, intracellular mechanical activities are of little concern and are blind in the current field of cellular research. The various physiological and pathological activities of cells depend on internal mechanical activities such as cell growth and division, contraction, edema, differentiation, nerve polarization, invasion and metastasis of malignant tumors and the like, and the morphological change of cells is necessarily the result of internal mechanical traction.
Although microwire and microtubule drag forces based on motiles (myostatin, dynein and kinesin) and crystal osmotic pressure based on ion channels have long been recognized and become important drivers for the regulation of intracellular mechanical activity. However, the force is a physical vector characteristic index, and different from a scalar quantity, the force and the direction are different, and it is not clear how the intracellular mechanical action realizes vector (direction) change.
The depolymerization of the microwire and the microtubule can not only eliminate the traction tension of the microwire and the microtubule depending on the implementation of dynamic molecules, because the content of depolymerized monomers (beta-myosin, alpha, beta-tubulin) in cells is very rich, each monomer accounts for about 1-10% of the total protein amount in the cells, the formed biological macromolecular particles and the polymer thereof have the size between 4-100nm, can effectively form colloid osmotic pressure, induce the cells to expand outwards, reverse the inward traction tension of the microwire and the microtubule, realize the vector transformation of intracellular mechanical activity, and form a new form and a new regulation mechanism of the intracellular mechanical activity.
Disclosure of Invention
The invention aims to solve the defects of identifying the osmotic pressure of living cells and intracellular colloids in the prior art, and provides a biomacromolecule optical detection method related to the osmotic pressure of intracellular colloids.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
an optical detection method of biomacromolecule related to intracellular colloid osmotic pressure, which optically or imagewise detects the size, shape, position and quantity of biomacromolecule particles or particle aggregates such as myosin, tubulin and the like which are abundant in cells or the density and intensity of protein markers thereof so as to evaluate the change of intracellular colloid osmotic pressure and the regional location, and the method comprises the following steps:
a) breaking cells, extracting cytoplasm, carrying out light intensity detection by using imaging light with sufficient resolution, determining the size and the number of biomacromolecule particles or particle aggregates, and establishing a corresponding relation between colloid osmotic pressure change and colloid optical difference by combining an osmotic pressure value detected by an osmometer;
b) imaging and optical intensity detection of cells or tissue cells with imaging light of sufficient depth resolution to determine the location, number of biomacromolecule particles or particle aggregates or the density and intensity of their protein markers; the regional variation of the colloid osmotic pressure is presumed by means of the established relationship between osmotic pressure and optical difference.
According to the invention, according to the properties of protein colloid, namely the tyndall effect, a dark field microscopy technology is adopted to observe a cell or cytoplasm extracting solution and detect scattered (backward, vertical and forward) or transmitted light to determine the particle position, size and quantity ratio. The measurement of the protein colloidal particles is specifically carried out using dark field microscopes such as come, olympus and other brands of dark field microscopes.
Detecting the cytoplasmic extract by an optical detector with sufficient depth resolution based on the tyndall effect, evaluating protein particles or protein markers thereof associated with oncotic pressure, and quantifying the intensity or fluorescence emitted by intracellular protein particles or fluorescent substances thereof. And correlating its properties and quantities with values of colloid osmotic pressure as a function of the evaluated colloid osmotic pressure. The specific adopted method comprises the step of detecting the absorbance or turbidity, the particle size and the quantity of the Nano-particles by scattering or transmitted light by using a Nano-particle size tester Zetasizer Nano ZS90 and a Nano-particle size tester of the same type. To increase the detection sensitivity, the protein particles can be labeled with fluorescent labels, which in combination with fluorescence excitation and corresponding fluorescence detection, improves the accuracy of the nanoparticle assay.
Fluorescent molecules capable of being combined with biological macromolecules are combined with protein particles rich in intracellular content, the fluorescent particles have the characteristic of nanoparticles, and characteristic fluorescent signals can be emitted only when the fluorescent molecules are combined with the biological macromolecules. Part of the fluorescent substance is able to penetrate the cell membrane and bind to the protein particles of the cell. Or the nano metal particles can penetrate through the microvascular barrier to realize the labeling of biomacromolecules in specific cells at the level of animals and tissues, and the super-resolution fluorescence microscopic imaging technology is combined to position and count the fluorescently-labeled macromolecules. Firstly, fluorescent dye or nano metal particles are selected according to specific research contents. Wherein the fluorescent substance is a fluorescent molecule selected from the group consisting of: oxazine dye AOI987, curcumin derived substance, thioflavin S, thioflavin T, Congo red, any combination thereof and any physiologically compatible derivative thereof; the fluorescent protein gene coupled with the protein particles can also be transferred into cells to mark single molecule protein particles; or selecting nanometer metal particle fluorescent substance, such as silicon sphere core coated by polyethylene glycol. Second, selecting a super-resolution fluorescence microscopy imaging technique with nm resolution, comprising: random optical reconstruction microscopy (STORM/dSTORM), light-activated positioning microscopy (PALM), fluorescence photosensitive positioning microscopy (FPALM), scattering type near-field scanning optical microscopy (s-SNOM), stimulated emission depletion microscopy (STED), interference light-activated positioning microscopy (iPALM), rotating disk confocal microscopy (SDCM), super-resolution optical fluctuation imaging (SOFI), Saturated Structure Illumination Microscopy (SSIM), reversible saturated light transition (RESOLFT), Scanning Angle Interference Microscopy (SAIM), and the like.
