CN113295885B - Micro-nano flow fluorescence bleaching speed measurement method and system based on camera imaging - Google Patents
Micro-nano flow fluorescence bleaching speed measurement method and system based on camera imaging Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01P—MEASURING LINEAR OR ANGULAR SPEED, ACCELERATION, DECELERATION, OR SHOCK; INDICATING PRESENCE, ABSENCE, OR DIRECTION, OF MOVEMENT
- G01P5/00—Measuring speed of fluids, e.g. of air stream; Measuring speed of bodies relative to fluids, e.g. of ship, of aircraft
- G01P5/26—Measuring speed of fluids, e.g. of air stream; Measuring speed of bodies relative to fluids, e.g. of ship, of aircraft by measuring the direct influence of the streaming fluid on the properties of a detecting optical wave
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01P—MEASURING LINEAR OR ANGULAR SPEED, ACCELERATION, DECELERATION, OR SHOCK; INDICATING PRESENCE, ABSENCE, OR DIRECTION, OF MOVEMENT
- G01P1/00—Details of instruments
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01P—MEASURING LINEAR OR ANGULAR SPEED, ACCELERATION, DECELERATION, OR SHOCK; INDICATING PRESENCE, ABSENCE, OR DIRECTION, OF MOVEMENT
- G01P13/00—Indicating or recording presence, absence, or direction, of movement
- G01P13/02—Indicating direction only, e.g. by weather vane
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- G—PHYSICS
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- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses a micro-nano flow fluorescence bleaching speed measurement method and a micro-nano flow fluorescence bleaching speed measurement system based on camera imaging, which are applied to the technical field of micro-nano flow field speed measurement, and the method comprises the following steps: collecting fluorescent light spot images of the fluorescent solution in a static flow field, a standard flow field and a flow field to be measured; carrying out light spot identification under a static flow field to construct a light spot area; measuring the relation between the fluorescence signal intensity and the flow velocity in the area under the standard flow field to obtain a speed calibration curve; calculating the fluorescence signal intensity in the region in the flow field to be measured, and calculating the corresponding flow velocity by using a velocity calibration curve; the change of the barycenter coordinates of the fluorescent distribution in the region in the flow field to be measured relative to the barycenter coordinates of the fluorescent distribution in the region in the static flow field is calculated to obtain an x-direction vector component and a y-direction vector component of the velocity vector of the region, so that the velocity vector of the light spot region is obtained. The invention has simple structure, can simultaneously measure the speed and the direction of the fluid, supports multipoint measurement, and has high precision and easy operation.
Description
Technical Field
The invention relates to the technical field of micro-nano flow field speed measurement, in particular to a micro-nano flow fluorescence bleaching speed measurement method and system based on camera imaging.
Background
The fluorescent bleaching speed measurement technology is a novel micro-nano flow field speed measurement technology, and the most commonly used traditional fluorescent bleaching speed measurement technology at present is a laser-induced fluorescent bleaching speed measurement (Laser Induced Fluorescence Photobleaching Anemometer, LIFPA for short) technology, is mainly applied to micro-nano flow control chip design and research on micro-nano fluid dynamics mechanism, and can realize measurement of the micro-nano flow field speed with high time and spatial resolution. The advantage of the LIFPA technique is that it can achieve both spatial resolution of tens to hundreds of nm and temporal resolution of up to 3 mus, which is difficult to reach with other velocimetry techniques.
However, the conventional LIFPA technology can only measure the fluid velocity at a single location, and cannot distinguish the direction of the fluid velocity, which is a significant drawback for the fluid velocity measurement technology. Even in the improved LIFPA technology, for example: the LIFPA technology based on the optical fiber bundle can realize measurement of the fluid speed direction, but the device has a complex structure, a measurement subsystem (a single measurement subsystem comprises a high-sensitivity photomultiplier, a signal amplifying and filtering device and the like) with the same number of fiber cores as the optical fiber bundle is needed, and the position of the optical fiber bundle needs to be adjusted before the use, so that the receiving end of the optical fiber bundle always aims at the fluorescent center in a static flow field, and the fluorescent light spots are required to be in strict Gaussian distribution. This is disadvantageous for structural optimization of the experimental setup, for practical operation of the experimental procedure and for the stability of the measurements. More importantly, the LIFPA technology based on optical fiber clusters is still a single point measurement technology, which limits the application scope of the technology. Therefore, it is a need for a simple method and system for fluorescence bleaching speed measurement that facilitates operation and execution, is identifiable in direction and is capable of detecting multiple points.
