CN111617390A

CN111617390A - Device for regulating and controlling red blood cells in living animal blood vessel and application thereof

Info

Publication number: CN111617390A
Application number: CN202010582545.3A
Authority: CN
Inventors: 刘晓帅; 李宝军; 李宇超; 高晴; 张垚
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2020-09-04
Anticipated expiration: 2040-06-23
Also published as: CN111617390B

Abstract

The invention provides a device for regulating and controlling red blood cells in a living animal blood vessel and application thereof, belonging to the technical field of photoelectricity; the device comprises a laser emission component, an acousto-optic modulation component, a light beam broadening component, a short-wave-pass dichroic mirror and an inverted objective lens along a laser path in sequence; the device also comprises an image input and output component, a convex lens, a condenser and a white light emitting component which are arranged on the imaging optical path; laser emitted by the laser emission component sequentially passes through the acousto-optic modulation component and the beam broadening component and then irradiates the short-wave-pass dichroic mirror to change the laser transmission direction; the laser with the changed transmission direction is focused to the sample chamber through the inverted objective lens. Utilize laser emission subassembly outgoing laser will with acousto-optic modulation subassembly interact, realize the dynamic scanning of laser focus, the accurate static distribution of laser focus in a plurality of positions can be realized to quick scanning, and then is applied to stable the catching of a plurality of erythrocytes, accurate range and rotatory control.

Description

Device for regulating and controlling red blood cells in living animal blood vessel and application thereof

Technical Field

The invention relates to the technical field of photoelectricity, in particular to a device for regulating and controlling red blood cells and application thereof.

Background

It is known that there is a complex vascular network system in animals, which supplies fresh oxygen and nutrients to tissues and organs through continuously flowing blood and takes away metabolic products such as carbon dioxide to maintain normal physiological balance in the body. Deep understanding of the physiological mechanism of blood flow, accurate non-contact intervention, and multifunctional control of blood flow can provide potential ideas and alternatives for further clinical diagnosis and treatment, drug delivery and anticancer treatment.

Currently, magnetic, ultrasound, and electric field based techniques have begun to be applied to the dynamic regulation of blood flow. However, the above schemes require invasive implantation of external materials, such as magnetic nanoparticles, ultrasound source and metal electrode implanted into blood vessels as manipulation markers or excitation source, and the above materials are inherently poor in compatibility with living organisms, are liable to induce immune system feedback in vivo, and reduce the precision and stability of manipulation.

Disclosure of Invention

The invention aims to provide a device for regulating and controlling red blood cells and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a device for regulating and controlling red blood cells in living animal blood vessels, which sequentially comprises a laser emission component, an acousto-optic modulation component, a light beam broadening component, a short-wave-pass dichroic mirror, an inverted objective and a sample chamber along a laser path; the device also comprises an image input and output component, a convex lens, a condenser and a white light emitting component which are arranged on the imaging optical path;

laser emitted by the laser emission component sequentially passes through the acousto-optic modulation component and the beam broadening component and then irradiates the short-wave-pass dichroic mirror to change the laser transmission direction; the laser with the changed transmission direction is focused to the sample chamber through the inverted objective lens;

the white light emitting component emits white light, and the white light is focused to the image input and output component through the condenser, the sample chamber, the inverted objective lens, the short-wave-pass dichroic mirror and the convex lens; the image input and output assembly observes and acquires image information of the sample chamber.

Preferably, the image input and output assembly includes a CCD camera connected to an electronic device.

Preferably, the short-wave pass dichroic mirror is a short-wave pass dichroic mirror which allows the wavelength band of 430-800 nm to transmit and does not allow the wavelength of 800-1300 nm to transmit.

Preferably, the laser emitted by the laser emitting assembly is single-mode laser; the wavelength of the emergent laser is 1000-1100 nm.

Preferably, the scanning frequency of the acousto-optic modulation component is 0-100 KHz.

Preferably, the beam broadening assembly comprises a first convex lens and a second convex lens; the first convex lens is arranged at the closer end of the acousto-optic modulation component; the second convex lens is arranged at the far end of the acousto-optic modulation component; the ratio of the curvature radii of the first convex lens and the second convex lens is 1: 10; the distance between the first convex lens and the second convex lens is the sum of the focal lengths of the first convex lens and the second convex lens.

The invention provides application of the device in the scheme in stable capture, accurate arrangement or controllable rotation of erythrocytes in blood vessels of living animals with non-therapeutic purposes.

Preferably, the application comprises the following steps:

1) fixing the live animal in a sample chamber after anesthesia;

2) opening a white light emitting component to emit white light, and focusing the white light to an image input and output component through a condenser, a sample chamber, an inverted objective lens, a short-wave-pass dichroic mirror and a convex lens;

3) observing and collecting image information of the living animal through the image input and output assembly, determining the position of red blood cells to be controlled of the living animal in the image information, and establishing a two-dimensional coordinate system by taking the lower left corner of an observation visual field as a coordinate origin; inputting coordinate values of the red blood cells to be controlled in the image information in the acousto-optic modulation component, and setting the coordinate values as the focal positions of the laser;

4) the laser emitting component emits laser, the focus position is scanned by the acousto-optic modulation component, the light beam broadening component broadens, the short wave-pass dichroic mirror turns and the inverted objective lens focuses, the laser is focused on the living animal of the sample chamber to form an optical trap, and the control of the red blood cells is realized by manually controlling the movement of the optical trap.

