CN111337469B - Method for acquiring microscopic motion information under shear field by utilizing fluorescence wide field imaging - Google Patents
Method for acquiring microscopic motion information under shear field by utilizing fluorescence wide field imaging Download PDFInfo
- Publication number
- CN111337469B CN111337469B CN202010342935.3A CN202010342935A CN111337469B CN 111337469 B CN111337469 B CN 111337469B CN 202010342935 A CN202010342935 A CN 202010342935A CN 111337469 B CN111337469 B CN 111337469B
- Authority
- CN
- China
- Prior art keywords
- light intensity
- sample
- detected
- imaging
- field
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Landscapes
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Optics & Photonics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a method for acquiring microscopic motion information under a shear field by utilizing the light intensity distribution of a fluorescent wide-field imaging track. The method is a measurement and analysis method for obtaining motion information under single molecular scale through light intensity distribution of a fluorescent particle motion track. The method adopts a polymer micro-rheological spectrometer which is self-developed by a subject group, combines a macroscopic shearing application and rheological measurement technology with a microscopic fluorescence wide-field microscopic imaging technology, and has single-molecule-level sensitivity and resolution; and the light intensity distribution characteristics can be analyzed through software, and the particle motion information and the change of the particle motion information along with an external shear field can be successfully obtained from a microscopic level. The method can be widely applied to the field of basic research of polymer physics under a complex flow field, and can acquire microscopic physical images and motion information.
Description
Technical Field
The invention belongs to the field of polymer physics, and relates to a method for acquiring microscopic motion information under a shear field by utilizing fluorescent wide-field imaging.
Background
In the field of polymer physics basic research, the rheological properties and the association with microscopic motion and structure of shear flow field characteristics and molecules, particles and polymer chains such as multi-charge soft substances under a shear field have been the hot research in academia and industry, and are also difficult issues. The research on the aspect not only is helpful for better understanding the microscopic physical process under the flow field, but also has important guiding significance for the research in the biological field and the design and manufacture of high-end microfluidic devices.
The traditional measurement under a shear flow field usually adopts a contact type or sampling type, so that not only can the real-time information inside the flow field not be obtained in detail, but also the flow field is interfered, and the reliability of the measurement result is low. The non-contact measurement method based on the laser technology has the advantages of no interference to a flow field, real-time in-situ measurement, high space-time resolution and the like. With the rapid development of modern laser technology, fluorescence spectrum, image information acquisition hardware and technology, the method has wide application prospect. For motion under an applied shear field, Daniel Bon et al, Amsterdam university, measured the flow velocity distribution of simple yielding fluids using a combination of confocal microscopy and rheology (Fall A, Paredes J, Bonn D.YIELDING and shear banding in soft glass materials, physical review letters.2010Nov 23; 105(22): 225502.); in order to obtain the motion information of single molecules (single particles) in the rheological process from the microscopic molecular detail level and directly relate the motion information with the parameters of the macroscopic rheological properties, the chemical research institute of the Chinese academy of sciences, the river and the like combine the Zhao high-resolution and high-sensitivity single-molecule fluorescence spectrum technology with a commercial rotary rheometer to develop a high-molecular microscopic rheological spectrometer, and the method can synchronously measure the macroscopic rheological properties and the microscopic structural characteristic parameters of a soft substance system such as a high polymer and the like (CN105675560B is a method for obtaining the fluorescence emission spectrum information of the single polymer molecules under a shear field); and the high-speed motion information of the fluorescence labeling polymer molecules under high shear rate is successfully obtained by utilizing the fluorescence fluctuation correlation spectrum method, and the specific value is in the range of 6000-18000 mu m/s (CN201610031737.9A a method for obtaining the diffusion and directional motion information of single polymer molecules under a shear field).
However, since the fluorescence correlation spectroscopy method cannot directly acquire real microscopic images, and has fitting model dependence: on one hand, when the flow characteristic time caused by steady-state shearing is close to the self diffusion characteristic time of the fluorescent probe in a low-speed flow field, the model is difficult to obtain the motion information in the low-speed steady-state shearing field; on the other hand, it is difficult to obtain the micro motion state and information under more common complex shearing modes such as oscillatory shearing through model fitting.