Disclosure of Invention
In view of the above, the invention provides a micro-nano flow fluorescence bleaching speed measurement method and system based on camera imaging.
In order to achieve the above object, the present invention provides the following technical solutions:
a micro-nano flow fluorescence bleaching speed measurement method based on camera imaging comprises the following steps:
step 1: collecting fluorescent light spot images of the fluorescent solution in a static flow field, a standard flow field and a flow field to be measured respectively;
step 2: identifying fluorescent light spots in the fluorescent light spot image under the static flow field, constructing a light spot area, and calibrating the position of each fluorescent light spot;
step 3: calculating barycentric coordinates of fluorescence distribution in each light spot area under a static flow field;
step 4: measuring the relation between the fluorescent signal intensity and the flow velocity in each light spot area under the standard flow field, and obtaining a speed calibration curve at the fluorescent light spot through numerical fitting;
step 5: calculating the fluorescence signal intensity in each light spot area in the flow field to be detected, and calculating the flow velocity corresponding to the fluorescence signal intensity by using a speed calibration curve at the light spot area;
step 6: calculating the barycenter coordinates of the fluorescent distribution in each light spot area in the flow field to be measured, and obtaining an x-direction vector component and a y-direction vector component representing the speed vector of the light spot area by calculating the change of the barycenter coordinates of the fluorescent distribution in the light spot area in the flow field to be measured relative to the barycenter coordinates of the fluorescent distribution in the light spot area under the static flow field;
step 7: and obtaining the actual speed vector in each spot area under the to-be-measured flow field according to the x-direction vector component and the y-direction vector component of the speed vector of each spot area.
And executing the steps on each fluorescent light spot to obtain the speed and the direction of the flow field in each light spot area.
Furthermore, the method for collecting the fluorescent light spot image in the step 1 is that the fluorescent light spot image is collected through a camera, specifically, a laser beam is generated through a light source, the laser beam is subjected to parallel collimation through a beam collimation device to obtain an excitation beam, the excitation beam is subjected to excitation on a fluorescent solution in a flow field through a dichroic mirror and an objective lens to generate a fluorescent light spot, the fluorescent light spot is received by the camera through the objective lens and the dichroic mirror and then through a tube mirror, a fluorescent light spot image is obtained, and a single fluorescent light spot exists in the fluorescent light spot image.
Further, after passing through the beam collimation device, the laser beam is further modulated by using a light field modulation device to generate a multi-focus beam; the fluorescent light spots pass through the objective lens and the dichroic mirror and then are transmitted to the tubular mirror to be received by the camera, so as to obtain fluorescent light spot images, wherein a plurality of fluorescent light spots are arranged in the fluorescent light spot images. By introducing the light field modulation device, the flow velocity and direction in the flow field of the fluorescent light spot areas can be measured.
Further, the method for identifying the fluorescent light spots in the step 2 specifically includes: the fluorescent light spots in the fluorescent light spot image under the static flow field are automatically identified through the area identification algorithm, so that the fluorescent light spots in the image can be quickly and accurately identified.
Further, in the step 6, the specific method for obtaining the x-direction vector component and the y-direction vector component of the velocity vector representing the light spot area by calculating the change of the barycenter coordinate of the fluorescent distribution in the light spot area in the flow field to be measured relative to the barycenter coordinate of the fluorescent distribution in the light spot area in the static flow field comprises the following steps:
the kth spot area is counted as A k Spot area a under stationary flow field k Is G (x) k0 ,y k0 ) Spot area a in flow field to be measured k Is G (x) km ,y km ) The x-direction vector component is:
the y-direction vector component is:
wherein the method comprises the steps ofRepresenting the x-direction vector component of the coordinate axis,/-, and>representing the vector component in the y-direction of the coordinate axis.
Further, in the step 7, the specific method for obtaining the actual velocity vector in each spot area under the to-be-measured flow field according to the x-direction vector component and the y-direction vector component of the velocity vector of each spot area is as follows:
with the kth spot area A k For example, light spot area A under flow field to be measured is measured k The flow velocity in the internal is u k Spot area a under the flow field to be measured k The actual velocity vector within the range is given by,
in the step 1, besides collecting the fluorescent light spot images of the fluorescent solution under the static flow field, the standard flow field and the flow field to be measured, the fluorescent light spot images of different positions are also collected, so that the fluid speed and the fluid direction of different positions in the flow field can be measured.