Preferably, the coordinate value of each of the red blood cells to be controlled in step 2) includes two coordinate values corresponding to two ends of the red blood cell.

Preferably, the red blood cells are replaced with other cells having no color characteristics; the other cells without color features are subjected to fluorescent labeling treatment.

The invention has the beneficial effects that: the invention provides a device for regulating and controlling red blood cells in living animal blood vessels, which sequentially comprises a laser emission component, an acousto-optic modulation component, a light beam broadening component, a short-wave-pass dichroic mirror and an inverted objective lens along a laser path; the device also comprises an image input and output component, a convex lens, a condenser and a white light emitting component which are arranged on the imaging optical path; laser emitted by the laser emission component sequentially passes through the acousto-optic modulation component and the beam broadening component and then irradiates the short-wave-pass dichroic mirror to change the laser transmission direction; the laser with the changed transmission direction is focused to the sample chamber through the inverted objective lens; the white light emitting component emits white light, and the white light is focused to the image input and output component through the condenser, the sample chamber, the inverted objective lens, the short-wave-pass dichroic mirror and the convex lens; the image input and output assembly observes and acquires image information of the sample chamber. Observing and collecting image information of a living animal through an image input and output assembly, determining the position of red blood cells to be controlled of the living animal in the image information, and establishing a two-dimensional coordinate system by taking the lower left corner of an observation visual field as a coordinate origin; inputting coordinate values of the red blood cells to be controlled in the image information in the acousto-optic modulation component, and setting the coordinate values as the focal positions of the laser; the laser emitting component emits laser, the focus position is scanned by the acousto-optic modulation component, the light beam broadening component broadens, the short wave-pass dichroic mirror turns and the inverted objective lens focuses, the laser is focused on the living animal of the sample chamber to form an optical trap, and the control of the red blood cells is realized by manually controlling the movement of the optical trap.

The invention utilizes the laser emitting component to emit laser to irradiate the acousto-optic modulation component, realizes the dynamic scanning of the fixed point laser focus according to the setting of the coordinate system, can realize the quasi-static distribution of the laser focus at a plurality of positions by fast scanning, and is further applied to the stable capture, the precise arrangement and the rotation control of a plurality of cells. The device provided by the invention can control the behavior of the red blood cells, so that the red blood cells are assembled into the optical flow cell switch (optical trap), the movement of the red blood cells is controlled by manually controlling the movement of the optical trap, and the accurate regulation and control of the blood flow direction and the blood flow speed in the blood vessel can be realized. The optical flow cell switch assembled by rotating red blood cells is used for increasing local flow rate to further cause blood pressure reduction, destroying the initial blood pressure balance state, inducing and driving the generation of blood flow, and further realizing the rotation driving of naturally blocking the blood flow in blood vessels.

The invention utilizes the erythrocytes naturally existing in the blood vessel to assemble the optofluidic cell switch, does not need to inject external functionalized particles, avoids complex micro-nano particle manufacturing and potential immune feedback, and has good biocompatibility; by utilizing the advantages of non-contact and no damage of the optical tweezers, the position of the focus in the z-axis direction is changed by moving the focusing lens (the convex lens on the light source path) up and down, the laser focusing depth can be regulated and controlled, so that erythrocytes in blood vessels with different depths under the epidermis can be captured and controlled, invasive implantation of electrodes or magnetrons is not needed, and potential physical damage is avoided; by utilizing the rapid scanning of the laser focus, a plurality of red blood cells can be controlled and accurately arranged at the same time, no additional optical regulating and controlling element is required, the system integration level is high, and the operation is convenient; the assembled optical flow cell switch can realize multifunctional regulation and control of blood flow in a target blood vessel, and the regulation precision can reach the level of a single cell.

Drawings

FIG. 1 is a schematic diagram of the application of the device of the present invention in red blood cell regulation; a in FIG. 1 indicates that optofluidic cell switches close branches II and III, and that blood is diverted and flows entirely into branch I; b in fig. 1 represents a cross-sectional view of a in fig. 1;

FIG. 2 is an exploded view of the apparatus of the present invention, wherein 1. laser emitting assembly; 2. an acousto-optic modulation component; 3. a beam broadening assembly; 4. a short wave pass dichroic mirror; 5. inverting the objective lens; 6. a sample chamber; 7. an illumination light source; 8. a condenser; 9. a lens; 10. a CCD camera connected with a computer;

FIG. 3 is a schematic illustration of angular orientation localization and dynamic migration of a plurality of red blood cells in a blood vessel; in FIG. 3, a represents the random distribution of erythrocytes (labeled 1-10) in the blood vessel; a2 in FIG. 3 indicates that by manipulating the laser focus, the first five red blood cells (labeled 1-5) are aligned end-to-end and uniformly oriented, while the other five cells (labeled 6-10) are aligned with their long axes perpendicular to the vessel wall; b1 in fig. 3 indicates that red blood cells are arranged into pentagons by manipulating the laser focus; b2 in FIG. 3 indicates that red blood cells are arranged in a letter "J" shape by manipulating the laser focus; b3 in FIG. 3 indicates that red blood cells are arranged in the letter "N" by manipulating the laser focus; b4 in FIG. 3 indicates that red blood cells are arranged in a letter "U" shape by manipulating the laser focus;

c in fig. 3 indicates that by scanning the laser focus along a circular trajectory, a controlled revolution of a single red blood cell is achieved (i.e. rotation about an axis other than the cell itself); d in FIG. 3 indicates that controlled autorotation (i.e., rotation about the cell center) of individual red blood cells can be achieved by fixing the cell center and scanning the laser focus along the cell diameter;