Disclosure of Invention
The invention aims to provide a method for acquiring microscopic motion information under a shear field by utilizing fluorescent wide-field imaging. The method can implement accurate shear field, has extremely high resolution and sensitivity, and can directly obtain motion state and parameters through imaging trajectory analysis.
The method for acquiring the microscopic motion information under the shear field comprises the following steps: and carrying out data processing on the sample A to be detected or the sample B to be detected by utilizing the track information obtained by the fluorescence wide field imaging to obtain the micro-motion information of the sample to be detected in the shearing field.
In the method, the sample A to be detected is a fluorescence-labeled sample C to be detected;
the sample C to be detected is a substance which cannot generate fluorescence per se; specifically at least one selected from silica, polystyrene microspheres and polymer chains; the polymer chain is specifically selected from at least one of sodium polystyrene sulfonate, polyethylene oxide and polystyrene;
the sample B to be detected is a substance which can generate fluorescence per se; in particular from dye molecules; more specifically at least one selected from rhodamine 6G, Alexa633 NHS, FITC Isothiocyanate and Atto 620.
Specifically, the method for obtaining the track information by using the fluorescence wide field imaging on the sample a to be detected or the sample B to be detected comprises the following steps:
1) preparing a dilute solution;
the dilute solution consists of the sample A to be detected or the sample B to be detected and a solvent;
2) applying a shear field to the dilute solution obtained in the step 1), irradiating laser on the dilute solution through an optical system while applying the shear field, and selecting an imaging mode to perform real-time imaging to obtain the track light intensity distribution of the sample to be detected.
In the step 1) of the above method, the concentration of the dilute solution is less than 10 nM; specifically 5 nM;
the solvent is at least one selected from water, glycerol, ethanol and tetradecane;
the excitation wavelength of the fluorescence is specifically 532 nm.
The fluorescence labeling method is chemical reaction bonding;
the position of the fluorescent marker is the surface or the inside of the sample particle to be detected;
the fluorescent molecules used for the fluorescent labeling are fluorescent dyes; specifically at least one selected from rhodamine 6G, Alexa633 NHS, FITC Isothiocyanate and Atto 620;
the structural formula of the rhodamine 6G is shown as a formula I:
the method may further comprise: before the fluorescence labeling step, the fluorescence molecules for fluorescence labeling are subjected to acidification and hydrolysis.
The method may further comprise the step of removing residual unlabeled fluorescent dye after the labeling, such as by ultrafiltration membranes and centrifugation or other separation methods.
In the step 2), the shear field is applied by a rheometer capable of performing precise external shear; the rheometer is specifically a rotary rheometer;
the imaging mode is selected from at least one of a total internal reflection mode, an inclined angle emergent mode and a vertical emergent mode;
in the optical system, the exciting light optical window is a quartz glass slide specifically; the thickness of the quartz glass slide is specifically 0.13-0.17 mm; thereby enabling the optical window to have high transmittance and short working distance;
the optical imaging hardware platform is an optical microscope;
the imaging unit is a chip amplification EMCCD camera with high enough quantum efficiency and large gain;
the laser is generated by a laser; the laser can be a continuous laser or a femtosecond pulse laser, the aim is to excite more fluorescent molecules, and the wavelength of the selected laser needs to be matched with the excited fluorescent probe; laser emitted from the laser is respectively reflected or transmitted by a plurality of reflectors and the diaphragm in turn and is subjected to two-stage beam expansion by the first beam expander and the second beam expander in turn to enlarge the diameter of a laser beam to about 2.0cm so as to ensure that the size of the laser beam is larger than that of a light inlet hole of a microscope objective lens of the optical microscope unit.
The track information is selected from at least one of track length, light intensity, and exposure time.