The invention also discloses a micro-nano flow fluorescence bleaching speed measurement system based on camera imaging, which comprises:
the image acquisition module is used for acquiring fluorescent light spot images of the fluorescent solution in a static flow field, a standard flow field and a flow field to be measured respectively;
the fluorescent light spot identification module is used for identifying fluorescent light spots in the fluorescent light spot image under the static flow field, constructing a light spot area and calibrating the position of each fluorescent light spot;
the fluorescent light spot gravity center calculating module is used for calculating gravity center coordinates of fluorescent distribution in each light spot area under the static flow field;
the speed calibration curve acquisition module is used for measuring the relationship between the fluorescence signal intensity and the flow velocity in each light spot area under the standard flow field and obtaining a speed calibration curve at the fluorescence light spot through numerical fitting;
the flow velocity calculation module is used for calculating the fluorescence signal intensity in each light spot area in the flow field to be measured, and calculating the flow velocity corresponding to the fluorescence signal intensity by utilizing a velocity calibration curve at the light spot area;
the speed vector calculation module is used for calculating the barycenter coordinates of the fluorescent distribution in each light spot area in the flow field to be measured, and obtaining an x-direction vector component and a y-direction vector component representing the speed vector of the light spot area by calculating the change of the barycenter coordinates of the fluorescent distribution in the light spot area in the flow field to be measured relative to the barycenter coordinates of the fluorescent distribution in the light spot area under the static flow field; and obtaining the actual speed vector in each spot area under the to-be-measured flow field according to the x-direction vector component and the y-direction vector component of the speed vector of each spot area.
Further, the image acquisition module comprises a laser light source, a light beam adjusting device, a spatial modulator, a dichroic mirror, an objective lens, a tubular mirror and a camera, wherein laser beams generated by the light source are subjected to parallel collimation by the light beam collimating device to obtain excitation light beams, the excitation light beams enter a flow field fluorescent solution through the dichroic mirror and the objective lens, and generated fluorescent light spots enter the camera through the objective lens, the dichroic mirror and the tubular mirror.
Further, the image acquisition module further comprises a light field modulation device, wherein the light field modulation device is positioned between the light beam adjustment device and the dichroic mirror and is used for further modulating the laser beam to generate a multi-focus light beam.
Compared with the prior art, the micro-nano flow fluorescence bleaching speed measuring method and system based on camera imaging provided by the invention have the following beneficial effects:
(1) The FPV system of the invention can be realized based on an inverted fluorescence microscopic imaging device and a camera, wherein the inverted fluorescence microscopic imaging device consists of a microscope body, a light source, a light field modulation device, a dichroic mirror, an objective lens, a tube mirror and a camera, and the inverted fluorescence microscopic imaging device can also comprise a filter. In LIFPA systems with multiple measurement points, only when the number of measurement points is increased to 4, a complex fluorescence signal separation optical path, 4 confocal modules and 16 sets of measurement subsystems (including a high-sensitivity photomultiplier, a signal amplifying and filtering device and the like) are required for the four-core optical fiber bundle LIFPA technology. For the present invention, the huge number of optical devices can be replaced by only one camera.
(2) The invention not only can measure the speed of the fluid in the flow field, but also can measure the speed direction of the fluid at the same time, and overcomes the limitation that the traditional method only supports measuring the speed of the fluid.
(3) The method can simultaneously measure the speed at more than 40 x 40 points by combining the beam modulation technology, realizes the multi-point speed measurement in the flow field, and overcomes the limitation of single-point measurement in the traditional method; meanwhile, the flow velocity and direction of different positions of the flow field can be measured by collecting fluorescent light spot images of different positions in the flow field.
(4) The invention has simple and easy operation and provides great convenience for the use of professionals.