FIG. 4 is a schematic diagram of dynamic closure of a branch flow;

FIG. 5 is a schematic view of directional focusing of blood flow;

FIG. 6 is a schematic view of controlled steering of blood flow;

FIG. 7 is a schematic view of the rotational drive of blood flow; a in fig. 7 shows that the blood vessel is blocked at natural pressure equilibrium without blood flow when the rotary switch is not provided; b in fig. 7 shows that by setting the rotary switch to lower the blood pressure value at the inlet of branch I, the driving blood flow is successfully generated in branch I, and red blood cells continuously flow out; c in fig. 7 shows that as the rotation speed of the rotary switch increases, the driving blood flow rate increases, i.e., the number of red blood cells per unit time is increased; d in fig. 7 indicates that the rotary switch is closed and the drive blood flow in branch I is lost, i.e. no more red blood cells are flowing out; e in fig. 7 denotes a restart of the rotary switch; in fig. 7, f indicates that the red blood cells continuously flow out of the branch I again after the switch is restarted.

Detailed Description

The invention provides a device for regulating and controlling red blood cells in living animal blood vessels, which sequentially comprises a laser emission component, an acousto-optic modulation component, a light beam broadening component, a short-wave-pass dichroic mirror, an inverted objective and a sample chamber along a laser path; the device also comprises an image input and output component, a convex lens, a condenser and a white light emitting component which are arranged on the imaging optical path; laser emitted by the laser emission component sequentially passes through the acousto-optic modulation component and the beam broadening component and then irradiates the short-wave-pass dichroic mirror to change the laser transmission direction; the laser with the changed transmission direction is focused to the sample chamber through the inverted objective lens; the white light emitting component emits white light, and the white light is focused to the image input and output component through the condenser, the sample chamber, the inverted objective lens, the short-wave-pass dichroic mirror and the convex lens; the image input and output assembly observes and acquires image information of the sample chamber.

An exploded view of an apparatus according to one embodiment of the present invention is shown in fig. 2, wherein 1. a laser emitting assembly; 2. an acousto-optic modulation component; 3. a beam broadening assembly; 4. a short wave pass dichroic mirror; 5. inverting the objective lens; 6. a sample chamber; 7. an illumination light source; 8. a condenser; 9. a lens; 10. and a CCD camera connected with a computer.

The distance between the components of the device is not particularly limited in the present invention and may be set at a distance that is conventional in the art.

In the invention, the outgoing laser of the laser emission component is preferably single-mode laser; the wavelength of the emitted laser is preferably 1000-1100 nm, more preferably 1064nm, the biological tissue absorbs less at 1000-1100 nm, and the biological tissue can be prevented from being damaged by a strong thermal effect; the power of the emergent laser is preferably 0-4W and is continuously adjustable.

In the invention, the angle of the emergent laser of the laser emitting component incident on the surface of the acousto-optic modulation component is preferably vertical; the invention adjusts the deflection direction of incident light by controlling the sound wave frequency based on the acousto-optic effect; in the specific implementation process of the invention, different coordinate values are input into the coordinate system of the acousto-optic modulation component to change the position of the focus, so that the dynamic scanning of the laser focus is realized; the scanning frequency of the acousto-optic modulation component is preferably 0-100 KHz; taking 100KHz as an example, the method can realize that the laser focus scans 100K positions on the focal plane within 1s, and the method can automatically scan and focus on the coordinate value points after inputting different coordinate values; the acousto-optic modulation assembly of the present invention preferably includes a scanning optical tweezers system; the scanning optical tweezers system is preferably available from Tweez250si, available from aristice technologies, inc (Aresis).

In the invention, the laser passing through the acousto-optic modulation component irradiates onto the light beam widening component, and the light beam is widened to completely cover the entrance pupil of the inverted objective lens, so that the optimal focusing of the laser is realized; as an embodiment of the present invention, the beam broadening assembly includes a first convex lens and a second convex lens; the first convex lens is arranged at the closer end of the acousto-optic modulation component; the second convex lens is arranged at the far end of the acousto-optic modulation component; the ratio of the radii of curvature of the first convex lens and the second convex lens is preferably 1: 10; the distance between the first convex lens and the second convex lens is the sum of the focal lengths of the first convex lens and the second convex lens.

In the invention, the laser emission component, the acousto-optic modulation component and the light beam widening component are positioned on the same straight line so as to ensure that emergent laser of the laser emission component sequentially passes through the acousto-optic modulation component and the light beam widening component; the direction of the laser light uptake is preferably vertical to ensure an optimal modulation effect.

In the invention, the short-wave-pass dichroic mirror is used for irradiating the broadened incident laser on the short-wave-pass dichroic mirror so as to change the transmission direction and enable the laser to enter the inverted objective mirror.

In the invention, the short-wave-pass dichroic mirror is a short-wave-pass dichroic mirror which allows the wavelength band of 430-800 nm to transmit and does not allow the wavelength of 800-1300 nm to transmit. When laser (1064nm) is incident on the surface of the short-wave-pass dichroic mirror at an angle of 45 degrees, total reflection occurs, the steering angle is changed by 90 degrees, and the laser enters a focusing lens and is irradiated to the surface of a sample. And the corresponding wavelength of the white light illuminating light source is 430-800 nm, and scattered light after striking the surface of the sample can directly pass through the short wave pass dichroic mirror, is focused by the lens and then converges on the surface of the CCD camera to finish imaging.

