CN116679081A - Multi-focus particle detection system and method based on metalens array - Google Patents

Abstract

The invention discloses a multi-focus particle detection system and method based on a metalens array, including an illumination module, an imaging module, an image acquisition module, an analysis and calculation module and a microfluidic device, wherein the illumination module includes The set laser, collimator lens, beam expander lens and dielectric metalens array first generate parallel beams through the laser, collimator lens and beam expander lens, and make the parallel beams cover the entire aperture of the dielectric metalens array, and then pass through the dielectric metalens array. A set of focal point arrays is generated by a structured lens array; the microfluidic device is placed between the illumination module and the imaging module to carry the solution sample containing the particles to be measured; the image acquisition module is located at the rear end of the focal plane of the dielectric metalens array; the analysis The calculation module is used to obtain the relevant physical quantities of the particles to be measured in the solution. The invention utilizes the meta-lens array to generate a focus array within a very short working distance, simplifies the optical path, and facilitates integration with microfluidic devices.

Description

Multi-focus particle detection system and method based on metalens array

technical field

The invention belongs to the technical field of particle flow velocity and density measurement in a flow field, and in particular relates to a multi-focus particle detection system and method based on a meta-lens array.

Background technique

Correlation spectroscopy is a powerful quantitative technique for particle dynamics, which has been widely used in many fields such as biomedicine, biophysics and chemistry. By recording the light intensity fluctuations caused by the free diffusion or directional flow of particles in the focused beam, and performing correlation analysis on the fluctuation signals that change with time, physical parameters such as particle concentration and diffusion coefficient can be quantitatively measured. Fluorescence correlation spectroscopy (FCS) uses fluorescent dyes or fluorescent proteins to label the biological particles to be tested, and by recording the fluctuation of the fluorescence intensity of the sample to be tested under the excitation of the focused spot, the relevant physical parameters of the particles can be obtained. Fluorescent labeling allows the specific analysis of biomacromolecules of interest in biological samples. In addition, the interaction between different biomacromolecules can also be studied by using two-color fluorescence correlation spectroscopy technology by fluorescently labeling different particles with different colors. However, due to the phototoxicity and photobleaching properties of fluorescent substances, fluorescence correlation spectroscopy is difficult to measure biological samples for a long time, and many kinds of particulate matter in life cannot be fluorescently labeled. Therefore, people are eager to explore a method of correlation spectroscopy without fluorescent labels, which uses the reflection or scattering signals of target particles to perform intensity correlation analysis, and obtains the flow rate of particles in free diffusion or in directional flow, in which the concentration of particles, Diffusion coefficient and other related physical quantities.

In addition, the traditional correlation spectroscopy technique obtains the change of the intensity information of the particle in the moving state with time by generating a single focus. This technique needs to know the spatial volume of the generated focus in order to quantitatively calculate the physical parameters of the particle. In fluorescence correlation spectroscopy, a bifocal fluorescence correlation spectroscopy technique has been proposed: a differential interference prism is added to the confocal microscope to separate two beams of orthogonally polarized laser light into two focal points, and simultaneously record the two laser beams with a certain lateral distance. Intensity fluctuations produced by particles in two focus observation areas are calculated by using autocorrelation function and cross-correlation function to obtain the relevant information of the physical parameters to be measured in two focus areas. Compared with the traditional single-focus correlation spectroscopy, this technique only needs to know the lateral distance between the two focuses in advance to quantitatively obtain the physical parameters and dynamic information of the particles. Nevertheless, the size and distribution of the observation area corresponding to the particle information in the sample under the traditional correlation spectroscopy technology is very limited, and it is impossible to perform higher throughput and more comprehensive measurements of the particles to be measured.

Contents of the invention

In order to solve the above-mentioned problems in the prior art, the present invention provides a multi-focus particle detection system and method based on a metalens array. The technical problem to be solved in the present invention is realized through the following technical solutions:

The present invention provides a multi-focus particle detection system based on a metalens array, including an illumination module, an imaging module, an image acquisition module, an analysis and calculation module, and a microfluidic device, wherein,

The illumination module, the imaging module and the image acquisition module are sequentially coupled and connected along the beam transmission direction;

The illumination module includes a laser, a collimator lens, a beam expander lens, and a dielectric meta-lens array arranged in sequence along the beam transmission direction, firstly a parallel beam is generated by the laser, the collimator lens, and the beam expander lens, and Make the parallel beam cover the entire aperture of the dielectric metalens array, the polarizer and the quarter wave plate are used to control the polarization state of the beam, and then generate a set of focal points through the dielectric metalens array array;

The microfluidic device is placed between the illumination module and the imaging module, and is used to carry a solution sample containing particles to be tested;

The imaging module includes an imaging objective lens;

The image acquisition module is located at the rear end of the focal plane of the dielectric metalens array, and is used for imaging the particle to be measured or performing secondary imaging on the focal point array on the back focal plane of the dielectric metalens array, and then using the obtained focal point array To observe the particles to be measured;

The analysis and calculation module is used to perform statistics on the light intensity value of the particles to be measured observed in real time, and perform autocorrelation and cross-correlation analysis and calculation to obtain the relevant physical quantities of the particles to be measured in the solution.