(5) The invention can realize the adjustment of the size of the spot area by adjusting the spot radius r, thereby realizing higher spatial resolution and measurement accuracy.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a micro-nano flow fluorescence bleaching velocimetry method based on camera imaging according to the invention;
FIG. 2 is a block diagram of a micro-nano flow fluorescence bleaching velocimetry system based on camera imaging according to the present invention;
FIG. 3 is a schematic diagram of an image acquisition module in one embodiment;
FIG. 4 is a schematic diagram of an image capturing module according to another embodiment;
FIG. 5 is a schematic diagram of a data processing software interface in one embodiment;
FIG. 6 (a) is a schematic diagram of a data processing software cursor calibration options interface;
FIG. 6 (b) is a schematic diagram of a data processing software speed calibration options interface;
FIG. 6 (c) is a schematic diagram of a data processing software speed field calculation options interface;
FIG. 7 is a diagram showing the change of the barycentric coordinates of the fluorescent distribution in the spot area of the flow field to be measured relative to the barycentric coordinates of the fluorescent distribution in the spot area of the stationary flow field;
wherein, light source 1, light beam adjusting device 2, dichroic mirror 3, objective lens 4, tube mirror 5, camera 6, light field modulating device 7, flow field 8.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a micro-nano flow fluorescence bleaching speed measurement method and a micro-nano flow fluorescence bleaching speed measurement system based on camera imaging, which utilize the speed measurement principle that:
calculating the intensity of the fluorescence signal acquired by the camera according to the algorithm by utilizing the characteristic that the intensity of fluorescence increases along with the increase of the flow velocity, so as to obtain the velocity of the fluid at the light spot; the velocity measurement method or system obtains the velocity direction of the fluid on the plane perpendicular to the optical axis by calculating the relative shift of the center of gravity of the fluorescence signal by using the distribution rule that the fluorescence intensity is reduced along the flow direction. Namely: the invention adopts a two-dimensional flow velocity vector measuring mode, and simultaneously measures the velocity and the direction of fluid flowing on a plane perpendicular to an optical axis.
One micro-nano flow fluorescence bleaching velocimetry method based on camera imaging is shown in fig. 1, and comprises the following steps:
step 1: collecting fluorescent light spot images of the fluorescent solution in a static flow field, a standard flow field and a flow field to be measured respectively;
step 2: identifying fluorescent light spots in a fluorescent light spot image under a static flow field, constructing a light spot area, and calibrating the position of each fluorescent light spot, wherein the light spot area is preferably a circular light spot area, and can also be square or triangular and the like, and the invention is not limited to the circular light spot area;
step 3: calculating barycentric coordinates of fluorescence distribution in each light spot area under a static flow field;
step 4: the standard flow field is generated by a flow rate control device (such as a pressure pump), the flow rate is controlled by the flow rate control device, so that the flow rate under the standard flow field is known, the relationship between the fluorescent signal intensity and the flow rate in each light spot area under the standard flow field is measured, and a speed calibration curve at the fluorescent light spot is obtained through numerical fitting;
step 5: calculating the fluorescence signal intensity in each light spot area in the flow field to be detected, and calculating the flow velocity corresponding to the fluorescence signal intensity by using a speed calibration curve at the light spot area;
step 6: calculating the barycenter coordinates of the fluorescent distribution in each light spot area in the flow field to be measured, and obtaining an x-direction vector component and a y-direction vector component representing the speed vector of the light spot area by calculating the change of the barycenter coordinates of the fluorescent distribution in the light spot area in the flow field to be measured relative to the barycenter coordinates of the fluorescent distribution in the light spot area under the static flow field;
step 7: and obtaining the actual speed vector in each spot area under the to-be-measured flow field according to the x-direction vector component and the y-direction vector component of the speed vector of each spot area.
And executing the steps on each fluorescent light spot to obtain the speed and the direction of the flow field in each light spot area.
The barycentric coordinate calculation formula for calculating the fluorescence distribution in the light spot area in the image is as follows:
wherein W is x 、W y The x and y coordinates of the center of gravity of the spot area are respectively represented, I is a gray matrix of the image, m, n represents the size of the image matrix, for example, m=1024, n=2048 for an image with size 1024×2048.