In the present invention, the inverted objective comprises a water mirror with a magnification of preferably 60 ×; the numerical aperture of the inverted objective is preferably 1.0, so that the effective focusing of laser is realized; the inverted objective lens functions to eliminate aberrations and reduce scattering due to refractive index mismatch.

In the present invention, the length of the sample chamber is preferably 5cm, the width is preferably 2.5cm, the height is preferably 1cm, the lower layer is a glass slide, the two sides are cushioned by agar gel, and the uppermost layer is sealed by the glass slide.

In the present invention, the apparatus includes a white light emitting component and a condenser; the light source is focused to the image input and output component through the condenser, the sample chamber, the inverted objective lens, the short-wave-pass dichroic mirror and the convex lens, and a clear visual field is provided for observing blood vessels and red blood cells in the sample and imaging; the light source is preferably a white light source. In the present invention, the light source direction of the white light emitting element is opposite to the direction of the laser light transmitted through the inverted objective lens. The laser light propagates from bottom to top, and the illumination light source irradiates from top to bottom. The short wave-pass dichroic mirror allows illumination light to enter the CCD camera for imaging, and laser is reflected on the surface of the short wave-pass dichroic mirror and cannot enter the CCD camera.

In the invention, the image input and output component observes and collects the image information of the sample chamber through a convex lens, a short-wave-pass dichroic mirror and an inverted objective lens; the image input and output assembly comprises a CCD camera connected with the electronic equipment and is used for real-time observation and video recording; the electronic device is preferably a computer; the convex lens has the function of focusing light beams, and then the light beams are all irradiated onto a sensing chip of the image input and output assembly to realize the optimal imaging quality. In the invention, the light beam is the light beam of the light source passing through the condenser, and the light beam cannot be turned due to full transmittance when passing through the short-wave-pass dichroic mirror.

In the invention, the focusing lens (the convex lens on the light source path) is moved up and down, the position of the focus in the z-axis direction is changed, the laser focusing depth can be regulated and controlled, and then the red blood cells in blood vessels with different depths under the epidermis are captured and controlled.

The present invention relies on the optical gradient force of a focused light beam on red blood cells to achieve stable capture of the cells: the laser beam is focused after passing through the focusing lens, the gradient of the light field near the laser focus is maximum, and then large optical gradient force is generated to capture target red blood cells to the laser focus. On the path that the laser passes through, because the optical field gradient is small, the corresponding received optical force of the cell is negligible, so that the cell cannot be captured.

In the present invention, the size of the microscope field of view taken by the CCD camera is preferably 93 μm × 93 μm; the imaging principle of the CCD camera is as follows: the light scattered by the object is converged by the lens and then is emitted to a photosensitive chip of the CCD to be converted into an electric signal, and the electric signal is finally converted into a visible video image through current amplification and analog-to-digital conversion.

As shown in FIG. 1, in the practice of the present invention, a live animal is anesthetized and then fixed in a sample chamber; observing and collecting image information of the living animal through a CCD camera connected with electronic equipment, and determining the position of red blood cells to be controlled of the living animal in the image information; establishing a two-dimensional coordinate system by taking the lower left corner of the observation visual field as a coordinate origin; clicking and inputting coordinate values corresponding to the red blood cells on a software (TWEEZ software in TWEEZ250 si) interface through a mouse, and setting the coordinate values as the focal positions of the laser; then the acousto-optic modulator is driven by software to drive the laser to the corresponding position, and an optical trap is formed. The set optical trap is manually dragged on the software picture through a mouse, so that the optical trap can be adjusted in real time.

In the invention, the laser can capture the red blood cells because of the action of light, namely, micro-nano particles and cells (the refractive index is larger than the surrounding environment) are subjected to the force pointing to the laser focus near the laser focus, and then are successfully captured. In addition to red blood cells, white blood cells, platelets, E.coli or yeast can also be manipulated by laser stable capture.

Preferably, the application comprises the following steps:

1) fixing the live animal in a sample chamber after anesthesia;

Firstly, anesthetizing a living animal and fixing the animal in a sample chamber; the invention has no special limitation on the anesthesia mode, and the conventional anesthesia mode in the field is adopted according to the living animals; the immobilization preferably comprises immobilization with 2% agarose; the 2% agarose is preferably obtained by dissolving 2g of agar in 100mL of water, maintaining the liquid state for several hours at 37 ℃, and continuously cooling to solidify and form gel.

After the living animal is fixed in the sample chamber, the white light emitting component is opened to emit white light, and the white light is focused on the image input and output component through the condenser, the sample chamber, the inverted objective lens, the short wave-pass dichroic mirror and the convex lens.

After white light is focused on the image input and output assembly, the invention observes and collects the image information of the living animal through the image input and output assembly, determines the position of the red blood cell to be controlled of the living animal in the image information, and establishes a two-dimensional coordinate system by taking the lower left corner of the observation visual field as the origin of coordinates; inputting coordinate values of the red blood cells to be controlled in the image information in the acousto-optic modulation component, and setting the coordinate values as the focal positions of the laser; the coordinate value of each red blood cell to be controlled preferably includes two coordinate values corresponding to both ends of the red blood cell. The invention can realize the specific orientation and stable movement of the red blood cells by inputting two coordinate values corresponding to two ends of each red blood cell to be controlled.