In one embodiment of the present invention, the dielectric metalens array is composed of a substrate and a periodic uniform array of structural units located above the substrate, wherein,

The material of the substrate is silicon dioxide, resin or aluminum oxide. The size and period of the structural units in the periodic uniform array of the structural units are smaller than the wavelength of the electromagnetic wave in vacuum. The shape of the structural units includes a cube Waveguide, cylindrical waveguide, elliptical cylindrical waveguide, the material of the structural unit is selected from silicon, gallium nitride or titanium dioxide.

In one embodiment of the present invention, the illumination module includes a laser, a beam expander lens, a collimator lens, a polarizer, a quarter-wave plate, and a dielectric metalens array arranged in sequence along the beam propagation direction. The fluidic device is located at the common focal plane of the dielectric metalens array and the imaging objective lens, and the plane where the microfluidic device is located is perpendicular to the beam propagation direction, and the image acquisition module is a CCD camera.

In one embodiment of the present invention, a converging lens is set between the dielectric metalens array and the microfluidic device for secondary imaging of the focal point array generated by the dielectric metalens array, and the The light beam after secondary imaging converges in the sample area of the microfluidic device.

In an embodiment of the present invention, the image acquisition module is also used for performing multi-frame continuous image acquisition of the particles to be measured in the flow state, and adjusting relevant parameters of the image sequence obtained by multi-frame continuous acquisition to ensure that the Particles in the sampled area are distinguished from the background.

In one embodiment of the present invention, the analysis and calculation module is specifically used for:

Extracting the intensity information inside one or more focus areas generated by each frame in the image sequence, integrating and summing the signal intensity values in the areas containing particle swarm flow information, and obtaining a set of time-varying light intensity data curves ;

Perform numerical compensation on the obtained light intensity data curve to eliminate the influence of the signal-to-background ratio drop;

Image correlation calculation is performed on the intensity information after numerical compensation to obtain the autocorrelation result of the intensity information of each focal area and the cross-correlation result of the intensity information between the focal areas;

The data curves of the obtained autocorrelation results and cross-correlation results are fitted and calculated to obtain the particle flow velocity.

Another aspect of the present invention provides a multi-focus particle detection method based on a metalens array, including:

S1: According to the multi-focus particle detection system based on the metalens array described in any one of the above-mentioned embodiments, a multi-frame continuous image sequence of the particles to be measured in a flowing state is obtained;

S2: Carry out image correlation spectroscopy calculation on the image sequence to obtain single-focus autocorrelation curves and multi-focus cross-correlation curves, and obtain flow velocity and concentration parameters of the particles to be measured by function fitting.

In one embodiment of the present invention, said S1 includes:

S1.1: Turn on the power supply of the lighting module, adjust the intensity of the light source, and ensure that the image formed by the CCD camera for the metalens array is clearly visible and the exposure is moderate;

S1.2: Adjust the positional relationship between the dielectric metalens array, the imaging objective lens, and the CCD camera, so that the focal point generated by the dielectric metalens array can be clearly imaged on the CCD camera;

S1.3: Fix the microfluidic device between the dielectric metalens array and the imaging objective lens, and move the position of the microfluidic device axially to make the observation area in the microfluidic device clearly imaged, and to The microfluidic device injects the solution containing the particles to be tested at a constant speed;

S1.4: When the solution flow is in a steady state, perform multi-frame continuous image acquisition of the particles to be measured in the sample, and adjust relevant parameters for the image sequence obtained by multi-frame continuous acquisition to ensure that the images captured by the CCD camera can Clearly distinguish the particles to be measured from the background information.

In one embodiment of the present invention, the step S2 includes:

S2.1: Extract the intensity information inside any one or more focus areas generated by each frame in the image sequence, integrate and sum the signal intensity values in the area containing the particle swarm flow information, and obtain a set of time-varying The intensity curve of each focus area corresponds to a curve of intensity information data;

S2.2: Perform numerical compensation on the obtained intensity curve to eliminate the influence of the decrease of signal-to-background ratio;

S2.3: Carry out image correlation calculation on the intensity information after numerical compensation, and obtain the autocorrelation result of the intensity information of each focus area and the cross-correlation result of the intensity information between focus areas;

S2.4: Fitting and calculating the data curves of the obtained autocorrelation results and cross-correlation results to obtain particle flow velocity.

In one embodiment of the present invention, said S2.4 includes:

The data curves of the obtained autocorrelation results and cross-correlation results are fitted and calculated to obtain particle flow velocity and concentration, where the physical model used for fitting is:

Among them, G _a () is the result of autocorrelation calculation of the intensity information in one focus area, G _C () is the result of cross correlation calculation of intensity information in two focus areas, and N is the flow rate in the observation area τ _d is the diffusion time parameter caused by the Brownian motion of the particle itself, τ _f is the time parameter related to the particle flow caused by the external force, d ₀ represents the actual distance between any two selected focal points, r ₀ means the radius value of the current focus;

The particle flow velocity is obtained by using the formula v _f =r ₀ / _f , and the particle concentration is obtained by dividing N by the volume of the observation area.

Compared with prior art, the beneficial effect of the present invention has:

1. The present invention does not need a fluorescent label, and uses the reflection or scattering characteristics of the target particles to carry out correlation analysis of the image intensity value, and can measure the physical quantity of the particles in the natural state; the medium metalens array adopted in the present invention makes the conventional The multi-focus correlation spectroscopy measurement system is more compact, and can flexibly change the number of focal points generated in the rear by controlling the polarization state of the electromagnetic wave irradiated on the metalens array.