The calculation formula for the fluorescence signal intensity in the fluorescence region is:
with the kth spot area A k For example, the specific process of calculating the flow field velocity and direction in the region is as follows:
calculating a facula area A under a static flow field k Center of gravity coordinate G (x) k0 ,y k0 )；
Spot area a under measuring standard flow field k Intensity of fluorescent signal I in k And the flow velocity U k By numerical fitting to obtain the fluorescent spot A k Velocity calibration curve I of (2) k -U k ；
Calculating a facula area A in a flow field to be measured k Intensity of fluorescent signal I in k,m By means of spot area A k Velocity calibration curve I of (2) k -U k Calculating the intensity I of fluorescent signals k,m Corresponding flow rate size u k U is k Namely the facula area A in the flow field to be measured k The flow rate in the inner part;
calculating a facula area A in a flow field to be measured k Barycentric coordinates G (x) km ,y km ) By calculating the facula area A in the flow field to be measured k Center of gravity coordinate G (x) km ,y km ) With respect to the barycentric coordinates G (x) k0 ,y k0 ) Is changed to obtain a representative spot area A k X-direction vector component of velocity vector of (2)And y-direction vector component->The specific formula is that,
wherein the method comprises the steps ofRepresenting the x-direction vector component of the coordinate axis,/-, and>a vector component representing the y-direction of the coordinate axis;
according to the light spot area A k X-direction vector component of velocity vector of (2)And y-direction vector component->Obtaining a light spot area A k The specific formula of the actual velocity vector in the model is +.>
In conclusion, it can be seen that u k Namely the facula area A in the flow field to be measured k The flow velocity in the inner part is greater than the flow velocity in the outer part byThe spot area A can be known k The flow field direction in the inner part.
Referring to fig. 2, a micro-nano flow fluorescence bleaching velocimetry system based on camera imaging comprises:
the image acquisition module is used for acquiring fluorescent light spot images of the fluorescent solution in a static flow field, a standard flow field and a flow field to be measured respectively;
the fluorescent light spot identification module is used for identifying fluorescent light spots in the fluorescent light spot image under the static flow field, constructing a light spot area and calibrating the position of each fluorescent light spot;
the fluorescent light spot gravity center calculating module is used for calculating gravity center coordinates of fluorescent distribution in each light spot area under the static flow field;
the speed calibration curve acquisition module is used for measuring the relationship between the fluorescence signal intensity and the flow velocity in each light spot area under the standard flow field and obtaining a speed calibration curve at the fluorescence light spot through numerical fitting;
the flow velocity calculation module is used for calculating the fluorescence signal intensity in each light spot area in the flow field to be measured, and calculating the flow velocity corresponding to the fluorescence signal intensity by utilizing a velocity calibration curve at the light spot area;
the speed vector calculation module is used for calculating the barycenter coordinates of the fluorescent distribution in each light spot area in the flow field to be measured, and obtaining an x-direction vector component and a y-direction vector component representing the speed vector of the light spot area by calculating the change of the barycenter coordinates of the fluorescent distribution in the light spot area in the flow field to be measured relative to the barycenter coordinates of the fluorescent distribution in the light spot area under the static flow field; and obtaining the actual speed vector in each spot area under the to-be-measured flow field according to the x-direction vector component and the y-direction vector component of the speed vector of each spot area.
In one embodiment, the image acquisition module in the system corresponds to step 1 in the method, and specifically comprises a light source 1, a light beam adjusting device 2, a dichroic mirror 3, an objective lens 4, a tube mirror 5 and a camera 6, and the connection mode is shown in fig. 3, wherein the light source 1 is a laser light source and is used for generating a laser beam, the generated laser beam is subjected to parallel collimation through the light beam collimation device 2, the excitation beam enters a fluorescent solution of a flow field 8 through the dichroic mirror 3 and the objective lens 4, the generated fluorescent light spot enters the camera 6 through the objective lens 4, the dichroic mirror 3 and the tube mirror 5, and the camera 6 is a high-sensitivity camera. The apparatus of fig. 3 is used to acquire a single fluorescent light spot.
In another embodiment, the image acquisition module further comprises a light field modulation device 7, the connection manner of which is shown in fig. 4, wherein the laser beam generated by the light source 1 is modulated by the beam collimation device 2 and the light field modulation device 7, and a multi-focus beam is output, and the multi-focus beam enters a fluorescent solution of the flow field 8 through the dichroic mirror 3 and the objective lens 4, and the generated fluorescent light spot enters the camera 6 through the objective lens 4, the dichroic mirror 3 and the tube mirror 5. By introducing the light field modulation device 7, the apparatus of fig. 4 can obtain a plurality of fluorescent light spots. The light field modulation device 7 may use a spatial light modulator.
In a specific implementation process, the tube mirror 5 can be further split into a combination of the tube mirror and the reflecting mirror, so that light transmission is realized.