After the focal position of the laser is obtained, the laser emitting component emits the laser, the focal position is scanned by the acousto-optic modulation component, the light beam is widened by the light beam widening component, the short wave-pass dichroic mirror is turned and focused by the inverted objective lens, the laser is focused on the living animal of the sample chamber, and the control on the red blood cells is realized by manually controlling the movement of the optical trap; the control of the red blood cells preferably includes angular orientation positioning, dynamic migration, precise alignment and dynamic rotation; in the process of controlling the red blood cells, the invention preferably utilizes an image input and output assembly to observe and record the regulation of the red blood cells.

The device can be used for stably capturing and accurately arranging single or multiple red blood cells, and the captured multiple red blood cells can be assembled into the optofluidic cell switch.

In the present invention, the optofluidic cell switch is capable of closing a blood vessel branch, and the principle is as follows: laser is focused on a specific branch inlet by using scanning optical tweezers, a plurality of red blood cells are captured and arranged at the same time, an optical flow cell switch is assembled to obtain and is gathered on the branch inlet, the blood flow speed is reduced to cause continuous reduction of blood pressure, finally, the blood pressure in the blood vessel branch reaches new balance, the flow speed is reduced to 0ms, and the blood vessel branch is closed. After the control laser is removed, the captured red blood cells are immediately released, the assembled optical flow switch is opened, and the closed specific branch inlet is synchronously opened; the diameter range of the closable blood vessel is preferably 50 μm or less.

In the invention, the optofluidic cell switch can realize directional aggregation of blood flow, and the principle is as follows: the red blood cells are captured at the inlet of the blood flow branch and the optical flow cell switch is assembled, the diameter of the blood flow distribution is continuously reduced through the assembled optical flow cell switch, namely, the blood starts to focus through a narrower blood vessel at the moment, and the corresponding flow velocity is increased, so that the directional concentration of the blood flow is realized.

In the present invention, the optofluidic cell switch can control the direction of blood flow in a blood vessel, and the principle is as follows: the red blood cells are captured at the inlet of the blood flow branch and the optical flow cell switch is assembled, the assembled optical flow cell switch can dynamically close the blood flow branch, and blood flows into other branches, so that the blood flow direction in the blood vessel is dynamically controlled, and the method has potential application in the aspect of targeted transportation of drug particles.

When the device of the invention is used for closing a blood vessel branch, directionally gathering blood flow or controlling the direction of blood flow in the blood vessel, the blood vessels with different thicknesses capture a row of cells, but the larger the diameter of the blood vessel is, the larger the number of red blood cells needing to be captured is; to ensure a stable closure of the vessel, the ratio of the number of red blood cells to the diameter of the vessel is preferably greater than 5 red blood cells/10 μm, i.e. the number of red blood cells captured by a 10 μm diameter vessel is at least 5, the number of red blood cells captured by a 20 μm diameter vessel is at least 10, and so on.

The device of the invention can be used for controlled rotation of single or multiple red blood cells.

The shape of the laser scanning track is preferably circular, the ratio of the number of laser focuses on each circular track to the diameter of the circular track is more than 30/mum, namely when the diameter of the rotating track is 10μm, at least 300 laser focuses are arranged on the circular track; when the diameter of the rotating track is 20 μm, a minimum of 600 laser focal points are arranged on the circular track.

The invention captures a plurality of red blood cells, moves the optical trap to make the optical trap synchronously rotate to form a rotary switch; the red blood cells in the rotary switch keep moving synchronously with the laser focus under the action of light force; when the laser focus scans anticlockwise, the red blood cells rotate anticlockwise; when the laser focus scans clockwise, the red blood cells will rotate in the clockwise direction; the center coordinate of the rotary switch preferably corresponds to the center of the blood vessel; the radius of the rotary switch is preferably larger than that of red blood cells and smaller than that of blood vessels; the dynamic regulation of the blood flow driving speed can be realized by controlling the rotating speed of the red blood cells in the rotary switch; the invention adjusts the rotating speed of the red blood cells by adjusting the scanning frequency; the number of revolutions per second of the red blood cells was calculated using the following formula: the number of revolutions of the red blood cell per second is the number of scanning frequencies/scanning points. In certain blood disorders, the blood vessel may become occluded under natural pressure equilibrium, i.e., when there is no blood flow within the blood vessel. If the red blood cells are rotated at the entrance of the blood vessel, the local flow rate is increased, so that the blood pressure is reduced, the initial blood pressure balance is destroyed, and the generation of the driving blood flow is induced.

In addition to the revolution of the cells, the present invention is capable of controlling the autorotation of the red blood cells within the blood vessel, i.e., the rotation around the center of the red blood cells themselves. In the specific implementation process of the invention, the coordinate value of the center of the red blood cell to be controlled is input, a static light trap is arranged to fix the center of the red blood cell, meanwhile, an annular scanning light trap (the diameter is equal to the red blood cell) is applied, the scanning method of the annular scanning light trap is set, and the red blood cell keeps rotating synchronously with the laser focus under the action of light force; the rotation speed of the red blood cell rotation is preferably 6 π rad/s.

In the present invention, the red blood cells can be replaced by other cells without color characteristics; and the other cells without the color characteristics are subjected to fluorescence labeling treatment, so that the fluorescence imaging and tracking of the other cells without the color characteristics are realized. In the invention, whether the cells have colors or not has no influence on the control, and the cells in white blood cells or other blood can be controlled without fluorescent labeling; however, the number of other cells is small, and the purpose of the fluorescent labeling is to easily distinguish the target cells and realize fluorescent imaging in a dark field.