The protein particle concentration is measured in terms of the specific absorbance that a particular protein has. Such as actin, tubulin, which are present in large amounts in the cell. The two proteins are rich in tyrosine and tryptophan, which have maximum absorption at 280nm, so that actin and tubulin are measured by measuring absorbance value of protein solution at 280nm, which is called ultraviolet absorption method.
The invention solves the defect of identifying the osmotic pressure of living cells and intracellular colloid in the prior art and provides an optical detection and optical imaging method. The method for optically detecting the biomacromolecule related to the intracellular colloid osmotic pressure analyzes the change of the intracellular colloid osmotic pressure and analyzes the composition and the function of the crystal osmotic pressure and the colloid osmotic pressure in the intracellular osmotic pressure by combining an osmometer.
Another objective of the invention is to construct a relevant drug screening cell platform for screening drugs relevant to the regulation of colloid osmotic pressure according to the research of the optical detection of the invention.
Compared with the prior art, the invention has the beneficial effects that:
the biomacromolecule optical detection method related to the intracellular colloid osmotic pressure can analyze the change of the intracellular colloid osmotic pressure, and analyzes the composition and the function of the crystal osmotic pressure and the colloid osmotic pressure in combination with an osmometer, so that the biomacromolecule optical detection method has important research significance. Therefore, a relevant drug screening cell platform can be constructed, and the drugs relevant to the regulation of the colloid osmotic pressure can be screened.
Drawings
FIG. 1: the absorbance, particle size and quantity of the particles were measured by using a nanosizer zetasizer nanozs90 oblique or scattered light.
FIG. 2: the osmolarity of myosin or tubulin particles or aggregates thereof versus Count rate (kcps) curve.
FIG. 3: image J software-processed cell structure corrected maps.
FIG. 4: standard curves determined by using the uv absorbance method for actin or tubulin concentrations.
Detailed Description
Various embodiments and aspects of the invention are set forth in detail below.
The following description and drawings are illustrative of the invention and are not to be construed as limiting the invention. Numerous specific details are described to provide a thorough understanding of various embodiments of the invention. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present inventions.
The term "comprising" as used herein is to be interpreted as being inclusive and open-ended, and not exclusive. Particularly, as used in this specification including the claims, "comprises/comprising" and variations thereof means that a particular feature, step or ingredient is included therein. The terms are not to be interpreted to exclude the presence of other features, steps or components.
The term "exemplary" as used herein means "serving as an example, instance, or illustration," and should not be construed as preferred or advantageous over other configurations disclosed herein.
As used herein, the terms "about" and "approximately," when used in connection with a particle size range, a mixture composition, or other physical properties and characteristics, are intended to encompass minor variations that may exist in the upper and lower limits of the size range, so as not to exclude embodiments in which an average majority of the sizes meet, but statistically, sizes outside of the range may exist. This application is not intended to exclude embodiments such as these.
The protein particles such as myosin, tubulin and the like can be depolymerized and generated from a cell skeleton structure and can also be synthesized and increased by itself, the polymerization and the regional change of the protein particles become important mechanisms for regulating and controlling the outward tension of cells, and the outward stretching force of the cells is formed to drive outward mechanical activities of cell forms.
The use of the tyndall phenomenon allows quantification of the number of intracellular protein particles and their size. Dark field imaging techniques are used to determine subcellular localization changes of protein particles within cells. Meanwhile, coupled fluorescent protein is adopted to research the polarization front of cells, such as growth cones of neurons, the distribution of biomacromolecule particles and the change of colloid osmotic pressure thereof. In addition, the distribution of biomacromolecules located in nerve cells was studied using high resolution imaging techniques. This morphology is essentially consistent with dark field microscopy for cellular localization of intracellular protein particles. Therefore, the development and application of high-resolution imaging technology are beneficial to the observation and detection of intracellular biomacromolecules.
Intracellular protein particle markers were analyzed. Alternative labels include fluorescent substances, spectral signals, differential polarization signals, optical path differences, or scattered light differences. These signals may be from intracellular protein particles. The region of the expected area showing the presence of the marker may then be magnified with a higher magnification and then the shape and size characteristics of the marker and the intensity and spatial distribution signal evaluated within the previously identified region.
A first possible label includes fluorescent molecules that are non-toxic to body cells, including, but not limited to, smart optical probes (that emit a characteristic fluorescent signal only upon binding to protein particles) and other fluorescent dyes, such as the near-infrared fluoroxazine dye muoi 987, the curcumin derivative CRANAD-2, thioflavin T, thioflavin S or its derivatives, rhodamine, or congo red. If fluorescence is used, methods of tissue and cell imaging include wavelengths that excite selected molecules with single or two-photon excitation, a filtering system that separates incident light and fluorescence, and a detector that is sensitive to fluorescence. The detector filter and detector are selected to highlight the fluorescence wavelength of the label bound to the protein molecule so that background tissue fluorescence is not apparent.
Some of the fluorescent markers that can be used are markers that are capable of crossing the blood brain barrier. This allows intravenous injection to be performed during animal level testing.
Fluorescent dyes delivered by liposome encapsulation may also be selected. The dye can be localized in central nerve cells after being released in brain nerve cells. In the case of single or two-photon excitation of fluorescence, an excitation beam can penetrate into the multilayer cell, excite the fluorochrome and return fluorescence. The fluorescence can also be used to determine the structure and shape of protein particles. Also, the protein particles may be labeled with a fluorescent dye.