2. The present invention uses a metalens array with a thickness of only sub-wavelength scale to generate single or multiple focal points within a short working distance to illuminate and observe the particles to be measured in the solution, and is easy to integrate with microfluidic devices , the optical system built based on this is compact in structure; at the same time, the number of focal points generated by the metalens array is flexible and adjustable, which can simultaneously measure multiple points in the sample, and the particle can be obtained by recording the image sequence and performing subsequent calculation processing Compared with the traditional correlation spectroscopy measurement system, this method uses the metalens array to generate multiple focal points within a very short working distance, and can measure multiple points in the sample at the same time, which simplifies the optical path and facilitates Integrated with microfluidic devices; and the number of focal points is flexible and adjustable.

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

Description of drawings

Fig. 1 is a block diagram of a multi-focus particle detection system based on a metalens array provided by an embodiment of the present invention;

Fig. 2 is a schematic diagram of the optical path of a multi-focus particle detection system based on a metalens array provided by an embodiment of the present invention;

Fig. 3 is a metalens array designed in a multi-focus particle detection system based on a metalens array provided by an embodiment of the present invention obtained under the irradiation conditions of left-handed circularly polarized light, right-handed circularly polarized light, and elliptically polarized light respectively. Actual images of 4-focus mode, 9-focus mode and 13-focus mode;

Fig. 4 is the 1500th frame, the 3000th frame, the 4500th frame and the 6000th frame in the 6000 frame image sequence captured by the CCD camera in the multi-focus particle detection system based on the metalens array provided by the embodiment of the present invention frame image;

Fig. 5 is a time-varying curve of light intensity values corresponding to different focal points obtained by a multi-focus particle detection system based on a metalens array provided by an embodiment of the present invention;

Figure 6 shows two sets of experimental data and fitting curves at different speeds obtained from autocorrelation calculations for a single focus data, where V _sta represents the actual sample flow rate value obtained from experimental measurements, and V _fit represents the sample obtained through fitting calculations Velocity value;

Figure 7 shows two sets of experimental data and fitting curves at different speeds obtained by cross-correlation calculations for multiple focus data, where V _sta represents the actual sample flow rate value obtained from experimental measurements, and V _fit represents the value obtained through fitting calculations Sample flow rate value;

Figure 8 is the experimental data and fitting curves of three groups of different concentrations obtained by performing autocorrelation calculations on multiple focus data, where C _sta represents the actual sample concentration value obtained from experimental measurement, and C _fit represents the value obtained through fitting calculation Sample concentration value.

Explanation of reference signs:

1-laser; 2-beam expander lens; 3-collimating lens; 4-polarizer; 5-quarter wave plate; 6-dielectric meta-lens array; 7-microfluidic device; 8-imaging objective lens; 9-CCD camera.

Detailed ways

In order to further explain the technical means and effects of the present invention to achieve the intended purpose of the invention, the multi-focus particle detection system and method based on the metalens array proposed according to the present invention will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

The aforementioned and other technical contents, features and effects of the present invention can be clearly presented in the following detailed description of specific implementations with accompanying drawings. Through the description of specific embodiments, the technical means and effects of the present invention to achieve the intended purpose can be understood more deeply and specifically, but the accompanying drawings are only for reference and description, and are not used to explain the technical aspects of the present invention. program is limited.

It should be noted that in this document, relational terms such as first and second etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is a relationship between these entities or operations. There is no such actual relationship or order between them. Furthermore, the terms "comprises", "comprises" or any other variation are intended to cover a non-exclusive inclusion such that an article or device comprising a set of elements includes not only those elements but also other elements not expressly listed. Without further limitations, an element defined by the phrase "comprising a" does not exclude the presence of additional identical elements in the article or device comprising said element.

Embodiment one

Please refer to FIG. 1 . FIG. 1 is a block diagram of a multi-focus particle detection system based on a metalens array provided by an embodiment of the present invention. The multi-focus particle detection system includes an illumination module, an imaging module, an image acquisition module, an analysis calculation module and a microfluidic device 7, wherein the illumination module, the imaging module and the image acquisition module are sequentially coupled along the beam transmission direction.

The illumination module includes a laser 1, a collimator lens 2, a beam expander lens 3, a polarizer 4, a quarter-wave plate 5, and a dielectric meta-lens array 6 arranged in sequence along the beam transmission direction. First, the laser 1, the collimator lens 2 and the beam expander lens 3 produce parallel beams, the incident linearly polarized light can be converted into circularly polarized light or elliptically polarized light through the polarizer 4 and the quarter-wave plate 5, and the parallel beam covers the dielectric meta-lens The entire aperture of the array 6 generates a group of focus arrays through the dielectric meta-lens array 6; the microfluidic device 7 is placed between the illumination module and the imaging module, and is used to carry the solution sample containing the particles to be measured; the imaging module includes an imaging objective lens 8. The image acquisition module is located at the back end of the focal plane of the dielectric metalens array, and is used for imaging the particle to be tested or performing secondary imaging on the focal point array on the back focal plane of the dielectric metalens array, and then using the obtained focal point array to be tested Particles are observed; the analysis and calculation module is used to count the light intensity value of the particles to be measured in real time, and perform autocorrelation and cross-correlation analysis and calculation to obtain the relevant physical quantities of the particles to be measured in the solution.