In the method, the steps 2-7 correspond to a fluorescent light spot identification module, a light spot area determination module, a speed calibration curve acquisition module, a flow velocity calculation module and a speed vector calculation module in the system, in a specific embodiment, the modules are integrated into data processing software, after a camera 6 of the image acquisition module acquires a fluorescent light spot image, the fluorescent light spot image is input into the data processing software for processing, the data processing software comprises a cursor calibration option, a speed calibration option and a speed field calculation option, and an interface is shown in fig. 5, and the specific interface is shown in the figure 5:
in the cursor calibration option, referring to fig. 6 (a), a spot image under a static flow field is imported, and the number of fluorescent spots to be calibrated in the spot image and the radius r of a spot area are set through the options of fitting number and fitting multiplying power; automatically identifying fluorescent light spots of the light spot image under the static flow field through a fitting calibration option of the light spot calibration module to obtain a light spot circle center and an identification radius R, and constructing a light spot area by taking the light spot circle center as the center and a set calculation radius R, namely the light spot calibration area; calculating the barycenter coordinates of the fluorescent signals in the area to obtain the barycenter coordinates of the light spots in the light spot calibration area under the static flow field; the set calculated radius R is less than or equal to R, the size of the light spot area is adjustable, the spatial resolution of the system is determined, and the light spot area can be one or a plurality of light spot areas.
In the speed calibration option, see fig. 6 (b), the fluorescence spot images of different flow rates under the standard flow field are imported; and selecting a light spot image in the file list window, inputting the corresponding flow velocity in the flow velocity input window, measuring the relation between the fluorescence signal intensity in each light spot area under the standard flow field and the flow velocity at the light spot area, and obtaining a velocity calibration curve at the corresponding light spot through numerical fitting.
In the speed field calculation option, referring to fig. 6 (c), a fluorescent light spot image of the flow field to be measured is introduced, the flow velocity and the actual velocity vector of the area in the flow field to be measured are obtained by calculation, and fig. 7 is a schematic diagram of the change of the barycenter coordinates of the fluorescent distribution in the light spot area of the flow field to be measured relative to the barycenter coordinates of the fluorescent distribution in the light spot area of the stationary flow field.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. The micro-nano flow fluorescence bleaching speed measurement method based on camera imaging is characterized by comprising the following steps of:
collecting fluorescent light spot images of the fluorescent solution in a static flow field, a standard flow field and a flow field to be measured respectively;
identifying fluorescent light spots in the fluorescent light spot image under the static flow field, constructing a light spot area, and calibrating the position of each fluorescent light spot;
calculating barycentric coordinates of fluorescence distribution in each light spot area under a static flow field;
measuring the relation between the fluorescent signal intensity and the flow velocity in each light spot area under the standard flow field, and obtaining a speed calibration curve at the fluorescent light spot through numerical fitting;
calculating the fluorescence signal intensity in each light spot area in the flow field to be detected, and calculating the flow velocity corresponding to the fluorescence signal intensity by using a speed calibration curve at the light spot area;
calculating the barycenter coordinates of the fluorescent distribution in each light spot area in the flow field to be measured, and obtaining an x-direction vector component and a y-direction vector component representing the speed vector of the light spot area by calculating the change of the barycenter coordinates of the fluorescent distribution in the light spot area in the flow field to be measured relative to the barycenter coordinates of the fluorescent distribution in the light spot area in the static flow field, wherein the specific method comprises the following steps:
the kth spot area is counted as A k Spot area a under stationary flow field k Is G (x) k0 ,y k0 ) Spot area a in flow field to be measured k Is G (x) km ,y km ) The x-direction vector component is:
the y-direction vector component is:
wherein the method comprises the steps ofRepresenting the x-direction vector component of the coordinate axis,/-, and>a vector component representing the y-direction of the coordinate axis;
and obtaining the actual speed vector in each spot area under the to-be-measured flow field according to the x-direction vector component and the y-direction vector component of the speed vector of each spot area.
2. The micro-nano flow fluorescence bleaching velocimetry method based on camera imaging according to claim 1 is characterized in that the method for collecting fluorescence spot images is to collect by a camera, specifically, a laser beam is generated by a light source (1), the laser beam is collimated by a beam collimation device (2) to obtain a parallel collimated excitation beam, the excitation beam excites a fluorescence solution in a flow field after passing through a dichroic mirror (3) and an objective lens (4) to generate fluorescence spots, and the fluorescence spots pass through the objective lens (4) and the dichroic mirror (3) and then pass through a tube mirror (5) and are received by a camera (6) to obtain fluorescence spot images, wherein single fluorescence spots exist in the fluorescence spot images.