The application principle of the device of the invention in red blood cell regulation is shown in figure 1: blood flowing from above will encounter three vessel branches at the bifurcation: namely branch I, branch II and branch III. Under normal conditions, blood flow will flow into each branch non-selectively under the drive of blood pressure, and random blood flow distribution is realized. An optofluidic cell switch can be assembled to turn off branches II and III by focusing the laser at the entrance of a particular branch using scanning optical tweezers while trapping and aligning a plurality of red blood cells. At this point, the blood will automatically turn and flow entirely into branch I, as indicated by the arrow a in fig. 1. After removal of the steering laser, the captured red blood cells are released immediately and the closed branches II and III are opened simultaneously. Of course, the vessel branch to be closed can be adjusted by changing the position of the laser focus, for example, the present invention can close branches I and III simultaneously, so that blood flows into vessel branch II completely. In addition, the closing time of a particular branch can also be dynamically regulated. The directional gathering and the rotary driving of the blood flow are realized by integrating the function of closing part of blood vessels and controlling the rotation.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1. The device of the present invention was built around a scanning optical tweezers system (Tweez250si, Aresis corporation) as shown in fig. 2. The laser wavelength was set to 1064 nm. Firstly, the emergent laser interacts with the acousto-optic polarizer to realize the dynamic scanning of the laser focus, and the scanning frequency is 100 KHz. After being regulated by the acousto-optic polarizer, the laser beam is widened by a beam widening device consisting of double lenses and is focused to the surface of a sample through a 60 multiplied water immersion objective lens. The experiment process is observed in real time and recorded in a video mode through a CCD camera connected with a computer. The requirements for each element in the experimental setup are as follows:

1) a laser: emitting single-mode laser with wavelength of 1064 nm. The power requirement is continuously adjustable within 0-4W;

2) an acousto-optic modulator: the emergent laser is vertically incident to the surface of the acousto-optic modulator, and the deflection direction of the incident laser is adjusted by controlling sound waves with different frequencies based on the acousto-optic effect. The frequency is adjusted to 100 KHz.

3) The beam broadening device comprises: the laser after passing through the acousto-optic modulator is applied to a beam broadening device consisting of two identical convex lenses, and the distance between the lenses is two times of focal length

Note: the three parts of elements are required to be on the same straight line in the horizontal direction, namely emergent laser sequentially passes through the acousto-optic modulator and the light beam widening device;

4) short-wave pass dichroic mirror: and irradiating the broadened incident laser in the horizontal direction on the short wave-pass dichroic mirror, so that the short wave-pass dichroic mirror is transmitted in the vertical direction and enters the inverted objective mirror.

5) Inverting the objective lens: a water mirror with the magnification of 60X is adopted, and the numerical aperture is 1.0;

6) light source: a white illumination light source is adopted, and the white illumination light source passes through a condenser and then is focused on the surface of the sample.

The test sample selected in the experiment was zebrafish, and the schematic and optical pictures are shown in inset images I and II in fig. 2. As a model organism, zebra fish (4-6 cm) is widely applied to the research of the fields of cancers and nano medicines. Its tail and fin are optically transparent, and blood vessel and red blood cell can be clearly seen. Through the transgenic technology processing, specific cells in the tissues can be fluorescently labeled, the living body imaging of single cell precision is realized, and an excellent research platform is provided for the characterization of the optofluidic cell switch in vivo.

Experimental animals Red blood cells of adult zebra fish having a 90-day age were selected, and the adult zebra fish was first placed in a culture dish containing a tricaine solution (concentration 150mg/L) and anesthetized for 8 min. A small amount of agar gel was applied to the surface of the slide, and then the anesthetized zebrafish was placed on the slide (size: 15X 50mm) and fixed with 2% agarose (2g of agar dissolved in 100mL of water, poured into a glass cup to melt, and allowed to cool further to solidify into gel). In addition, the agar gel was continuously coated on both sides of the fish to be slightly higher than the thickness of the fish in the vertical direction. The anesthetic liquid is injected around the fish by using a syringe, so that it is in a stable liquid environment. Excess solution was removed through filter paper and finally carefully covered with a coverslip (on two strips of agar gel to achieve effective fixation of the fish) to prevent rapid evaporation of water.

3. Technical operation

1) Characterizing optical flow switch performance

In order to characterize the flexibility of the optical flow switch, the invention carries out a series of functional operations on the red blood cells in the living body blood vessel.

First, the angular orientation and dynamic migration of a plurality of red blood cells in a capillary vessel.

As shown in FIG. 3 at a1, ten erythrocytes (labeled 1-10) in a blood vessel are initially randomly distributed with their long axes oriented differently. By manipulating the laser focus distribution (a 2 in fig. 3), the first five red blood cells (labeled 1-5) are aligned end-to-end and uniformly oriented, i.e., the long axis is parallel to the vessel wall; while the other five cells (labeled 6-10) are aligned with their long axes perpendicular to the vessel wall. By regulating more red blood cells to be parallel to the blood vessel wall, the blood flow of the blood capillary which is blocked at present can be recovered.

In order to achieve the effect of a2 in fig. 3, two laser focuses are required to be respectively set at two ends of each erythrocyte, and for the first five erythrocytes (marked as 1-5), the connecting line of the laser focuses at two ends of each erythrocyte is parallel to the blood vessel wall, so that the erythrocytes are oriented parallel to the blood vessel wall, and the specific setting coordinates of the focuses are as follows (25,39), (33, 37); (35,39), (41, 43); (44,46), (49,50), (53,52), (59, 58); (63,59), (70, 63); for the last five red blood cells (marked as 6-10), a connecting line of laser focuses at two ends of each red blood cell is vertical to the vessel wall, so that the red blood cells are oriented to be vertical to the vessel wall, and the specific set coordinates of the focuses are (69,68), (76, 65); (74,69), (80, 66); (77,71), (84, 68); (82,72), (87, 68); (84,74),(91,70).