Specifically, the design wavelength of the dielectric metalens array in this embodiment is 635nm, so a continuous laser with a wavelength of 635nm is used in the illumination module, and after being collimated by a beam expander lens, it is shaped into a diameter sufficient to cover the entire metalens array. Structured parallel light beams; the dielectric metalens array is behind the lighting module to modulate the incident parallel light beams to form multiple converging beams to generate multiple focal points; the microfluidic device is used as a container for the solution in which the particles to be measured are placed, It is placed in the optical path of the imaging system, and the state of the particle to be measured flowing through the channel of the microfluidic device can be observed in real time through the analysis and calculation module, and it can be photographed and recorded. In other embodiments of the present invention, the microfluidic The device can also be replaced by a living biological sample; the image acquisition module is located at the back end of the back focal plane of the dielectric metalens array, and the image acquisition module performs secondary imaging on the focal point array on the back focal plane of the metasurface and at the same time captures the particles to be measured for direct imaging.

The dielectric metalens array used in this embodiment is a metasurface capable of generating multiple focal points, and each sub-metalens in the array functions as a convex lens. Please refer to FIG. 2 . FIG. 2 is a schematic diagram of an optical path of a multi-focus particle detection system based on a metalens array provided by an embodiment of the present invention. The dielectric metalens array contains 13 sub-metalenses in total, each sub-metalens has a focal length of 10 microns, and the length and width of the entire sub-metalens are 30×30 microns. The sub-metalens is composed of a substrate of low dielectric constant material and a periodic uniform array of structural units with high dielectric constant. Wherein, the material selected for the base part of the sub-metalens prepared in this example is silicon dioxide, and the material used for the surface structure unit on the base is silicon. The thickness of the substrate is a non-characteristic parameter and has no significant impact on the performance of the sub-metalens device, and an appropriate thickness is designed to ensure the stability of the prepared sub-metalens device. The size and period of the structural units in the periodic uniform array of structural units are both smaller than the wavelength of electromagnetic waves in vacuum, and the selected structural units are cubic waveguides with rectangular cross-sectional shapes. The metasurface of the entire dielectric metalens array adopts the PB phase regulation method to control the phase of the beam, that is, the plumb center axis of the structural unit is used as the rotation axis, and the nearly linear regulation of the phase delay can be achieved by controlling the rotation angle. The specific relationship Can be expressed as: where, /> Indicates the amount of phase delay that needs to be provided at different positions on the metasurface, and θ is the angle rotated by the structural unit at any position relative to the reference position. However, the relationship between the phase delay required for each position in each sub-metalens and the coordinates of each position needs to satisfy:

Among them, λ represents the working wavelength of the sub-metalens, f represents the focal length of the sub-metalens, and R represents the radial coordinate on a single sub-metalens in the metalens array. The sub-metalens designed according to the above relationship can achieve the effect of converging parallel light into points. By integrating multiple sub-metalenses on the entire metalens array, it can realize the function of shaping a single laser beam into multiple converging beams, thereby performing Multi-point sampling.

As shown in Figure 2, the illumination module of this embodiment includes a laser 1, a beam expander lens 2, a collimator lens 3, a polarizer 4, a quarter wave plate 5 and a dielectric metalens array arranged in sequence along the beam propagation direction 6. Wherein, the laser wavelength emitted by the laser 1 is 635nm, which is the working wavelength designed for the metasurface of the dielectric metalens array. The imaging module is an imaging objective lens 8; the image acquisition module is a CCD camera 9, and the microfluidic device 7 is placed between the imaging objective lens 8 and the CCD camera 9, and the microfluidic device 7 is used as a container for carrying the solution where the particles to be measured The particles to be detected are imaged.

Specifically, the function of the polarizer 4 and the quarter-wave plate 5 is to convert the polarization state of the laser light emitted by the laser 1 into circular polarization or elliptical polarization, so as to ensure that the metasurface based on PB phase regulation can work normally. Optionally, a second group of polarizers and a quarter-wave plate (not shown in the drawings) are arranged behind the microfluidic device 7 to adjust the polarization state of the outgoing light, so as to more flexibly adjust the polarization state in the system. In this embodiment, only one group is used for the working mode of the dielectric metalens array.

In this embodiment, the collimator lens 3 is located before the dielectric metalens array 6, and it is necessary to ensure that the collimated light beams generated by it can be completely irradiated on the metasurface of the dielectric metalens array 6, and the transmitted light forms the focal point array directly The light is irradiated in the microfluidic device 7, and the light modulated by the metalens array 6 and the sample is collected and imaged by the imaging objective lens 8 and the CCD camera 9 at the rear end. It should be noted that, in another embodiment of the present invention, a converging lens is added on the basis of the optical path in FIG. The light beam after secondary imaging is converged in the sample area of the microfluidic device 7, and the imaging objective lens 8 is still placed behind the microfluidic device 7 and is coaxial with it, so that the light beam emitted from the converging lens is transmitted to the entrance of the imaging objective lens 8. Hitomi.

On the other hand, the dielectric metalens array 6 of this embodiment realizes the polarization multiplexing function by combining the propagation phase and the PB phase two phase control modes in combination with the imaging system, and can complete a more flexible focus generation scheme. Specifically, the The metasurface can control the polarization state of the incident light by rotating the polarizer 4, and then realize different working modes: see Figure 3 for details. When the incident light is in the right-handed polarization state, four focal points arranged in 2×2 can be generated. When When the incident light is a left-handed polarization state, nine focal points arranged in 3×3 can be generated, and when the incident light is a specific elliptically polarized light, the above two modes can be simultaneously excited.