A second possible marker for protein particles is the spectrum. This includes, but is not limited to, raman spectroscopy, absorption spectroscopy, fluorescence correlation spectroscopy, NMR spectroscopy, quasi-elastic light scattering spectroscopy, circular dichroism spectroscopy, and fourier transform spectroscopy, the spectral signals may be generated from the enriched protein particles alone or from protein particles bound to a dye.
As a third possible marker, intracellular protein particles are visualized by differential absorption, transmission, scattering or reflection of polarized light (i.e. optically active), or by polarized spectroscopy, or by differential reflection of polarized light from intracellular protein particles. In addition, optically active dyes may be used. The reason for this is that they have different effects on the polarization properties of the light by known polarization imaging methods (e.g. confocal laser scanning microscopy modified by means of mueller matrix polarimetry) or detection structures which enhance the alignment of structures with different polarization properties.
A fourth possible marker is light with scattering characteristics and differential endogenous properties of protein particles, including the following. Visualization can be by dark field imaging or by super resolution microscopy.
In addition to the use of the above markers, other optical techniques can be used to assess the density of intracellular protein particles. Other methods of measuring particles of about 4nm to several hundred nm in size by scattered light may be used, as each intracellular protein particle will form differentially scattered light. These methods include the aforementioned light scattering characteristics or differential endogenous optical properties of the protein particles in order to determine the protein particles.
According to the principles of the present inventors' post-labeling measurement or direct evaluation of proteins, the present inventors have used the following techniques or instruments to image, localize and quantify protein particles: dark field microscope, super-resolution fluorescence microscopic imaging technology, nanometer particle size analyzer using dynamic light scattering as main principle, micro turbidimeter, and protein particle concentration measuring method based on specific light absorption of specific protein.
The first instrument is a nanometer particle size analyzer based on dynamic light scattering, such as Zetasizer Nano ZS90 and its similar nanometer particle size analyzer products.
The protein particles in the solution can make the scattered light intensity fluctuate with time due to Brownian motion to form dynamic state. When an interfering light passes through the cytoplasm, actin and tubulin particles that encounter the solution scatter in all directions. If the particles do random Brownian motion, the scattered light intensity can randomly fluctuate near the average light intensity along with time, the frequency of the scattered light can also slightly change, and the change of the light intensity autocorrelation function along with time can be obtained according to the fluctuation condition of the light intensity under a certain angle, so that the sizes and the distribution of actin and tubulin particles are calculated.
In order to achieve the object of detecting the change in the value of intracellular colloid osmotic pressure, the present invention provides a method of plotting a particle ratio of myosin or tubulin particles or polymers thereof to a corresponding curve of colloid osmotic pressure by using a nano-particle size meter, thereby presuming the change in intracellular colloid osmotic pressure and being useful for diagnosis of diseases associated with colloid osmotic pressure and screening of therapeutic drugs.
The detection method comprises the following steps:
1. cells were stimulated for 15min with different doses (1, 1/2, 1/4, 1/8, 1/16, 0 fold) of microfilament or microtubule disaggregating agents to induce the formation of intracellular myosin or tubulin particles and their aggregates to varying degrees. Wherein the microfilament depolymerizing agent is Cytochalasin D (cytoD), and the initial concentration is 5 mu M; the microtubule disaggregating agent was nocodazole (noc) at an initial concentration of 2 μ M.
2. The culture medium in the cell culture flask was poured off and washed twice with an appropriate amount of 4 ℃ PBS.
3. The cells were pelleted by centrifugation (12000rpm, 30min, 4 ℃), the supernatant medium was discarded and the cell pellet was retained.
4. And carrying out ultra-speed crushing for 15-30 seconds.
5. Centrifugation at 40000rpm was carried out for 15min, and the supernatant, i.e., cytoplasm (20-40. mu.l) was aspirated.
6. The cytoplasmic osmolarity is measured using an osmometer, such as the Advanced freezing point osmometer of the United states.
7. The absorbance, particle size and quantity of the particles were measured by using a nanosizer zetasizer nanozs90 oblique or scattered light (as shown in fig. 1).
In order to make the data obtained when detecting the solution to be detected more accurate, the concentration of the solution to be detected can be further defined when preparing a sample for the detection of Zetasizer NanoZS90, the minimum concentration is 0.5g/l, and the maximum concentration is based on the condition that the protein particles are not aggregated and gelled with each other.
8. The composition of the cytoplasmic crystal (or ion) osmolarity is analyzed by plotting the osmolarity of myosin or tubulin particles or aggregates thereof against the Count rate (kcps). Glia cells were stimulated with drugs (microfilaments or microtubule depolymerants) for 15 min. The above steps (except the first step) are repeated. Linear Y-0.3149X +152.92 was obtained by treating with SPSS17.0 software using cytosolic osmolality as the Y-axis and actin or tubulin formation by depolymerization as the X-axis. When X is 0, Y is 152.9, indicating an intracellular osmolality of 152.9 Osm/Kg. Thus, the cytosolic colloid osmotic pressure under normal conditions is about 147.1 Osm/Kg. (see FIG. 2)
9. The corresponding relation between the colloid osmotic pressure and the Count rate can be directly obtained.
The second is a micro turbidimeter. Turbidimetry can employ a variety of methods including photoelectric turbidimetry, immunotransmission turbidimetry, immunoscattering turbidimetry, and resonance light scattering turbidimetry. The excitation light is irradiated through the cytoplasm extracting solution, and the scattering and absorption of actin or tubulin or other antigen-antibody complexes reduce the light transmittance. Optical density or transmittance was measured accurately using a turbidimeter. Within a certain range, the protein or antigen-antibody complex particle concentration is inversely proportional to the transmittance and directly proportional to the optical density (or scattered light). The optical density was determined using a series of suspensions of known particle numbers and a standard curve of optical density versus protein particle number was generated. Then, the amount of actin or tubulin was determined from the standard curve by the optical density measured in the sample liquid.