Specifically, the data processing includes:
3) starting coordinate (x) of light intensity accumulation through single particle trajectory0,y0) And endpoint coordinate (x)1,y1) Determining the light intensity accumulation baseline and the slope k of the corresponding equation1＝(y1-y0)/(x1-x0)；
The slope of the equation corresponding to the cumulative line of light intensity in the direction perpendicular to the base line is k2＝-1/k1；
In the light intensity accumulation process, the selected step length e and the step number n can cover the whole track area of the single particle;
4) algebraically adding the light intensity values of all pixel points through which the light intensity accumulation line passes in the step 3) to obtain a light intensity distribution diagram on the particle motion track of the sample A to be detected or the sample B to be detected;
in the light intensity distribution graph, the abscissa is the step number n; the ordinate is the algebraic sum of the light intensity values of all the pixel points on each step length;
if the light intensity distribution graph shows that the light intensity is uniformly distributed, the shearing mode is proved to be stable shearing, and the flowing state is simple laminar flow;
the moving speed v of the particles is defined by v ═ dphy/texAnd calculating to obtain:
wherein d isphyThe actual corresponding physical size of the light intensity track of the particle;
texexposure time for known real-time imaging;
and if the light intensity distribution graph shows that the light intensity is not uniformly distributed and the tail appears, the shearing mode is proved to be unstable shearing.
In the data processing method, the unsteady state shearing is oscillation shearing;
the length d of the light intensity accumulation baseline is determined by the product of the step length e and the step number n or by the termination point (x)1,y1) And the coordinates of the starting point (x)0,y0) Determining;
the width w between the light intensity accumulation baseline and the light intensity accumulation line is required to cover the whole track area of the particle;
d isphyThe actual physical size corresponding to the pixel point in the EMCCD is multiplied by the number of the pixel points contained in the track. Considering that the pixel points have actual physical sizes, the error in the accumulation process can be carried out through a threshold error of the area of a region cut by the light intensity accumulation lineSetting, in the accumulation process, omitting the pixel points of which the area of s1 is smaller than the error value in the graph 2; the threshold value error is specifically 0-0.5
In the above method, both the equation and the calculation can be obtained by using software processes such as MATLAB and Originlab which are generally known.
The measurement system capable of carrying out in-situ fluorescence wide field imaging under a shear field used in the method is a polymer micro-rheological spectrometer, the specific structure of which is shown in figure 1 and comprises an excitation module 1, a shear application and rheological measurement module 2 and an optical micro single module: a single-molecule fluorescence imaging unit EMCCD camera 14 and/or a spectrum measuring unit 13 and/or a fluorescence correlation spectrum measuring unit 12, wherein an exciting light optical window is arranged on the lower substrate at the bottom of the shear application and rheology measuring unit 2;
exciting light emitted by an exciting light source irradiates a sample to be detected, the sample to be detected is a polymer molecule and/or a colloidal particle, and a dye molecule is connected to a polymer molecular chain and the surface or the inside of the colloidal particle in a chemical bonding mode; the shear application and rheological measurement module 2 is used for applying an accurate shear field to a sample to be measured; exciting light emitted by the laser light source module 1 is introduced into a sample positioned under the shear flow field through the optical window 5, so that dye molecules in the sample to be detected are excited to generate fluorescent signals, and the generated fluorescent signals are collected and respectively emitted to the monomolecular fluorescence imaging unit 14, the monomolecular fluorescence emission spectrum measuring unit 13 and/or the fluorescence correlation spectrum measuring unit 12; the monomolecular fluorescence imaging unit 14 is used for performing real-time fluorescence imaging on a single fluorescent molecule in a system with a slower movement speed to obtain monomolecular real-space movement information in the system, namely obtaining the directional movement speed and the movement state information of the fluorescent molecule;
preferably, the shearing application and rheological measurement module 2 adopts a rotational rheometer capable of implementing accurate shearing, the lower substrate is provided with an excitation light optical window with high transmittance and short working distance, specifically a quartz glass slide with the thickness of 0.13-0.17 mm, the short working distance can be matched with microscope water or an oil immersion objective with high numerical aperture, and the working distance is not more than 0.2 mm.
Preferably, the excitation light source module 1 may employ a continuous laser or a femtosecond pulse laser, in order to excite more fluorescent molecules with different properties, and the wavelength of the selected laser needs to be matched with the excited dye molecules, and the excitation light source module 1 in this embodiment includes three lasers with different wavelengths (for this example, but not limited thereto, and may be selected according to the practical use), for example, the wavelengths of the three lasers may be 647nm, 532nm, and 473nm, respectively.
Preferably, the single-molecule fluorescence imaging unit 14 can employ a chip-amplified EMCCD camera with sufficiently high quantum efficiency and large gain.