3. The micro-nano flow fluorescence bleaching velocimetry method based on camera imaging according to claim 2, wherein the laser beam is modulated by a light field modulation device (7) after passing through a beam collimation device (2) to generate a multi-focus beam; the fluorescent light spots are transmitted to a tube mirror (5) after passing through the objective lens (4) and the dichroic mirror (3), and are received by a camera (6) to obtain fluorescent light spot images, wherein a plurality of fluorescent light spots are arranged in the fluorescent light spot images.
4. The micro-nano flow fluorescence bleaching velocimetry method based on camera imaging according to claim 1, wherein the method for identifying fluorescent light spots is specifically as follows: and automatically identifying the fluorescent light spots in the fluorescent light spot image under the static flow field through a region identification algorithm.
5. The micro-nano flow fluorescence bleaching velocimetry method based on camera imaging according to claim 1, wherein the specific method for obtaining the actual velocity vector in each spot area under the flow field to be measured according to the x-direction vector component and the y-direction vector component of the velocity vector of each spot area is as follows:
with the kth spot area A k For example, light spot area A under flow field to be measured is measured k The flow velocity in the internal is u k Spot area a under the flow field to be measured k The actual velocity vector within the range is given by,
6. the micro-nano flow fluorescence bleaching velocimetry method based on camera imaging according to claim 1 is characterized in that besides collecting fluorescence spot images of a fluorescent solution in a static flow field, a standard flow field and a flow field to be measured respectively, fluorescence spot images of different positions are also collected for measuring the fluid velocity and the fluid velocity direction of different positions in the flow field.
7. Micro-nano flow fluorescence bleaching velocimetry system based on camera imaging, characterized by comprising:
the image acquisition module is used for acquiring fluorescent light spot images of the fluorescent solution in a static flow field, a standard flow field and a flow field to be measured respectively;
the fluorescent light spot identification module is used for identifying fluorescent light spots in the fluorescent light spot image under the static flow field, constructing a light spot area and calibrating the position of each fluorescent light spot;
the fluorescent light spot gravity center calculating module is used for calculating gravity center coordinates of fluorescent distribution in each light spot area under the static flow field;
the speed calibration curve acquisition module is used for measuring the relationship between the fluorescence signal intensity and the flow velocity in each light spot area under the standard flow field and obtaining a speed calibration curve at the fluorescence light spot through numerical fitting;
the flow velocity calculation module is used for calculating the fluorescence signal intensity in each light spot area in the flow field to be measured, and calculating the flow velocity corresponding to the fluorescence signal intensity by utilizing a velocity calibration curve at the light spot area;
the speed vector calculation module is used for calculating the barycenter coordinates of the fluorescent distribution in each light spot area in the flow field to be measured, and obtaining an x-direction vector component and a y-direction vector component representing the speed vector of the light spot area by calculating the change of the barycenter coordinates of the fluorescent distribution in the light spot area in the flow field to be measured relative to the barycenter coordinates of the fluorescent distribution in the light spot area under the static flow field; specific: the kth spot area is counted as A k Spot area a under stationary flow field k Is G (x) k0 ,y k0 ) Spot area a in flow field to be measured k Is G (x) km ,y km ) The x-direction vector component is:
the y-direction vector component is:
wherein the method comprises the steps ofRepresenting the x-direction vector component of the coordinate axis,/-, and>a vector component representing the y-direction of the coordinate axis; and obtaining the actual speed vector in each spot area under the to-be-measured flow field according to the x-direction vector component and the y-direction vector component of the speed vector of each spot area.
8. The micro-nano flow fluorescence bleaching velocimetry system based on camera imaging according to claim 7, wherein the image acquisition module comprises a light source (1), a light beam adjusting device (2), a dichroic mirror (3), an objective lens (4), a tube mirror (5) and a camera (6), wherein a laser beam generated by the light source (1) is subjected to parallel collimation by the light beam collimation device (2), the excitation beam enters a flow field fluorescent solution through the dichroic mirror (3) and the objective lens (4), and a generated fluorescent light spot passes through the objective lens (4), the dichroic mirror (3) and the tube mirror (5) and enters the camera (6).
9. The micro-nano flow fluorescence bleaching velocimetry system based on camera imaging according to claim 8, wherein the image acquisition module further comprises a light field modulation device (7), the light field modulation device (7) being located between the beam adjustment device (2) and the dichroic mirror (3) for further modulating the laser beam to generate a multi-focal beam.
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