In order to achieve the object of detecting the change in the value of intracellular colloid osmotic pressure, the present invention provides a method of plotting a particle ratio of myosin or tubulin particles or polymers thereof to a corresponding curve of colloid osmotic pressure by using an immunoscattering turbidimetry, thereby presuming the change in intracellular colloid osmotic pressure and being useful for diagnosis of diseases associated with colloid osmotic pressure and screening of therapeutic drugs.
The nephelometry adopts IMMAGE full-automatic turbidimeter (BECKMAN COULTER. Inc.) and matched reagents and calibrators. The quality control product is a self-made cytoplasm extracting solution.
The detection method comprises the following steps:
1. the instrument was measured for both intra-batch and inter-batch precision. The coefficient of variation CV is less than or equal to 5%.
2. Selecting standard solution with known actin or tubulin or polymer concentration, and diluting to 5.1%, 7.5%, 10.5%, 13.5%, 15.0% sample solution by gradient dilution
3. The measurement of 6 samples was repeated by the nephelometry, and the measurement results were processed by the SPSS17.0 software to obtain a linear standard curve. Wherein Y is a nephelometric turbidimeter measurement and X is a protein particle concentration.
4. The change in intracellular colloid osmotic pressure can be estimated by measuring a sample to be measured by nephelometry and calculating the protein solution concentration using a standard curve.
The third kind of detecting instrument is dark field microscope, also called dark field microscope, which is designed based on the Tyndall effect principle and can observe the object to be detected under the condition of black background, can observe the very tiny object which can not be seen in bright field, and can distinguish the existence and movement of the particle with the diameter in the range of 4-200nm, so the dark field microscope can be used for measuring intracellular protein including actin and tubulin and polymer. Dark field microscopes use a central light shield or dark field condenser (usually a parabolic condenser) to block the central beam of the light source from passing down and up through the specimen into the objective lens, thereby redirecting the light to impinge obliquely on the cells to be observed, which are reflected or scattered by the light, and the scattered light is injected into the objective lens, so that the entire field of view is dark. The diffraction light image of the intracellular protein particles (not the protein particles) is observed in the dark field, and the existence and movement of the protein particles can be observed, so that the intracellular proteins and polymers including intracellular actin and tubulin can be relatively quantified and positioned.
In order to realize the purpose of detecting the change of the intracellular colloid osmotic pressure value, the invention provides a method for dynamically imaging and relatively quantifying myosin or tubulin particles or polymers thereof in living cells by using a dark-field microscope together with total internal reflection illumination, so as to estimate the change of the intracellular colloid osmotic pressure and be used for diagnosing diseases related to the colloid osmotic pressure and screening therapeutic drugs.
The method for dynamic imaging and relative quantification of myosin or tubulin particles or aggregates thereof in living cells by using a dark-field microscope in combination with total internal reflection illumination comprises the following steps:
1. all cells were cultured at 37 ℃ with 5% carbon dioxide.
2. During cell growth and experiments, cells were suspended in cell culture medium supplemented with 10% fetal bovine serum (Dulbecco's modified medium (DMEM)).
3. A glass-bottom plate of type ibidi GmbH, Martinsried, Germany, a coverslip of type ibidi μ -Dish 1.5h (170 μm +/-5 μm) was used. And thoroughly cleaning the culture dish with the glass bottom and the cover glass. Prior to each experiment, cells were detached from the cell culture flask, transferred to a glass-bottom dish, and covered with a cover slip.
4. A parallel, linearly polarized 488nm laser beam deflected from a two-axis tilted scan mirror is used. A 488mm laser excites the sample to look for a suitable area for imaging.
5. An acousto-optic tunable filter is placed at the beginning of a light beam channel to ensure that the laser power is constant when P is 5.0 mW. A 20x beam expander expands the beam diameter to 14 mm and then to the scan mirror.
6. Specific membranes were placed in the polypropylene to constitute dark field detection, blocking all totally internally reflected light. Only the scattered light passes through the diaphragm and lens L4 and forms an image on the sCMOS camera.
7. Image acquisition at a frame rate of 100Hz
8. In order to eliminate the constant background interference caused by the interface reflections of the microscope optics superimposed on each of the original images, a single background image must be subtracted from all of the original images to produce a clear final image of the reflection-corrected cell structure.
9. Image data processing is all completed in Image J software (see fig. 3).
According to the imaging principle, the brightness of a local area in an image is proportional to the protein particle concentration and inversely proportional to the transmittance. The protein particle concentration in the high brightness (low transmittance) region is higher than that in the low brightness (high transmittance) region. By means of relative quantification, the position, the number or the density and the strength of protein markers of biological macromolecular particles or particle aggregates such as myosin, tubulin and the like in cells can be detected so as to evaluate the change of intracellular colloid osmotic pressure and the regional positioning.