Further, the multi-focus particle detection in this embodiment can realize the measurement of particle properties in the flowing solution, and the analysis and calculation module can measure the flow velocity information of the particles to be measured in the solution. Specifically, the analysis and calculation module in this embodiment is specifically used for:

extracting the intensity information inside any one or more focus areas generated by each frame in the image sequence, and integrating and summing the signal intensity values in the areas containing particle swarm flow information to obtain a set of time-varying intensity curves;

Perform numerical compensation on the obtained intensity curve to eliminate the influence of the signal-to-background ratio drop;

Image correlation calculation is performed on the intensity information after numerical compensation to obtain the autocorrelation result of the intensity information of each focal area and the cross-correlation result of the intensity information between the focal areas;

The data curves of the obtained autocorrelation results and cross-correlation results are fitted and calculated to obtain the particle flow velocity.

In the actual process, the operation process of the multi-focus particle detection includes the following steps:

Step 1: Turn on the power of the laser 1, adjust the light intensity of the outgoing laser, and ensure that the CCD camera 9 can clearly image the medium metalens array 6 and the exposure is moderate. Overexposure will lead to the loss of effective information of the particles to be measured in the observation area;

Step 2: Fix the microfluidic device or biological sample at the common focal point of the converging lens and the imaging objective lens, ensure that the plane where the microfluidic device or biological sample is located is perpendicular to the optical axis of the optical system, and at the same time ensure that the light beam can completely pass through the micro Effective detection area for fluidic devices or biological samples. In this embodiment, the aqueous solution of PMMA microsphere powder is used as the sample to be tested. In order to reduce the agglomeration effect of the particles and ensure that there are always uniform PMMA beads flowing through the observation area, it is necessary to ensure the concentration ratio of the suitable PMMA beads solution as much as possible. The PMMA microsphere powder in the solution used is 0.025g, and the water solvent is about 1ml;

Step 3: Move the position of the microfluidic device 7 axially to adjust the focus, so that the observation area of the microfluidic device 7 can be clearly imaged. At this time, inject the solution to be tested into the inlet end of the microfluidic device 7 at a constant speed, and flow The solution in the channel part flows out from the outlet port of the microfluidic device. In this example, the sample of the solution to be tested is poured into the syringe, and the syringe is pushed by a microfluidic pump. After the flow state of the solution in the microfluidic device is in a steady state, the observed particle groups flowing in the solution can be as valid image data for computation;

Step 4: Adjust the frame rate of the image to about 200 frames per second through the image acquisition software associated with the CCD camera on the computer side, perform multi-frame continuous acquisition and storage of the particle sample in the flow state, and monitor the CCD camera in the software The exposure time, gain, and gamma value of the collected images are adjusted to ensure high contrast and appropriate brightness between the particles in the observation area and the background. In this embodiment, a total of 6000 frames were taken continuously for about 30s, and the actual image shown in Figure 4 can be obtained. Here, the 1500th frame and the 3000th frame are selected when the metalens array is in the 4-focus mode. Frame, the 4500th frame and the 6000th frame are shown as images, and the passing PMMA spheres can be clearly seen in the detection area formed by the four focal points.

Step 5: For the summation of the signal strength in each observation area in the image sequence obtained in step 4, a set of intensity versus time curves with a duration of 30s can be obtained, and numerical compensation is performed on it to eliminate the signal-to-background ratio fluctuation. Then, the image correlation spectrum is calculated based on one or more sets of intensity integral-time curves, and the autocorrelation curve of single focus and the cross-correlation curve of multi-focus are obtained. Finally, the particle flow velocity is obtained through physical function fitting calculation. accurate value.

The specific process of step 5 includes:

S5.1: Extract the intensity information inside any one or more focus areas generated in each frame of the image sequence, and integrate and sum the signal intensity values in the observation area containing the particle swarm flow information to obtain a set of total The time-varying intensity integral curve of 30s, as shown in Figure 5, the image focus 1 and the image focus 2 respectively correspond to the intensity integral change curves over time in the two observation areas where the PMMA balls flow successively;

S5.2: Perform numerical compensation on the obtained intensity curve to eliminate the impact of signal-to-background ratio fluctuations. In this embodiment, firstly, the 6000 intensity integral data obtained in step S5.1 are averaged based on the equidistant The method is scaled to 100 data, and then combined with the following functions for fitting:

Among them, a, b, and c are the three coefficients to be fitted respectively, x represents the independent variable time, y(x) represents the light intensity value of the dependent variable changing with time, i represents the i-th item in the fitting polynomial, and then Expand the independent variable data of the scaled data into an array with a length of 6000 using the nearest neighbor interpolation method, and then use the function obtained by fitting to obtain the corresponding fitting intensity value. The intensity integral curve of the final compensation result is as follows Figure 6 shows.

S5.3: Introduce the delay amount τ, and perform image spectral correlation calculation on the above array of intensity values obtained through fitting correction:

G _a (τ)＝<I _C (t)I _C (t+τ)>/<I _C (t)> ² -1

G _c (τ)＝<I ₁ (t)I ₂ (t+τ)>/<I ₁ (t)><I ₂ (t)>-1

Among them, G _a is the result of autocorrelation calculation of the intensity information in a certain focus area, and G _C is the result of cross correlation calculation of the intensity information in a certain two focus areas, both of which are functions related to the time delay, I _C (t) is the image intensity value of a certain focal point at any time point t, I _C (t+τ) is the corresponding image intensity value at t+τ time, I ₁ and I ₂ correspond to two The two selected focal areas must be the area through which the same particle group flows sequentially along a specific direction.