Preferably, the system also carries a high-precision displacement platform of the prior art, and the rotational rheometer can be placed above the microscope objective 3 when needed, so that the precise linkage of all the devices is ensured.
The specific operation of imaging by using the polymer micro-rheological spectrometer can be as follows:
a. opening an excitation light source (532nm), adjusting and collimating the expanded excitation light beam to form a parallel light beam, entering a rear lens barrel lens of the microscope, and emitting the parallel light beam again at the lens;
b. moving the rheometer to a position right above a microscope objective lens through a high-precision displacement platform;
c. zero calibration is carried out on the rheometer;
d. switching to a water immersion objective lens, adding 40 mu L of ultrapure water on the microscope objective lens, and adjusting the microscope objective lens upwards to reach a proper focusing position;
e. adding the sample into the right center of the glass slide;
f. moving the rotor of the rheometer to a testing position, and opening rheometer control software to implement a steady-state shearing testing method;
g. and opening the fluorescence imaging unit for in-situ imaging.
The invention has the following beneficial effects:
the measuring method provided by the invention adopts a macromolecular rheological microscope spectrometer, and combines a macroscopic shearing application and rheological measurement technology with a monomolecular fluorescence imaging technology. The method has single molecule level sensitivity, and the concentration of the measured sample can reach nM level. By realizing the application of steady-state shearing and oscillating shearing with wide frequency range, different amplitudes and different frequencies to the sample, the in-situ single-molecule fluorescence microscopic imaging observation can be carried out on the sample applied with the shearing force field: the movement of a single fluorescent probe molecule (or fluorescent particle) in a sample is directly observed at the single molecule level, and movement information at the single molecule scale is obtained and analyzed.
Drawings
FIG. 1 is a schematic diagram of an experimental apparatus "high molecular rheomicroscope" used in the present invention;
each label is as follows: 1. the device comprises an excitation light source, 2 a shearing application and rheological measurement module, 3 a microscope objective, 4 a dichroic mirror, 5 a quartz glass slide, 6 a sample, 7 an optical filter, 8 a focusing lens, 9a pinhole, 10 a beam expander, 11 a reflector, 12 a single photon detector, 13 a single molecule fluorescence spectrometer, 14 an EMCCD camera and 15 a diaphragm.
Fig. 2 is a schematic diagram showing a mechanism of performing an accumulation process on the light intensity of the trajectory to obtain a light intensity distribution.
FIG. 3 is a graph of low shear rate (0.1 s)-1) Under the steady state shear field, the motion track of the particles under the micro scale is obtained.
FIG. 4 is a graph of low shear rate (0.1 s)-1) Under the steady-state shear field, the obtained motion trail is processed by images and the light intensity distribution is obtained.
Fig. 5 is a particle motion trajectory at a microscopic scale obtained under an oscillating shear field.
Fig. 6 is a diagram illustrating image processing of the obtained motion trajectory and obtaining light intensity distribution in the oscillating shear field.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified. Rhodamine 6G, 99%, analytically pure, purchased from sigma reagent, inc.
The measuring system which can perform in-situ fluorescence wide field imaging under a shear field used by the method of the invention is a polymer micro-rheological spectrometer, the specific structure of which is shown in figure 1 and comprises an excitation module 1, a shear application and rheological measuring module 2 and an optical micro single module: a single-molecule fluorescence imaging unit EMCCD camera 14 and/or a spectrum measuring unit 13 and/or a fluorescence correlation spectrum measuring unit 12, wherein an exciting light optical window is arranged on the lower substrate at the bottom of the shear application and rheology measuring unit 2;
exciting light emitted by an exciting light source irradiates a sample to be detected, the sample to be detected is a polymer molecule and/or a colloidal particle, and a dye molecule is connected to a polymer molecular chain and the surface or the inside of the colloidal particle in a chemical bonding mode; the shear application and rheological measurement module 2 is used for applying an accurate shear field to a sample to be measured; exciting light emitted by the laser light source module 1 is introduced into a sample positioned under the shear flow field through the optical window 5, so that dye molecules in the sample to be detected are excited to generate fluorescent signals, and the generated fluorescent signals are collected and respectively emitted to the monomolecular fluorescence imaging unit 14, the monomolecular fluorescence emission spectrum measuring unit 13 and/or the fluorescence correlation spectrum measuring unit 12; the monomolecular fluorescence imaging unit 14 is used for performing real-time fluorescence imaging on a single fluorescent molecule in a system with a slower movement speed to obtain monomolecular real-space movement information in the system, namely obtaining the directional movement speed and the movement state information of the fluorescent molecule;
preferably, the shearing application and rheological measurement module 2 adopts a rotational rheometer capable of implementing accurate shearing, the lower substrate is provided with an excitation light optical window with high transmittance and short working distance, specifically a quartz glass slide with the thickness of 0.13-0.17 mm, the short working distance can be matched with microscope water or an oil immersion objective with high numerical aperture, and the working distance is not more than 0.2 mm.