Further, the red blood cells are patterned in the vein vessel having a larger diameter. As shown in b in fig. 3, by optimizing the settings of the optical tweezers system (on the basis of capturing cells, by changing the position coordinates of specific focal points, different cells are placed at specific positions and can be controlled in orientation and arranged into different patterns), a plurality of red blood cells are simultaneously manipulated in a blood vessel with the diameter of 26 μm and are sequentially arranged into a pentagon (b 1 in fig. 3) and patterns of letters "J" (b 2 in fig. 3), "N" (b 3 in fig. 3) and "U" (b 4 in fig. 3), the flexible manipulation capability of the optical tweezers system on the red blood cells in the blood vessel of the living body is proved.

In addition to precise alignment, the present invention enables dynamic rotation of red blood cells within a blood vessel. As shown in c in figure 3, an optical trap which scans along a circular track is arranged in a blood vessel with the diameter of 16 μm, the anticlockwise rotation of a single red blood cell is successfully realized, and the rotation speed and the direction can be flexibly adjusted. In addition to single cell rotation, the present invention can also achieve simultaneous rotation of multiple cells, such as simultaneous rotation of the "red blood cell pentagons" arranged in b1 in fig. 3.

In order to realize the controllable rotation of the red blood cells, a laser focus is required to be arranged at the position for capturing the red blood cells to dynamically scan along a circular track. The process is as follows: firstly, writing a scanning track file by using MATLAB according to the rotation radius, wherein the scanning track is circular, the coordinates of the circle center are (20,56), the radius is 5 mu m, and 400 scanning points are arranged on the scanning track at equal intervals. After the red blood cells are captured, the red blood cells move along 400 scanning points along with the laser focus in sequence, and the rotation of the determined track is completed.

In addition to the revolution of the cell, the present invention can control the autorotation of the red blood cell in the blood vessel, i.e., the rotation around the center of the cell itself. As shown in d of FIG. 3, the present invention sets a stationary optical trap to fix the center of the cell while applying a circular scanning optical trap (diameter equal to the red blood cell) at which the captured red blood cell starts to rotate counterclockwise at a rotation speed of 6 π rad/s.

2) Dynamic closure of branch blood flow

Based on the controlled red blood cells, the invention assembles an optofluidic cell switch at the entrance of a specific blood vessel branch to realize dynamic closing of the branch blood flow. The corresponding coordinate range of the branch I entry in the picture is firstly calculated, five capture positions (linear arrangement) are selected from the coordinate range, and the coordinate values of the five positions are input in a software interface. And then the laser is regulated and controlled to scan back and forth at 5 position points at the entrance, and the red blood cells are stably captured. As shown in FIG. 4, a plurality of red blood cells are captured simultaneously at the entrance of the blood vessel branch I and assembled into an optofluidic cell switch. The captured red blood cells will decrease the blood flow velocity and thus the blood pressure will decrease continuously, and finally the blood pressure in the blood vessel branch I will reach a new equilibrium and the flow velocity will decrease to 0 μm/s, i.e. the blood vessel branch I is closed. At time 6s the laser was removed and the captured red blood cells immediately flowed away with the blood and the assembled optical flow switch was opened. At this time, blood flow reappears in the blood vessel branch I, and the blood flow merges with the blood flow of the branch II and flows along the upper right. In order to verify the repeatability of the control, the laser is turned on again at the entrance of the branch I at 15s, the optical flow switch is successfully assembled again, and the secondary closing of the blood flow in the branch I is realized. The laser was turned off at 35s, and blood flow in vessel branch I was again restored. No vascular damage or cell rupture was observed during the experiment, demonstrating that the use of optofluidic cell switches allows for the controlled shut-off of blood flow in specific vascular branches without physiological damage to biological tissues.

3) Directional focusing of blood flow

In order to realize the directional focusing of blood flow, the invention uses red blood cells to block one part of a blood vessel (namely, the blood vessel is not blocked), namely ten coordinate points are selected at the entrance of the blood vessel, an input program is used for driving a laser focus to a preset coordinate position, so that the stable capturing of the red blood cells is realized, the blood flow is partially blocked, and the focusing is realized. The procedure for setting the scanning spot is as follows: firstly, obtaining the coordinates of the position of a cell to be captured, then inputting the coordinates on a software interface, and simultaneously capturing a plurality of red blood cells; and then the position of the red blood cells is manually adjusted, so that partial blockage of the blood vessel is realized. As shown in fig. 5, without intervention, the blood in vessel branch II sinks into branch I and flows together in the upward right direction. The white curve in the picture represents the streamline distribution within each vessel branch, which is obtained by statistical analysis of the spatiotemporal distribution of the red blood cell flow. It can be seen that the streamline diameters and branch vessel diameters in branch I and branch II were the same at time 0s, D1 ═ 42 μm and D2 ═ 30 μm, respectively. The invention then captures the red blood cells at the entrance of branch II and assembles the optofluidic cell switch. At the same time, the path of the blood flow is narrowed, and the corresponding streamline diameters are reduced to D123, 15, 8, 5m and D220, 14, 12, 9m at times of 3s, 6s, 9s and 12s, respectively. Thus, the diameter of the blood flow distribution is continuously reduced by the assembled optical flow switch, i.e. the blood starts to focus through a narrower vessel, with a corresponding increase in flow rate.