In this embodiment, the dielectric metalens array is set to work in the four-focus mode, and the left two focus points are selected as the observation areas. Firstly, the autocorrelation calculations are performed on the two areas from the integrated intensity curves obtained by statistics in step 5.2. At the same time, by adjusting the propulsion speed of the microfluidic pump, the flow speed of the solution sample is changed to obtain statistical results under different speed conditions. The experimental results obtained are shown in the dot-shaped marks in Figure 6, and the left and right pictures correspond to The speed set in the microfluidic pump is under the conditions of 4 μL/min and 5 μL/min. On the other hand, based on the integrated intensity curve obtained in step 5.2, the cross-correlation calculation of the two regions is performed, and the obtained experimental results are shown as dot-shaped marks in Fig. 7 . It should be noted that in order to prove that the measurable speed range of this method is large enough, the speed setting of the microfluidic pump was deliberately adjusted to 40 μL/min and 45 μL/min when calculating the cross-correlation results in Figure 7.

S5.4: Fit and calculate the data curves of the obtained autocorrelation results and cross-correlation results according to the following physical model or deformation based on it:

Among them, N is the average number of particles flowing through the observation area, τ _d is the diffusion time parameter caused by the Brownian motion of the particle itself, τ _f is the time parameter related to the particle flow caused by the external force, and d ₀ represents any two The actual distance of the observation area where the selected focal points are located, and r ₀ represents the radius value of a certain focal point. Subsequently, the particle flow velocity is obtained by using the formula v _f =r ₀ /τ _f , and the particle concentration is obtained by dividing N by the volume of the observation area.

In the present embodiment, at first the autocorrelation results obtained by two groups of different experiments at slower speeds are fitted according to the physical function corresponding to G _a (τ) in the above formula, and the measurement and fitting results are shown in Fig. 6, The parameters τ _f obtained by fitting under two speed conditions are 0.044s and 0.027s respectively, and the radius value of each circular observation area formed by the focal point is 11.2μm. Using the formula v _f =r ₀ /τ _f can be calculated The fitting speeds under the two slow conditions are 252 μm/s and 417 μm/s respectively. On the other hand, the cross-correlation results obtained from two groups of different faster experiments are fitted according to the physical function corresponding to G _c (τ) in the above formula. The measurement and fitting results are shown in Fig. 7. The fitting parameters τ _f under the condition of speed are 0.004s and 0.003s respectively. Similarly, the fitting speeds under the two fast conditions can be obtained as 2909μm/s and 4267μm/s respectively. The average error between the fitting result and the actual velocity is about 8.07%, so the method of the embodiment of the present invention can measure the particle flow velocity more accurately.

The present invention does not require fluorescent labels, uses the reflection or scattering characteristics of target particles to perform intensity correlation analysis, and can measure particles in the natural state; the dielectric metalens array used in the present invention enables traditional multi-focus correlation spectrum measurement The system is more compact, and can flexibly change the number of focal points generated in the rear by controlling the polarization state of the electromagnetic wave irradiated on the metalens array. The invention utilizes a metalens array whose thickness is only sub-wavelength scale to generate single or multiple focal points within a very short working distance to illuminate and observe the particles to be measured in the solution, and is easy to integrate with microfluidic devices, based on The constructed optical system has a compact structure; at the same time, the number of focal points generated by the metalens array is flexible and adjustable, which can simultaneously measure multiple points in the sample, and the flow velocity of the particles can be obtained by recording the image sequence and performing subsequent calculation processing and concentration information. , compared with the traditional correlation spectroscopy measurement system, this method uses a metalens array to generate focal points within a very short working distance, which simplifies the optical path and facilitates integration with microfluidic devices; moreover, the number of focal points is flexible and adjustable, and the sample Simultaneously measure multiple points.

Embodiment two

On the basis of Embodiment 1, this embodiment provides a multi-focus particle detection method based on a metalens array, which specifically includes:

S1: Using the multi-focus particle detection system based on the metalens array described in Embodiment 1 to obtain a multi-frame continuous image sequence of the particles to be detected in a flowing state.

Specifically, S1 of this embodiment includes:

S1.1: Turn on the power supply of the lighting module, adjust the intensity of the light source, and ensure that the image formed by the CCD camera for the metalens array is clearly visible and the exposure is moderate;

S1.2: Fix the microfluidic device at the common focal point of the dielectric metalens array and the imaging objective lens, ensure that the plane where the microfluidic device is located is perpendicular to the beam propagation direction, and at the same time ensure that the beam can completely pass through the microfluidic device effectively detection area;

S1.3: Move the position of the microfluidic device axially to make the observation area in the microfluidic device clearly imaged, inject the solution containing the particles to be measured into the microfluidic device at a constant speed and make the solution The flow state is in a steady state;

S1.4: Collect multiple frames of continuous images of the particles to be measured in the flow state, and adjust the relevant parameters of the image sequence obtained by continuous acquisition of multiple frames to ensure that there is a high contrast between the particles in the sampling area and the background and a suitable brightness.

S2: Carry out image correlation spectroscopy calculation on the image sequence to obtain single-focus autocorrelation curves and multi-focus cross-correlation curves, and obtain flow velocity and concentration parameters of the particles to be measured by function fitting.