The fourth detection technology is a super-resolution fluorescence microscopic imaging technology, and comprises a random optical reconstruction microscopy (STORM/dSTORM), a light-activated positioning microscopy (PALM), a fluorescence photosensitive positioning microscopy (FPALM), a scattering type near-field scanning optical microscope (s-SNOM), a stimulated emission depletion microscopy (STED), an interference light-activated positioning microscopy (iPALM) and the like. The following is a detailed list:
1) light activated Localization microscopy (PALM). The method is characterized in that the fluorescent protein is used for marking myosin or tubulin, the energy of a laser is adjusted, the cell surface is irradiated with low energy, only a few fluorescent molecules which are sparsely distributed in a visual field are activated at one time, then the laser irradiation is adjusted again to excite fluorescence, and the position of the myosin or tubulin is accurately positioned through Gaussian fitting. Laser irradiation is then used to bleach these fluorescent molecules that have been correctly positioned so that they are not reactivated by the next round of laser light. The laser is then cycled again to activate and bleach additional fluorescent molecules. After a number of cycles, most fluorescent molecules within the cell are precisely localized. The images of the molecules are synthesized on one image, excitation and detection are repeated continuously, and finally enough fluorescent molecules can be accurately positioned, and a myosin or tubulin super-resolution image is reconstructed by utilizing a plurality of sub-images, so that the protein quantification is realized. In addition, the PALM technique is combined with the principle of light interference, known as interferometric photosynthetically localized Microscopy (iPALM). iPALM can be used to visualize the nanoscaled protein microstructure, as can actin and tubulin assays as described above.
2) Random Optical Reconstruction microscopy (STORM). A light-converting fluorescent dye is used as a fluorescent probe. Cyanine dyes such as Alexa Fluor647, Alexa Fluor532 and ATTO532 are adopted to mark myosin or tubulin, and the fluorescent dye can realize light-dark conversion in a solution containing a sulfhydryl substance and can directly realize light conversion. The fluorescent molecules can be mutually converted between a bright state and a dark state by controlling the irradiation of exciting light with different wavelengths, and the adjacent fluorescent molecules are separated from each other, so that the aim of single-molecule imaging is fulfilled, and the nano-scale imaging of the labeled protein particles is realized. The STORM technology can reconstruct an ultrahigh resolution image of intracellular marker protein by repeatedly activating, positioning and quenching fluorescent molecules for hundreds to thousands of times, thereby realizing the measurement and the measurement of intracellular protein particles.
3) Fluorescent light sensitive positioning microscope (FPALM). Photoactivatable green fluorescent protein (PA-GFP) fluorescent protein is used to label myosin or tubulin, thousands of individual fluorescent molecules are analyzed per collection, and a small fraction of them are localized at a time with low excitation intensity. For these photoactivatable molecules, the rate of activation is controlled by the activation illumination; non-fluorescent, non-reactive molecules are activated by high frequency (405-nm) laser light and then fluoresce when excited at a lower frequency. Digital image sensor (CCD) cameras image the fluorescence and then these molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The speed of photobleaching is controlled by the intensity of the laser used to excite fluorescence, typically an Ar + ion laser is used. Since only a small number of fluorescent molecules are visible at a given time, their position can be accurately determined with a positioning accuracy that is 10 times higher than the resolution, achieving a spatial resolution better than 10 nm. Thus, the amount and distribution of intracellular proteins such as myosin or tubulin are determined.
4) Stimulated emission depletion microscopy (STED). Myosin or tubulin were labeled with fluorescent proteins. Electrons in fluorescent molecules are in a ground state, and are irradiated by excitation light, absorbing photons and jumping to an unstable excited state. The electrons in the excited state will transition to the ground state and emit autofluorescence. The excited radiation is generated when an electron of an excited state encounters a photon having a wavelength corresponding to the energy difference between the excited state and the ground state. The sample exciting light is combined with the loss light and focused on the protein sample to be detected through the objective lens. Then, a sample is excited by adopting light of combination of the depletion light and the excitation light, two beams of laser act on a fluorescent molecule together, the fluorescent molecule can spontaneously radiate fluorescence, and the parts irradiated by the depletion light can not generate fluorescence due to the stimulated depletion effect, so that the positioning and the metering of intracellular marker proteins such as myosin or tubulin are realized.
5) Scattering-type Scanning Near-field Optical microscope (s-SNOM). Near field refers to the dimension of the probe used as a physical observer and the distance between the probe and the observed object both being smaller than the wavelength of the radiation used for observation. The cytoplasm extracting solution sample to be detected is excited by a far field or an evanescent wave, and the SNOM needle point is used as a detection probe to collect optical signals. The probe scans point by point on the surface of the cytoplasm extracting solution, collects the change of various scattering light field information in the near-field range near the surface of the intracellular protein particles, records point by point and then images to obtain the appearance and optical information of protein particles such as actin, tubulin and the like in cytoplasm, and the like to form an optical image.
For the purpose of detecting a change in the value of intracellular colloid osmotic pressure, the present invention provides a method for imaging actin or tubulin by using the STORM technique, whereby a change in intracellular colloid osmotic pressure is presumed and used for diagnosis of diseases associated with colloid osmotic pressure and screening of therapeutic drugs.
The method for optically detecting intracellular actin or tubulin by using the STORM platform comprises the following steps:
1. stimulating cells with microfilament or microtubule depolymerizing agent for 15min to induce formation of intracellular myosin or tubulin particles and particle aggregates thereof to different degrees. Wherein the microfilament depolymerizing agent is Cytochalasin D (cytoD) with the concentration of 5 mu M; the microtubule disaggregating agent was nocodazole (noc) at a concentration of 2 μ M.
2. A #1.5 model 22mm by 22mm slide was selected. The slides and coverslips were thoroughly cleaned.
3. The cells are inoculated in a well-treated pore plate or culture dish for culture, and the cell density is not excessively high.