Preferably, the excitation light source module 1 may employ a continuous laser or a femtosecond pulse laser, in order to excite more fluorescent molecules with different properties, and the wavelength of the selected laser needs to be matched with the excited dye molecules, and the excitation light source module 1 in this embodiment includes three lasers with different wavelengths (for this example, but not limited thereto, and may be selected according to the practical use), for example, the wavelengths of the three lasers may be 647nm, 532nm, and 473nm, respectively.
Preferably, the single-molecule fluorescence imaging unit 14 can employ a chip-amplified EMCCD camera with sufficiently high quantum efficiency and large gain.
Preferably, the system also carries a high-precision displacement platform of the prior art, and the rotational rheometer can be placed above the microscope objective 3 when needed, so that the precise linkage of all the devices is ensured.
Example 1, the movement information of the fluorescent labeled particles in the mixed system of water and glycerol is obtained by using the fluorescent imaging track under the shear field.
1) Preparation of fluorescent labeling particles:
dispersing the silicon dioxide particles to be marked in ethanol, adding (3-aminopropyl) triethoxysilane with volume fraction of one thousandth, adding a small amount of ammonia water for catalytic reaction, and reacting at room temperature for 18 hours;
further reacting the product with carboxylated rhodamine 6G dye, using 4- (Dimethylamino) pyridine (Alfa Aesar, 99%) as a catalyst and 1-ethyl-3- (3-Dimethylamino) carbodiimide hydrochloride (Acros Organics, 98%) as a dehydrating agent, fully reacting amino groups and carboxyl groups, and bonding dye molecules to the particle surface by a chemical method;
washing the reacted particle dispersion liquid with water and ethanol for three times respectively, and centrifugally separating a product to remove free dye molecules to obtain rhodamine 6G dye-labeled silicon dioxide particles;
dispersing the rhodamine 6G dye-labeled silicon dioxide particles in a mixed system consisting of water and glycerol (the volume ratio is 4:1) to obtain a dilute solution, wherein the concentration of the dilute solution is 5 nM;
2) acquisition of motion information in a steady-state shear field
The operation steps are as follows:
opening an excitation light source (532nm), adjusting and collimating the expanded excitation light beam to form a parallel light beam, entering a rear lens barrel lens of the microscope, and emitting the parallel light beam again at the lens;
moving the rheometer to a position right above a microscope objective lens through a high-precision displacement platform;
zero calibration is carried out on the rheometer;
switching to a water immersion objective lens, adding 40 mu L of ultrapure water on the microscope objective lens, and adjusting the microscope objective lens upwards to reach a proper focusing position;
adding the sample into the right center of the glass slide;
moving the rotor of the rheometer to a testing position, and opening rheometer control software to implement a steady-state shearing testing method;
and opening the fluorescence imaging unit for in-situ imaging, wherein the motion track at a single molecular scale is shown in FIG. 3. The corresponding real physical size of each pixel point of the EMCCD camera is 0.267 mu m, and the exposure time tex0.2 s. As shown in a in fig. 4, the initial coordinate (219,449) is accumulated, the accumulation direction width w is 5, the step number n is 350, the step length e is 0.1, the software obtains that the baseline accumulation length d is n × e 35, which is enough to cover the whole particle light intensity area, and the threshold error is set to 0. The light intensity distribution of the track obtained by the accumulation in the step b in fig. 4 shows that the light intensity is uniformly distributed, the particles move at a uniform speed, and the fact that the shearing mode is steady shearing and the flowing state is simple laminar flow is proved. Actual physical dimension d corresponding to particle trajectory in cumulative lengthphy4.92 μm, and the movement velocity v-d at the microscopic scale is calculatedphyAnd/t, wherein v is 24.6 mu m/s.