In this embodiment, the S2 includes:

S2.1: Extract the intensity information inside any one or more focus areas generated by each frame in the image sequence, integrate and sum the signal intensity values in the area containing the particle swarm flow information, and obtain a set of time-varying The intensity curve of each focus area corresponds to a curve of intensity information data;

S2.2: Perform numerical compensation on the obtained intensity curve to eliminate the influence of the decrease of signal-to-background ratio;

S2.3: Introduce the delay amount τ, perform image correlation calculation on the intensity information after numerical compensation, and obtain the autocorrelation result of the intensity information of each focus area and the cross-correlation result of the intensity information between focus areas, wherein, the The expressions of the autocorrelation result and the cross-correlation result are respectively:

G _a (τ)＝<I _C (t)I _C (t+τ)>/<I _C (t)> ² -1

G _c (τ)=<I ₁ (t)I ₂ (t+τ)>/(<I ₁ (t)><I ₂ (t)>)-1

Among them, G _a (τ) is the result of autocorrelation calculation on the intensity information in one focus area, G _C (τ) is the result of cross-correlation calculation on the intensity information in two focus areas, I _C (t) is the image intensity value of a focal point at any time point t, I _C (t+τ) is the corresponding image intensity value at the time point t+τ, and I ₁ and I ₂ correspond to the intensities of two relevant focal points in the image respectively value, the two selected focal areas are the areas where the same particle group flows sequentially along a specific direction.

S2.4: Fitting and calculating the data curves of the obtained autocorrelation results and cross-correlation results to obtain particle flow velocity.

In the present embodiment, the above-mentioned experimental steps are respectively performed on three groups of samples with different concentrations, and the obtained autocorrelation results are fitted according to the physical function corresponding to G _a (τ) in the above formula. The specific results are shown in Fig. 8, Fig. The three subgraphs from left to right in the figure correspond to the measurement and fitting results under the three concentration conditions, respectively. The average particle number N obtained by fitting under the three concentration conditions is 0.82, 0.55 and 0.34 respectively, and the observation area of each circular focal spot formed by the focal point is 201 μm ² on the camera screen, and the thickness of the microfluidic device is is 100 μm, then the observation volume is 201 μm ² ×100 μm=0.201×10 ^-4 μL. Using c=N/V, the fitted concentrations under the two slow conditions were 4.10×10 ⁴ /μL, 2.76× ^{10 4} /μL, 1.67×10 ⁴ /μL, and the actual The statistical concentrations are 4.17×10 ⁴ /μL, 2.64 ^{×10 4} /μL, 1.83× ^{10 4} /μL, and the average measurement error is 4.93%. The calculation results can accurately reflect the actual fluid concentration.

The invention designs a multi-focus particle detection method based on a meta-lens array, and performs multi-point sampling on the particles to be measured in a solution through multiple focal points generated by the dielectric meta-lens array, and performs subsequent data processing and related calculations. Compared with the fluorescence correlation spectroscopy technique, there is no need to use fluorescent labels, and the observation process will not be affected by photobleaching, and the particles in the natural state can be measured. Compared with the traditional two-focal correlation spectroscopy imaging system, the introduction of the metalens array makes the optical system more compact, and it is easier to realize multi-point detection with more than two focal points. In this embodiment, at least 4 A correlation spectroscopy system with up to 13 focal points effectively enriches the information content of a single experimental data, and the number of focal points is flexible and adjustable.

In the several embodiments provided by the present invention, it should be understood that the device and method disclosed in the present invention can be implemented in other ways. For example, the device embodiments described above are only illustrative. For example, the division of the modules is only a logical function division. In actual implementation, there may be other division methods. For example, multiple modules or components can be combined or May be integrated into another system, or some features may be ignored, or not implemented.

In addition, each functional module in each embodiment of the present invention may be integrated into one processing module, each module may exist separately physically, or two or more modules may be integrated into one module. The above-mentioned integrated modules can be implemented in the form of hardware, or in the form of hardware plus software function modules.

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention.

Claims (10)