4. Fixing actin microfilaments of cells by adopting cytoskeleton Buffer solution (Cytoskeletal Buffer, CB, formula shown in the table) containing 3% of paraformaldehyde and 0.1% of glutaraldehyde; tubulin was fixed using phosphate buffer containing 4% paraformaldehyde.
5. Using a paraformaldehyde fixed sample, and washing the sample with a glycine solution containing 50mM to quench the autofluorescence generated by the sample; samples fixed with paraformaldehyde were washed with freshly prepared buffer containing 0.1% sodium borohydride to eliminate autofluorescence.
6. The membrane permeation is carried out by using a non-ionic cleaning agent such as TritonX-100 or NP-40, and the concentration range is 0.05% to 0.5% (v/v).
7. Actin or tubulin is fluorescently labeled.
Actin staining procedure (using 33mm diameter dish as an example):
(1) the cell sample was removed and the culture medium was aspirated. Cells were washed 2 times for 2 minutes each with 1mL of 37C pre-warmed CB buffer.
(2) Fixing: cells were fixed for 10 minutes in 1mL of a fixative containing 3% paraformaldehyde and 0.1% glutaraldehyde, and washed 3 times with 5 minutes each time after fixation using 2mL PBS.
(3) Membrane permeation: cell permeability was increased by adding 1mL of freshly prepared 0.25% Triton X-100 for 10 min at room temperature. PBS was washed 2 times for 5 minutes each.
(4) To reduce autofluorescence lmL freshly prepared 0.1% sodium borohydride solution was added and allowed to react for 7 minutes at room temperature. PBS rinse for 10 minutes each.
(5) And (3) sealing: cells were blocked for 1 hour by adding 1mL of freshly prepared 3% Bovine Serum Albumin (BSA) solution 37C to prevent non-specific adsorption of antibodies, followed by 3 washes with PBS.
(6) Dyeing: alexa Fluor 647-labeled phalloidin fluorescent antibody was diluted with 1% BSA in PBS
(7) Post-fixing: adding 1mL of 4% paraformaldehyde solution, repeating the fixation for 10 minutes, and washing with PBS; cells were washed 2 times 5 minutes each with 50mM glycine solution.
A tubulin staining step:
(1) taking out a cell sample, sucking out the culture solution, and washing the cells by using a PBS solution preheated by 37C;
(2) fixing cells for 10 minutes by taking 1mL of phosphate buffer solution containing 4% paraformaldehyde, and washing by PBS;
(3) membrane penetration and blocking reference actin experimental procedures;
(4) primary antibody incubation: the 2ug anti-a-tublin antibody was diluted in 1ml PBS solution, the cell sample was added and allowed to react for 1 hour at room temperature, and 2ml PBS was washed 3 times for 5 minutes each.
(5) And (3) secondary antibody incubation: 2ug Alexa Fluor 647-labeled coat Anti-Mouse antibody was diluted in 1mL PBS solution containing 1% BSA, incubated at 37 ℃ for 2 hours, and washed 3 times with PBS, each for 5 minutes.
(6) The post-fixation step is as above.
8. Fluorescent microsphere reference particles are added to a sample, the sample is fixed on a glass slide, and the image is calibrated by calculating the relative positions of the reference particles and the target fluorescent points. Stock solutions were diluted 1000-fold with PBS and stored at 4C. When in use, the fluorescent microspheres can be ultrasonically oscillated for 10 minutes to ensure that the fluorescent microspheres are uniformly distributed.
9. And after the sample is added with the reference particles, adding the imaging buffer solution into the sample and soaking for 1 minute, and then putting the sample into a microscopic imaging system for super-resolution imaging.
10. The sample was excited with a 640mm laser to find the appropriate area for imaging.
11. After a suitable imaging region is found (which must be guaranteed to contain the reference particles), the intensity of the excitation light is gradually increased until the sample has mostly switched to a dark state.
12. The addition of low intensity activating light (532nm) causes part of the fluorescent molecules to return from dark state to bright state, and the intensity of the activating light is generally set between 1.5% and 3%. When single-molecule fluorescent points in the image are uniformly distributed, the EMCCD can be used for collecting the image, and about 3 ten thousand frames of images are collected in one area.
13. The image data processing is completed in Matlab software.
14. The amount of intracytoplasmic protein was estimated and the cytosolic colloid osmotic pressure was calculated.
The fifth technique is to measure the protein particle concentration based on the specific absorbance that a particular protein has. Such as actin, tubulin, which are present in large amounts in the cell. The two proteins are rich in tyrosine and tryptophan, which have maximum absorption at 280nm, so that the determination of the absorbance value of cytoplasm at 280nm is a UV absorption method for measuring actin and tubulin. During measurement, cytoplasm to be measured is dripped on a micro optical detector, an absorbance value of 280nm is directly read on an ultraviolet spectrophotometer, and the approximate concentration of protein particles based on the specific absorbance of tyrosine and tryptophan is calculated.
In order to achieve the object of detecting the change in the value of intracellular colloid osmotic pressure, the present invention provides a method for measuring actin or tubulin concentration by using ultraviolet absorption method, thereby presuming the change in intracellular colloid osmotic pressure and being used for diagnosis of diseases associated with colloid osmotic pressure and screening of therapeutic drugs.