Acquisition of motion information under shock shear
Opening an excitation light source (532nm), adjusting and collimating the expanded excitation light beam to form a parallel light beam, entering a rear lens barrel lens of the microscope, and emitting the parallel light beam again at the lens;
moving the rheometer to a position right above a microscope objective lens through a high-precision displacement platform;
zero calibration is carried out on the rheometer;
switching to a water immersion objective lens, adding 40 mu L of ultrapure water on the microscope objective lens, and adjusting the microscope objective lens upwards to reach a proper focusing position;
adding the sample into the right center of the glass slide;
moving a rheometer rotor to a test position, and opening rheometer control software to implement a vibration shearing test method;
and opening the fluorescence imaging unit to perform in-situ real-time imaging, wherein the motion track of the single-molecule scale is shown in FIG. 5.
The light intensity of the trace shown in a in fig. 6 is accumulated, the initial coordinate is (354,367), the end coordinate is (376,393), the width w is 5, and the threshold error is set to 0. The b result in fig. 6 shows that the light intensity distribution is not uniform, the light intensity accumulation value is maximum at the maximum amplitude and is minimum at the equilibrium position, and the shear mode is the oscillation shear at this time.
Claims (13)
1. A method of obtaining microscopic motion information in a shear field, comprising: carrying out data processing on a sample A to be detected or a sample B to be detected by utilizing track information obtained by fluorescence wide field imaging to obtain micro motion information of the sample to be detected in a shear field;
the data processing comprises:
a. starting coordinate (x) of light intensity accumulation through single particle trajectory0,y0) And endpoint coordinate (x)1,y1) Determining the light intensity accumulation baseline and the slope k of the corresponding equation1=(y1-y0)/(x1-x0)；
The slope of the equation corresponding to the cumulative line of light intensity in the direction perpendicular to the base line is k2=-1/k1；
In the light intensity accumulation process, the selected step length e and the step number n can cover the whole track area of the single particle;
b. b, algebraically adding the light intensity values of all pixel points through which the light intensity accumulation line passes in the step a to obtain a light intensity distribution diagram on the particle motion track of the sample A or the sample B to be detected;
in the light intensity distribution graph, the abscissa is the step number n; the ordinate is the algebraic sum of the light intensity values of all the pixel points on each step length;
if the light intensity distribution graph shows that the light intensity is uniformly distributed, the shearing mode is proved to be stable shearing, and the flowing state is simple laminar flow;
velocity of movement of particlesvByv=d phy /texAnd calculating to obtain:
wherein, thed phy The actual corresponding physical size of the light intensity track of the particle;
texexposure time for real-time imaging;
if the light intensity distribution graph shows that the light intensity is unevenly distributed and the tail appears, the shearing mode is proved to be unstable shearing;
the sample A to be detected is a sample C to be detected which is marked by fluorescence;
the sample C to be detected is a substance which cannot generate fluorescence per se;
the sample B to be detected is a substance which can generate fluorescence per se.
2. The method of claim 1, wherein: the sample C to be detected is selected from at least one of silicon dioxide, polystyrene microspheres and polymer chains;
the sample B to be detected is selected from dye molecules.
3. The method of claim 2, wherein: the polymer chain is selected from at least one of sodium polystyrene sulfonate, polyethylene oxide and polystyrene;
the sample B to be detected is selected from at least one of rhodamine 6G, Alexa633 NHS, FITC Isothiocyanate and Atto 620.
4. The method of claim 1, wherein: the method for obtaining the track information by utilizing the fluorescence wide field imaging of the sample A to be detected or the sample B to be detected comprises the following steps:
1) preparing a dilute solution;
the dilute solution consists of the sample A to be detected or the sample B to be detected and a solvent;
2) applying a shear field to the dilute solution obtained in the step 1), irradiating laser on the dilute solution through an optical system while applying the shear field, and selecting an imaging mode to perform real-time imaging to obtain the track light intensity distribution of the sample to be detected.
5. The method of claim 4, wherein: in the step 1), the concentration of the dilute solution is less than 10 nM;
the solvent is at least one selected from water, glycerol, ethanol and tetradecane.