1. A multi-focus particle detection system based on a metalens array, characterized in that it includes an illumination module, an imaging module, an image acquisition module, an analysis calculation module and a microfluidic device (7), wherein, The illumination module, the imaging module and the image acquisition module are sequentially coupled and connected along the beam transmission direction; The illumination module includes a laser (1), a collimator lens (2), a beam expander lens (3), a polarizer (4), a quarter-wave plate (5) and a dielectric superstructure sequentially arranged along the beam transmission direction. Lens array (6), at first produces parallel beam by described laser (1), described collimating lens (2) and described beam expander lens (3), and makes described parallel beam cover medium metalens array (6 ) the entire aperture, and then produce a group of focus arrays through the medium metalens array (6); The microfluidic device (7) is placed between the illumination module and the imaging module, and is used to carry a solution sample containing particles to be tested; The imaging module includes an imaging objective lens (8); The image acquisition module is located at the rear end of the focal plane of the imaging objective lens (8), and is used for imaging the particle to be measured or performing secondary imaging on the focal point array on the back focal plane of the medium metalens array, and then utilizing the obtained focal point array To observe the particles to be measured; The analysis and calculation module is used to perform statistics on the light intensity value of the particles to be measured observed in real time, and perform autocorrelation and cross-correlation analysis and calculation to obtain the relevant physical quantities of the particles to be measured in the solution. 2. The multi-focus particle detection system based on a metalens array according to claim 1, wherein the dielectric metalens array is composed of a substrate and a periodic uniform array of structural units positioned above the substrate, wherein, The material of the base is silicon dioxide, resin or aluminum oxide. The characteristic size and period of the structural units in the periodic uniform array of the structural units are smaller than the wavelength of the electromagnetic wave in vacuum. The shape of the structural units includes Cubic waveguide, cylindrical waveguide, elliptical cylindrical waveguide, the material of the structural unit is selected from silicon, gallium nitride or titanium dioxide. 3. according to the multi-focus particle detection system based on metalens array in claim 1, it is characterized in that, described illumination module comprises laser device (1), beam expander lens (2), collimator arranged in sequence along beam propagation direction Lens (3), polarizer (4), quarter-wave plate (5), medium metalens array (6), described imaging module comprises imaging objective lens (8); Described image acquisition module is CCD camera ( 9), the microfluidic device (7) is located at the common focal plane of the dielectric metalens array (6) and the imaging objective lens (8), and the plane where the microfluidic device (7) is located and The direction of beam propagation is vertical. 4. according to the multi-focus particle detection system based on metalens array in claim 3, it is characterized in that, except using described medium metalens array (6) to carry out direct imaging to sample, in described medium metalens A converging lens is arranged between the array (6) and the microfluidic device (7), which can be used for secondary imaging of the focal point array generated by the dielectric metalens array (6), and then the secondary imaging The light beam is converged in the sample area of the microfluidic device (7). 5. according to the multi-focus particle detection system based on metalens array in claim 3, it is characterized in that, described image acquisition module is also used for carrying out multi-frame continuous image acquisition to the particle to be measured under the flowing state, and multiple The relevant parameters of the image sequence obtained by frame continuous acquisition are adjusted to ensure that the particles and background information in the sampling area can be clearly distinguished. 6. According to the multi-focus particle detection system based on the metalens array in claim 5, it is characterized in that, the analysis and calculation module is specifically used for: Extracting the intensity information inside one or more focus areas generated by each frame in the image sequence, integrating and summing the signal intensity values in the areas containing particle swarm flow information, and obtaining a set of time-varying light intensity data curves ; Perform numerical compensation on the obtained light intensity data curve to eliminate the influence of the signal-to-background ratio drop; Image correlation calculation is performed on the intensity information after numerical compensation to obtain the autocorrelation result of the intensity information of each focal area and the cross-correlation result of the intensity information between the focal areas; The data curves of the obtained autocorrelation results and cross-correlation results are fitted and calculated to obtain the particle flow velocity. 7. A multi-focus particle detection method based on a metalens array, characterized in that, comprising: S1: According to the multi-focus particle detection system based on the metalens array according to claims 1 to 6, a multi-frame continuous image sequence of the particles to be measured in a flowing state is obtained; S2: Carry out image correlation spectroscopy calculation on the image sequence to obtain single-focus autocorrelation curves and multi-focus cross-correlation curves, and obtain flow velocity and concentration parameters of the particles to be measured by function fitting. 8. The multi-focus particle detection method based on a metalens array according to claim 7, wherein said S1 comprises: S1.1: Turn on the power supply of the lighting module, adjust the intensity of the light source, and ensure that the image formed by the CCD camera for the metalens array is clearly visible and the exposure is moderate; S1.2: Adjust the positional relationship between the dielectric metalens array, the imaging objective lens, and the CCD camera, so that the focal point generated by the dielectric metalens array can be clearly imaged on the CCD camera; S1.3: Fix the microfluidic device between the dielectric metalens array and the imaging objective lens, and move the position of the microfluidic device axially to make the observation area in the microfluidic device clearly imaged, and to The microfluidic device injects the solution containing the particles to be tested at a constant speed; S1.4: When the solution flow is in a steady state, perform multi-frame continuous image acquisition of the particles to be measured in the sample, and adjust relevant parameters for the image sequence obtained by multi-frame continuous acquisition to ensure that the images captured by the CCD camera can Clearly distinguish the particles to be measured from the background information. 9. The multi-focus particle detection method based on a metalens array according to claim 6, wherein said step S2 comprises: S2.1: Extract the intensity information inside any one or more focus areas generated by each frame in the image sequence, integrate and sum the signal intensity values in the area containing the particle swarm flow information, and obtain a set of time-varying The intensity curve of , the intensity information data of each focal area corresponds to a curve; S2.2: Perform numerical compensation on the obtained intensity curve to eliminate the influence of the signal-to-background ratio drop; S2.3: Carry out image correlation calculation on the intensity information after numerical compensation, and obtain the autocorrelation result of the intensity information of each focus area and the cross-correlation result of the intensity information between focus areas; S2.4: Fitting and calculating the data curves of the obtained autocorrelation results and cross-correlation results to obtain particle flow velocity. 10. The multi-focus particle detection method based on a metalens array according to claim 9, wherein said S2.4 comprises: The data curves of the obtained autocorrelation results and cross-correlation results are fitted and calculated to obtain particle flow velocity and concentration, where the physical model used for fitting is: Among them, G _a (τ) is the result of autocorrelation calculation of intensity information in one focus area, G _C (τ) is the result of cross correlation calculation of intensity information in two focus areas, and N is the The average number of particles flowing through in , τ _d is the diffusion time parameter caused by the Brownian motion of the particle itself, τ _f is the time parameter related to the particle flow caused by the external force, and d ₀ represents the actual distance between any two focal points , r ₀ represents the radius value of the current focus; The particle flow velocity is obtained by using the formula v _f =r ₀ /τ _f , and the particle concentration is obtained by dividing N by the volume of the observation area.