Wherein, the detection method comprises the steps of drawing a standard curve and calculating the concentration of the corresponding protein according to the ultraviolet absorbance:
1. 1.0, 1.5, 2.0, 2.5, 3.0mL of 3.00mg/mL standard protein solution was pipetted into 5 10mL cuvettes, diluted to the scale with 0.9% NaCl solution and shaken well. The series of proteins are: 0.3, 0.45, 0.6, 0.75, 0.9 mg/mL.
2. The absorbance A278 of each standard solution was measured at 278nm using a 1cm quartz cuvette with 0.9% NaCl solution as a reference, and the data was recorded.
3. And drawing a standard curve by taking the concentration of the standard protein solution as an abscissa and the absorbance as an ordinate. (see FIG. 4)
4. Processing by SPSS17.0 software gave linear Y ═ 4.3298X + 51.795.
5. 50 μ L of the protein solution to be measured with a proper concentration was taken, and the absorbance at 278nm was measured in the above-mentioned manner, and the absorbance was measured in parallel three times.
6. And (5) measuring the protein content of the sample to be measured according to the standard curve.
Several specific examples are listed below:
example 1
Method for measuring myosin or tubulin particles and particle aggregates thereof by using nanometer particle size tester Zetasizer Nano ZS90 based on dynamic light scattering principle
It should be noted that the present inventors intend to explain the operation method and principle of observation of myosin or tubulin particle and particle aggregate thereof by a Nano particle size analyzer by this example, not limited to Zetasizer Nano ZS90 instrument, nor actin or tubulin and particle aggregate thereof, but apply to all Nano particle size analyzers and all proteins and other macromolecules based on the dynamic light scattering principle, and the above examples are only used to illustrate the technical solution of the present invention and not to limit it, although the present invention has been described in detail with reference to the preferred examples, those skilled in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention, and they should be covered in the claims of the present invention.
Drawing of Standard Curve (FIG. 2)
1. Stimulating cells for 15min with different dosages (1, 1/2, 1/4, 1/8, 1/16, 0 times) of microfilament or microtubule depolymerizing agent, inducing formation of intracellular myosin or tubulin particles and their granular aggregates to different extents
2. Centrifuging at 4 deg.C to precipitate cells, removing supernatant culture medium, retaining cell precipitate, and ultra-rapidly crushing for 15-30 s.
3. 40000g, centrifuged for 15min and the supernatant, i.e., the cytoplasm (20-40. mu.l) was aspirated.
4. Measuring cytoplasmic osmotic pressure value by osmometer
5. The absorbance, particle size and amount were measured by using a nanosizer zetasizer nanozs90 oblique or scattered light.
6. The osmolarity of myosin or tubulin particles or aggregates thereof is plotted against the Count rate (kcps). The composition of cytoplasmic crystal (or ion) osmolarity and the numerical correspondence between colloid osmolarity and Count rate are analyzed.
When X is 0, Y is 152.9, indicating an intracellular osmolality of 152.9 Osm/Kg; since the osmolality in normal cells is 300Osm/Kg, the oncotic pressure is about 147.1 Osm/Kg.
Measurement of
1. The drug (glutamic acid) is used to stimulate the glial cells for 15 min.
2. Repeating the above steps 2, 3, 5 and 6
7. Calculating the composition and value of cytoplasmic crystals and ion osmotic pressure according to the above formula
Note: the four groups of data are respectively the osmotic pressure values of normal cells, microfilament depolymerized cells, microtubule depolymerized cells, microfilaments and microtubule depolymerized cells and the corresponding protein particle ratios thereof.
Example 2
Total internal reflection dark field microscope for observing number and distribution of myosin or tubulin particles and particle aggregates thereof in cells
It should be noted that the present inventors intend to use the operation method and principle of observing myosin or tubulin particle and its particle aggregate by light and dark field microscope of this example, not limited to actin or tubulin and its particle aggregate, but all the proteins and other macromolecules, and the above examples are only used to illustrate the technical solution of the present invention and not to be limited thereto, although the present invention has been described in detail with reference to the preferred examples, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all the modifications and equivalents should be covered by the scope of the claims of the present invention.
1. The cells or extracted cytoplasm within the culture dish/cover glass were placed under a high resolution dark field condenser.
2. The target protein may optionally be stained, for example, by staining actin with conjugated rhodamine-phalloidin for about 1h, to facilitate visualization of actin particles.
3. The condenser aperture was adjusted to 1.4.
4. The aperture of the light source is maximized.
5. A large drop of cedar oil was placed on the condenser, the specimen was placed on the stage, and the condenser was rotated to bring the oil into contact with the slide (no bubbles could occur).
6. The object is aligned by light distribution with a low power objective lens and a 7 Xeyepiece. The height of the condenser is adjusted, firstly, an aperture with a black spot in the middle appears on the glass slide, and finally, the light spot is bright, the smaller the light spot is, the better the light spot is, so that the light spot is enlarged when the condenser moves up and down.
7. And changing the needed ocular lens and the high power lens, slowly lifting the objective lens for focusing until a luminous sample appears at the center of the visual field.
8. And (4) dropping a drop of cedar oil on the cover glass, rotating the oil lens to a position to adjust light distribution, and observing.
9. The recording of the image is performed using a CCD camera.
10. For each experiment, the condenser was rinsed 3 times with distilled water and 70% ethanol, and then sterilized with uv light for 30 minutes.
Example 3
Actin or tubulin particle concentration is determined based on the specific absorbance of a particular protein.