6. The method of claim 5, wherein: in the step 1), the concentration of the dilute solution is 5 nM;
the excitation light wavelength of the fluorescence is 532 nm.
7. The method of claim 1, wherein: the fluorescence labeling method is chemical reaction bonding;
the position of the fluorescent marker is the surface or the inside of the sample particle to be detected;
the fluorescent molecule used for the fluorescent labeling is a fluorescent dye.
8. The method of claim 7, wherein: the fluorescent molecule used for the fluorescent label is at least one selected from rhodamine 6G, Alexa633 NHS, FITC Isothiocyanate and Atto 620.
9. The method of claim 4, wherein: in the step 2), the shear field is applied by a rheometer capable of performing precise external shear;
the imaging mode is selected from at least one of a total internal reflection mode, an inclined angle emergent mode and a vertical emergent mode;
the optical imaging hardware platform is an optical microscope;
the imaging unit used is a chip amplification EMCCD camera with sufficiently high quantum efficiency and large gain.
10. The method of claim 9, wherein: in the step 2), the rheometer is a rotary rheometer;
in the optical system, the excitation light optical window is a quartz glass slide.
11. The method of claim 1, wherein: the track information is selected from at least one of a track length, a light intensity value, and an exposure time.
12. The method according to any one of claims 1 to 11, wherein: the unsteady state shearing is oscillation shearing;
the length of the light intensity accumulation baselinedDetermined by the product of the step size e and the number of steps n or by the end point (x)1,y1) And the coordinates of the starting point (x)0,y0) Determining;
the width w between the light intensity accumulation baseline and the light intensity accumulation line is required to cover the whole track area of the particle;
the above-mentionedd phy The product of the actual physical size corresponding to the pixel point in the EMCCD and the number of the pixel points contained in the track is obtained; the accumulated process error is set by a threshold error of the area of the region cut by the light intensity accumulation line.
13. The method of claim 12, wherein: the threshold error is 0-0.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010342935.3A CN111337469B (en) | 2020-04-27 | 2020-04-27 | Method for acquiring microscopic motion information under shear field by utilizing fluorescence wide field imaging |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010342935.3A CN111337469B (en) | 2020-04-27 | 2020-04-27 | Method for acquiring microscopic motion information under shear field by utilizing fluorescence wide field imaging |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111337469A CN111337469A (en) | 2020-06-26 |
| CN111337469B true CN111337469B (en) | 2021-06-08 |
Family
ID=71182898
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010342935.3A Active CN111337469B (en) | 2020-04-27 | 2020-04-27 | Method for acquiring microscopic motion information under shear field by utilizing fluorescence wide field imaging |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111337469B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546980A (en) * | 2003-12-16 | 2004-11-17 | 中国科学院长春应用化学研究所 | Portable temperature control conical shaped plate shearing pool |
| CN102183497A (en) * | 2011-02-15 | 2011-09-14 | 中国科学院化学研究所 | Measuring method for diffusion motion trail of single molecular chain in thin polymer film |
| CN104758058A (en) * | 2015-03-11 | 2015-07-08 | 苏州大学 | Synchronous pulse laser microimaging observation device for mechanical stress deformation of blood cells |
| CN105675560A (en) * | 2016-01-18 | 2016-06-15 | 中国科学院化学研究所 | Method for obtaining fluorescence emission spectrum information of single polymer molecule in shearing field |
| CN105675561A (en) * | 2016-01-18 | 2016-06-15 | 中国科学院化学研究所 | Method for acquiring diffusive and directional movement information of single polymer molecules in shear fields |
| CN105699344A (en) * | 2016-01-18 | 2016-06-22 | 中国科学院化学研究所 | Measuring system for obtaining single molecular spectrum and imaging under shear field |
| CN108072636A (en) * | 2016-11-14 | 2018-05-25 | 中国科学院化学研究所 | A kind of single annular polymeric molecule spreads the measuring method of information in solid/liquid interfaces |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11340214B2 (en) * | 2010-06-30 | 2022-05-24 | Anpac Bio-Medical Science Co., Ltd. | Apparatus for disease detection |