4) Controllable steering of blood flow

The present invention further enables controlled steering of blood flow using an assembled optofluidic cell switch. By obtaining the coordinate range at the entrance of branch I, coordinate values of five points (arranged linearly perpendicular to the vessel wall) are set up therein and input into the software interface. The laser would then scan through the five positions and capture the red blood cells. As shown in fig. 6, the blood flowing in below will encounter two vessel branches, branch I and branch II, at the vessel intersection. Normally, blood will flow randomly into a branch driven by blood pressure. For example, when the time is 0s, the blood flowing downward completely turns right and flows into branch II (as indicated by the arrow). In order to change the direction of blood flow, the present invention captures a plurality of erythrocytes at 6s and assembles an optofluidic cell switch, and simultaneously, branch II is closed, and all blood flowing in the lower direction flows upward along branch I. Further, the present invention turns off the laser at 21s, the trapped red blood cells are released, and the blood immediately resumes the original flow direction and re-flows into branch II. Therefore, by adjusting the optofluidic cell switch, the invention can dynamically control the blood flow direction in the blood vessel, and has potential application in the aspect of targeted delivery of drug particles.

5) Rotational drive of blood flow

In certain blood disorders, the blood vessel may become occluded under natural pressure equilibrium, i.e., when there is no blood flow within the blood vessel. If the red blood cells are rotated at the entrance of the blood vessel, the local flow rate is increased, so that the blood pressure is reduced, the initial blood pressure balance is destroyed, and the generation of the driving blood flow is induced. A circular track program is required to be arranged at the position of a rotary switch, namely 60 points are taken from a circle at equal intervals, the coordinates of the 60 points are simultaneously input into a software interface, a laser focus sequentially goes from a first point to a last point and then returns to the first point, and the circular scanning of the focus is realized through sequential circulation. After the corresponding red blood cells are captured, the laser goes from the first point for a circle according to the position of the laser focus and returns to the first point, and one rotation is completed. The larger the laser track is, the faster the laser speed is, and the larger the corresponding erythrocyte rotating track is, the faster the laser speed is. Thus, the present invention utilizes optofluidic cell switches to achieve a rotational drive that occludes blood flow within a vessel. As shown in fig. 7 a, the original blood vessel is in a natural occlusion state and no blood flows. Next, a plurality of red blood cells are captured at the entrance of branch I and synchronously rotated counterclockwise. At the same time, a priming blood flow occurs in branch I, and the red blood cells start to continuously flow from branch I into branch II, and then flow rightward together (b in FIG. 7 and c in FIG. 7). After the laser was removed, the red blood cells in the rotary switch immediately stopped moving and no more blood flowed out in branch I (d in fig. 7). At 53s the rotary switch is turned back on (e in fig. 7) and drive flow will again occur in branch I (f in fig. 7). In addition, by changing the rotating speed of the red blood cells in the rotary switch, the dynamic regulation of the driving blood flow speed can be realized.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A device for regulating and controlling red blood cells in living animal blood vessels sequentially comprises a laser emission component, an acousto-optic modulation component, a light beam broadening component, a short-wave-pass dichroic mirror, an inverted objective lens and a sample chamber along a laser path; the device also comprises an image input and output component, a convex lens, a condenser and a white light emitting component which are arranged on the imaging optical path;

2. The apparatus of claim 1, wherein the image input and output component comprises a CCD camera connected to an electronic device.

3. The device according to claim 1, wherein the short wave pass dichroic mirror is a short wave pass dichroic mirror that allows transmission of a wavelength band of 430-800 nm wavelength and does not allow transmission of 800-1300 nm wavelength.

4. The device according to any one of claims 1 to 3, wherein the laser light emitted from the laser emitting component is single-mode laser light; the wavelength of the emergent laser is 1000-1100 nm.

5. The device according to any one of claims 1 to 3, wherein the scanning frequency of the acousto-optic modulation component is 0 to 100 KHz.

6. The apparatus of any one of claims 1-3, wherein the beam broadening assembly comprises a first convex lens and a second convex lens; the first convex lens is arranged at the closer end of the acousto-optic modulation component; the second convex lens is arranged at the far end of the acousto-optic modulation component; the ratio of the curvature radii of the first convex lens and the second convex lens is 1: 10; the distance between the first convex lens and the second convex lens is the sum of the focal lengths of the first convex lens and the second convex lens.

7. Use of the device according to any one of claims 1 to 6 for stable capture, precise alignment or controlled rotation of erythrocytes in the blood vessels of living animals of non-therapeutic interest.

8. The application according to claim 7, characterized in that it comprises the following steps:

1) fixing the live animal in a sample chamber after anesthesia;

4) the laser emitting component emits laser, the focus position is scanned by the acousto-optic modulation component, the light beam broadening component broadens, the short wave-pass dichroic mirror turns and the inverted objective lens focuses, the laser is focused on the living animal of the sample chamber to form an optical trap, and the control of the red blood cells is realized by manually selecting and controlling the movement of the optical trap.

9. The use according to claim 8, wherein the coordinate values of each of the red blood cells to be controlled in step 2) comprise two coordinate values corresponding to two ends of the red blood cell.

10. Use according to claim 8 or 9, characterized in that the red blood cells are replaced by other cells without color features; the other cells without color features are subjected to fluorescent labeling treatment.