It should be noted that the present inventors intend to explain the operation method and principle of specific absorbance of a specific protein in the content measurement of target protein particles by this example, not limited to actin or tubulin, but applicable to all proteins and other macromolecules, and the above examples are only for illustrating the technical solution of the present invention and not for limitation, although the present invention has been described in detail with reference to the preferred examples, those skilled in the art should understand that modifications or equivalent substitutions can be made on the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and they should be covered in the claims of the present invention.
Drawing of Standard Curve (FIG. 4)
1. Stimulating cells for 15min with different dosages (1, 1/2, 1/4, 1/8, 1/16, 0 times) of microfilament or microtubule depolymerizing agent, inducing formation of intracellular myosin or tubulin particles and their granular aggregates to different extents
2. Centrifuging at 4 deg.C to precipitate cells, removing supernatant culture medium, retaining cell precipitate, and ultra-rapidly crushing for 15-30 s.
3. 40000g, centrifuged for 15min and the supernatant, i.e., the cytoplasm, was aspirated.
4. Measuring cytoplasmic osmotic pressure value by osmometer
5. And detecting the absorbance or the protein content by using 280nm ultraviolet light. Because beta-myosin or alpha-tubulin beta-tubulin is tryptophan-rich, its high content has a more pronounced absorbance value.
6. The osmolarity of myosin or tubulin particles or aggregates thereof is plotted against the protein content (mg/ml). And analyzing the composition of cytoplasmic crystals and ion osmotic pressure and the numerical correspondence between colloid osmotic pressure and protein content.
Measurement of
1. The drug (glutamic acid) is used to stimulate the glial cells for 15 min.
2. Repeating the above steps 2, 3, 5 and 6
7. The composition and values of cytoplasmic crystals and ion osmolarity were calculated according to the above formula.
Note: the four groups of data are respectively osmotic pressure values of normal cells, microfilament depolymerized cells, microtubule depolymerized cells, microfilaments and microtubule depolymerized cells and corresponding protein content ratios thereof.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. An optical detection method of biomacromolecules related to intracellular colloid osmotic pressure, which is characterized by comprising the following steps: optically or imagewise measuring the size, shape, location, quantity or density and intensity of protein markers of myoglobulin, tubulin biomacromolecule particles or particle aggregates abundant in cells to evaluate the change of intracellular colloid osmotic pressure and the regional localization, wherein the method comprises the following steps:
a) breaking cells, extracting cytoplasm, carrying out light intensity detection by using imaging light with sufficient resolution, determining the size and the number of biomacromolecule particles or particle aggregates, and establishing a corresponding relation between colloid osmotic pressure change and colloid optical difference by combining an osmotic pressure value detected by an osmometer;
b) imaging and optical intensity detection of cells or tissues using imaging light of sufficient depth resolution to determine the location, number of biomacromolecule particles or aggregates of particles or the density and intensity of their protein markers; the regional variation of the colloid osmotic pressure is estimated from the relationship between the osmotic pressure and the optical difference.
2. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: the detection is carried out by adopting cell or cytoplasm extracting solution, and dye and fluorescent dye mark and various fluorescent protein coupling mark samples can be adopted, so that the detection sensitivity is improved, and the observation and quantitative evaluation of living cell monomolecular or molecular polymerization fluorescence dynamic indexes are realized.
3. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: the excitation light source may be configured to use natural light, polarized light, fluorescent light of a particular wavelength, or a combination of polarized light and fluorescent light of a particular wavelength to highlight the amount of fluorescence emitted by the biomacromolecule particle or its combined fluorescent material within the cell and obscure any background fluorescence.
4. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: dark-field microscopic imaging or dark-field optical detection is adopted, and the scattered light and the transmitted light are observed by a high-sensitivity detector to detect the intracellular distribution, the particle number and the dynamic change of the micro-particles by adopting the Tyndall phenomenon; the change in optical signal of the micelles was measured to assess the change in quantity, distribution and their associated osmotic pressure.
5. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: the distribution of the nm-grade micro-particles in cells can be observed by utilizing the ultrahigh-resolution microscope for visualization; the change in optical signal of the micelles was measured to assess the amount and associated change in osmotic pressure.
6. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: the oxazine dye AOI987 with the characteristics of nanoparticles, curcumin derivative substances, thioflavin S, thioflavin T, Congo red and any physiologically compatible derivatives thereof are adopted to generate characteristic fluorescent signals when combined with biological macromolecules, and protein particles are adopted to couple with genes of fluorescent protein, transfer into cells and mark single-molecule protein particles.
7. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: the nano metal particle fluorescent substance can penetrate through a microvascular barrier, and the detection of biomacromolecule particles in specific cells at the level of animals and tissues and the measurement of related colloid osmotic pressure can be realized by combining dye labeling.
8. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: the labels are the result of the interaction of the protein particles with the imaging light and are detected by any of differential absorption, scattering, transmission, projection and reflection based on the optical activity of the protein particles.
9. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: and (3) distinguishing the compositions of ions and colloid osmotic pressure in cytoplasm and osmotic pressure effects generated by the ions and the colloid osmotic pressure in combination with the cytoplasm osmotic pressure numerical values detected by a freezing point, crystal and colloid osmometer, and quantitatively evaluating the relation between the biomacromolecule particle optical signal and the colloid osmotic pressure numerical value.
10. The method for the optical detection of biological macromolecules associated with the osmotic pressure of intracellular colloids as claimed in claim 1, wherein: and (3) presuming the regional imaging optical change according to the relation between the colloid osmotic pressure and the optical signal value of the biological macromolecular particles, and correspondingly, the colloid osmotic pressure value change.
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