-
2020
- 2020-04-27 CN CN202010342935.3A patent/CN111337469B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546980A (en) * | 2003-12-16 | 2004-11-17 | 中国科学院长春应用化学研究所 | Portable temperature control conical shaped plate shearing pool |
| CN102183497A (en) * | 2011-02-15 | 2011-09-14 | 中国科学院化学研究所 | Measuring method for diffusion motion trail of single molecular chain in thin polymer film |
| CN104758058A (en) * | 2015-03-11 | 2015-07-08 | 苏州大学 | Synchronous pulse laser microimaging observation device for mechanical stress deformation of blood cells |
| CN105675560A (en) * | 2016-01-18 | 2016-06-15 | 中国科学院化学研究所 | Method for obtaining fluorescence emission spectrum information of single polymer molecule in shearing field |
| CN105675561A (en) * | 2016-01-18 | 2016-06-15 | 中国科学院化学研究所 | Method for acquiring diffusive and directional movement information of single polymer molecules in shear fields |
| CN105699344A (en) * | 2016-01-18 | 2016-06-22 | 中国科学院化学研究所 | Measuring system for obtaining single molecular spectrum and imaging under shear field |
| CN108072636A (en) * | 2016-11-14 | 2018-05-25 | 中国科学院化学研究所 | A kind of single annular polymeric molecule spreads the measuring method of information in solid/liquid interfaces |
Non-Patent Citations (2)
| Title |
|---|
| Shear rheology of lipid monolayers and insights on membrane fluidity;Gabriel Espinosa et al;《PNAS》;20110412;第108卷(第15期);第6008-6013页 * |
| 获取剪切场下单个高分子的光谱信息-高分子流变显微光谱仪;郑锴锴 等;《2015年全国高分子学术论文报告会》;20151021;第498页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111337469A (en) | 2020-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2522988B1 (en) | Optical analysis device, optical analysis method, and computer program for optical analysis | |
| JP5877155B2 (en) | Method for detecting dilute particles in solution using luminescent probe | |
| EP2100127B1 (en) | Fast thermo-optical particle characterisation | |
| JP2011002415A (en) | Fluorescence correlation spectroscopic device | |
| EP2620762B1 (en) | Optical analysis method using the detection of a single light-emitting particle | |
| EP2749867B1 (en) | Optical analysing device and method using individual light-emitting particle detection | |
| Sergeeva et al. | Highly-sensitive fluorescent detection of chemical compounds via photonic nanojet excitation | |
| JP2010276380A (en) | Fluorescence correlation spectroscopic analyzer and method, and computer program therefor | |
| CN104502326A (en) | Enhanced SERS (surface enhanced raman scattering) signal quantitative analysis method and application thereof | |
| JP2008527375A (en) | Infrared transmitting substrate, semiconductor substrate, silicon substrate, fluid sample analysis device, fluid sample analysis method, and computer-readable recording medium | |
| CN105675560B (en) | A kind of method of single polymer molecule fluorescence emission spectrum information under acquisition shearing field | |
| CN111337469B (en) | Method for acquiring microscopic motion information under shear field by utilizing fluorescence wide field imaging | |
| CN105675561A (en) | Method for acquiring diffusive and directional movement information of single polymer molecules in shear fields | |
| Petrášek et al. | Simultaneous two‐photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures | |
| Ripple et al. | Protein Particles (0.1 µm to 100 µm) | |
| CN111610171A (en) | Method for obtaining diffusion movement and concentration information of polymer molecules on immiscible liquid interface | |
| CN113777008B (en) | Method for optical nondestructive detection of surface interface conversion of single nano particle | |
| KR100691528B1 (en) | Instrument of scanning surface plasmon microscope for the analysis of protein arrays | |
| JP4906789B2 (en) | Droplet spectroscopy system and spectroscopy | |
| Beuwer | Correlative microscopic characterization of nanoscale assemblies at interfaces | |
| CN117470826A (en) | Microfluidic surface-enhanced Raman test substrate and preparation method and application thereof | |
| Buehler et al. | The Integration of Single Molecule Detection Technologies into Miniaturized Drug Screening: Current Status and Future Perspectives | |
| Wang et al. | Optical methods of single molecule detection and applications in biosensors | |
| Huang et al. | Near-Surface Particle-Tracking Velocimetry | |
| BR112021006708A2 (en) | depositing microdots with analytes onto analysis chips |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |