WO2023092648A1

WO2023092648A1 - Optical path design method and device for fluorescence dispersion of flow cytometer

Info

Publication number: WO2023092648A1
Application number: PCT/CN2021/136155
Authority: WO
Inventors: 尤政; 晁子翕; 赵精晶
Original assignee: 清华大学
Priority date: 2021-11-23
Filing date: 2021-12-07
Publication date: 2023-06-01
Also published as: CN114136867B; CN114136867A

Abstract

An optical path design method and device for fluorescence dispersion of a flow cytometer, relating to the technical field of flow cytometers. The optical path design method for fluorescence dispersion of a flow cytometer comprises: determining total light transmittance required and material parameters of prisms used (110); establishing a prism dispersion model, the prism dispersion model determining, according to the total light transmittance and the material parameters of the prisms used, an optical path shape when the total dispersion rate value is the largest when light continuously enters at least one prism and then reaches a receiving screen (120); and determining the total path length of the optical path, and determining the size of the optical path shape according to the total path length of the optical path (130). According to the method and device, a shading slit required when light is incident in a grating dispersion system can be omitted, the utilization rate, the signal-to-noise ratio and the sensitivity of collected fluorescence energy are improved, and the optimal dispersion rate and light transmittance are achieved while effectively meeting a flow cytometer miniaturization requirement.

Description

Optical path design method and device for fluorescence dispersion of flow cytometer

Cross References to Related Applications

This application is based on a Chinese patent application with application number 202111393062.X and a filing date of November 23, 2021, and claims the priority of this Chinese patent application. The entire content of this Chinese patent application is hereby incorporated by reference into this application.

technical field

The present application relates to the technical field of flow cytometry, in particular to an optical path design method and device for fluorescence dispersion of flow cytometry.

Background technique

Flow cytometry is an instrument that can quickly detect the biophysical and biochemical information of each cell or biological particle in a cell population. It is widely used in scientific research, clinical testing and production activities; fluorescence detection is Flow cytometry is currently the main means of cell detection. Traditional flow cytometers use multi-channel filtering methods for fluorescence detection, while direct detection of full-spectrum fluorescence is a new fluorescence detection method that can greatly improve the accuracy of detection; considering the specific needs of flow cytometers, Especially in recent years, the demand for the miniaturization of flow cytometers has been newly generated. Prism dispersion is the main dispersion method suitable for micro flow cytometers. Therefore, there is an urgent need for an optical path design method for fluorescence dispersion of flow cytometers. It can realize the miniaturization requirement of the flow cytometer, and at the same time, can obtain the best dispersion rate and the best light transmittance.

Contents of the invention

This application aims to solve one of the technical problems in the related art at least to a certain extent.

For this reason, the first purpose of this application is to propose an optical path design method for the fluorescence dispersion of the flow cytometer, so as to solve the problem that the current optical path for the fluorescence dispersion of the flow cytometer cannot meet the needs of the miniaturization of the flow cytometer. At the same time, it is a technical problem to obtain the best dispersion rate and the best light transmittance.

The second purpose of the present application is to propose an optical path design device for fluorescence dispersion of a flow cytometer.

In order to achieve the above purpose, the embodiment of the first aspect of the present application proposes an optical path design method for fluorescence dispersion of a flow cytometer, including:

Determine the required total light transmittance and the material parameters of the prism used;

Establishing a prism dispersion model, the prism dispersion model determines the shape of the optical path when the total dispersion rate value is maximum when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used;

The total path length of the light path is determined, and the size of the shape of the light path is determined according to the total path length of the light path.

In some embodiments, the material parameter of the prism is the refractive index of the prism; the wavelength of light is determined according to the material parameter of the prism.

In some embodiments, the establishment of a prism dispersion model includes:

On a plane, when light enters any prism, take the vertex of the prism as the origin, the side of the vertex where the light first enters is the x-axis, and the direction perpendicular to the x-axis is the y-axis to ensure that the prism The direction in which the subject is located in the first quadrant is the x-axis and the positive direction of the y-axis to construct a plane Cartesian coordinate system, the vertex angle and the angle of incidence of the first incident light are acute angles, and the direction of the first incident light is the positive direction of the x-axis , the negative direction of the y-axis;

According to the angle of the vertex angle, the angle of incidence of the light for the first time and the abscissa of the intersection with the x-axis when the light is incident for the first time, the ordinate and the rotation angle of the intersection of the light-receiving plane and the y-axis are determined. The rotation angle of the light plane is the angle between the prism and the positive direction of the x-axis; the light-receiving plane is the receiving screen or the next prism to be injected by the light;

According to the angle of the vertex angle, the rotation angle of the light-receiving plane, the material parameters of the prism, the incident angle of the first incident light, the abscissa of the intersection point of the first incident light and the x-axis, the light-receiving plane and the y-axis The ordinate of the intersection point determines the position of the final falling point of the ray on the receiving plane.

In some embodiments, the prism dispersion model determines the shape of the optical path when the total dispersion rate value reaches the maximum value when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used, including:

When the light enters any prism and reaches the light-receiving plane, determine the relationship between the dispersion rate and the angle of the vertex angle of the prism and the angle of the first incident angle of the light according to the prism dispersion model;

Use the relationship between the dispersion rate and the angle of the prism vertex angle and the angle of the incident angle of the light for the first time to determine the abscissa of the intersection point with the x-axis when the light is incident for the first time and the distance from the position where the light exits the prism to the receiving screen The proportional value between the distances;

Determine the maximum value of the dispersion rate and the angle of the vertex angle of the prism at this time and the angle of the incident angle of the light incident for the first time according to the ratio value;

According to the angle of the vertex angle of the prism at the maximum value of the dispersion rate and the angle of the incident angle of the light incident for the first time, determine the light path when the dispersion rate value is the maximum when the light enters a prism and reaches the light receiving plane.

In some embodiments, the determining the relationship between the dispersion rate and the angle of the vertex angle of the prism and the angle of the first incident angle of the light according to the prism dispersion model includes: determining the light at the first time by the following formula The position of the final landing point on the receiving plane:

F＝ _x2 + _dr2

Among them, F is the final landing position of the light, _x2 is the abscissa of the light on the light-receiving plane, _r2 is the refraction angle when the light is emitted from the prism, and d is the distance from the position where the light is emitted from the prism to the light-receiving plane, α is the angle of the apex angle of the prism, n is the material parameter of the prism, i ₁ is the incident angle of the light incident for the first time, x ₁ is the abscissa of the intersection point with the x-axis when the light is incident for the first time, and k is an intermediate variable;

Determine the distance from the position where the light exits the prism to the light-receiving plane according to the rotation angle of the light-receiving plane and the ordinate of the intersection point of the light-receiving plane and the y-axis;

Determine the dispersion rate according to the position of the final drop point of the light on the light-receiving plane, and determine the dispersion rate by the following formula:

in,

is the dispersion rate,

is the partial derivative of k with respect to n,

is the partial derivative of r ₂ to n, F is the final landing position of the light, λ is the wavelength of the light, n is the material parameter of the prism, k is the intermediate variable, r ₂ is the refraction angle when the light exits the prism, and x ₁ is The abscissa of the intersection point of the ray and the x-axis when the ray is incident for the first time, d is the distance from the position where the ray exits the prism to the receiving plane;

Wherein, the partial derivative of k to n is determined by the following formula:

in,

is the partial derivative of k to n, k is an intermediate variable, α is the angle of the apex angle of the prism, n is the material parameter of the prism, and i ₁ is the incident angle of the light incident for the first time;

Wherein, the partial derivative of said r to _n is determined by the following formula:

in,

is the partial derivative of r ₂ to n, r ₂ is the refraction angle when the light exits the prism, α is the angle of the apex angle of the prism, n is the material parameter of the prism, and i ₁ is the incident angle of the first incident light;

Determine the abscissa of the point of intersection with the x-axis when the light is incident for the first time and the position from the position of the light exiting the prism to the light collection according to the position of the final landing point of the light on the receiving screen, the intermediate variable k, and the refraction angle when the light exits the prism Scale value between the distances of the planes.

In some embodiments, the prism dispersion model determines the light path when the total dispersion rate value is maximum when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used, including:

According to the optical path when the total dispersion rate value is the largest when the light enters at least one prism and reaches the receiving screen after the light enters each prism continuously, determine the total dispersion when the light continuously enters at least one prism and reaches the receiving screen The shape of the light path when the power value is maximum.

In some embodiments, the prism dispersion model determines the shape of the optical path when the total dispersion rate value reaches the maximum value when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used, including: The size, number and placement of the required prisms are determined according to the shape of the optical path.

In some embodiments, the determining the size, number and placement of the required prisms according to the shape of the optical path includes:

The size of the prism is larger than the size of the incident light rays, and the light rays incident on the prisms are close to but not located at the vertex corners of the prisms.

In some embodiments, the determining the size of the shape of the optical path according to the total path length of the optical path includes:

When light continuously enters at least one prism and reaches the receiving screen, the distance between light with different wavelengths in the light is _Δ θ ₁ (d ₁ +d ₂ +...+d _p )+ _Δ θ ₂ (d ₂ +d ₃ +...+d _p )+...+ _Δ θ _p d _p ;

Among them, △θp is the angle at which light with different wavelengths in the light is separated after the light passes through the pth prism; dp is the medium interface between the medium interface where the light exits the pth prism and the light receiving plane that the light reaches after exiting the pth prism the distance between the interfaces;

The size of the shape of the optical path, that is, the values of d1, d2...dp is determined according to the total path length of the optical path, and the values of d1, d2...dp are allocated in increments.

To sum up, the method proposed in the embodiment of the first aspect of the present application determines the required total light transmittance and the material parameters of the prism used; establishes a prism dispersion model, and the prism dispersion model is based on the total light transmittance and the used prism The material parameters of the prism determine the shape of the optical path when the total dispersion rate value is the largest when the light rays continuously enter at least one prism and reach the receiving screen; determine the total path length of the optical path, and determine the size of the optical path shape according to the total path length of the optical path. This application avoids the shading slit required when the light of the grating dispersion system enters, improves the utilization rate, signal-to-noise ratio and sensitivity of the collected fluorescent energy, and effectively realizes the miniaturization requirements of the flow cytometer while having the most Good dispersion and best light transmittance.

In order to achieve the above purpose, the embodiment of the second aspect of the present application proposes an optical path design device for fluorescence dispersion of flow cytometer, including:

Input module for determining the required total light transmittance and the material parameters of the prisms used;

The prism dispersion model module is used to establish a prism dispersion model, and the prism dispersion model determines when the total dispersion rate value is maximum when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used shape of the light path.

The determining module is configured to determine the total path length of the optical path, and determine the size of the shape of the optical path according to the total path length of the optical path.

To sum up, the device proposed in the embodiment of the second aspect of the present application determines the required total light transmittance and the material parameters of the prism used through the input module; the prism dispersion model module establishes the prism dispersion model, and the prism dispersion model is based on the total The light transmittance and the material parameters of the prisms used determine the shape of the optical path when the total dispersion rate value reaches the maximum after the light continuously enters at least one prism and reaches the receiving screen; The size of the optical path shape. The optical path obtained in this application has strong stability, can work only with general level mechanical positioning and fixing, can be used to realize an on-chip optical system, does not require frequent calibration during use, has a processing approach for one-time molding of the optical path, and is conducive to system integration, and It is more suitable for the miniaturization requirements of flow cytometer.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

Description of drawings

The above and/or additional aspects and advantages of the present application will become apparent and easy to understand from the following description of the embodiments in conjunction with the accompanying drawings, wherein:

FIG. 1 is a flowchart of an optical path design method for fluorescence dispersion of a flow cytometer provided in an embodiment of the present application;

Fig. 2 is a schematic diagram of the establishment process of the prism dispersion model provided by the embodiment of the present application;

Fig. 3 is the schematic structural diagram of the mathematical model of the single prism provided by the embodiment of the present application;

Fig. 4 is a schematic diagram of parameter scanning of a single prism provided by the embodiment of the present application;

Fig. 5 is a schematic diagram of the optimal value provided by the embodiment of the present application;

Fig. 6 is a schematic diagram of parameter scanning of multiple prisms provided by the embodiment of the present application;

7 is a schematic diagram of the optical path design of the discrete device provided by the embodiment of the present application;

FIG. 8 is a schematic diagram of the optical path design of the first on-chip dispersion provided by the embodiment of the present application;

FIG. 9 is a schematic diagram of the optical path design of the second on-chip dispersion provided by the embodiment of the present application;

FIG. 10 is a schematic structural diagram of an optical path design device for fluorescence dispersion of a flow cytometer provided in an embodiment of the present application.

Detailed ways

Embodiments of the present application are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary, are only for explaining the present application, and should not be construed as limiting the present application. On the contrary, the embodiments of the present application include all changes, modifications and equivalents falling within the spirit and scope of the appended claims.

When the flow cytometer performs fluorescence detection, it is necessary to focus on the two indicators of dispersion rate and light transmittance; first, the flow cytometer needs to detect the fluorescent signal emitted by the cells when performing fluorescence detection, but due to The light intensity of the signal is very weak, so a photomultiplier tube (PMT) needs to be used for detection, and the photomultiplier tube often has a large space size. In addition, because the fluorescence signal itself is very weak, the transmittance of the fluorescence signal is the key to the signal-to-noise ratio of fluorescence detection. Therefore, when designing the optical path for fluorescence dispersion in flow cytometry, it is necessary to pursue the best Good dispersion and best light transmittance.

Example 1

FIG. 1 is a flowchart of an optical path design method for fluorescence dispersion of a flow cytometer provided in an embodiment of the present application.

As shown in Figure 1, a kind of optical path design method for flow cytometer fluorescence dispersion provided by the embodiment of the present application comprises the following steps:

Step 110, determining the required total light transmittance and the material parameters of the prism used;

Step 120, establishing a prism dispersion model, the prism dispersion model determines the shape of the optical path when the total dispersion rate value is maximum when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used;

Step 130, determine the total path length of the optical path, and determine the size of the shape of the optical path according to the total path length of the optical path.

In the embodiment of the present application, the material parameter of the prism is the refractive index of the prism; the wavelength of light is determined according to the material parameter of the prism.

In the embodiment of this application, a prism dispersion model is established, including:

According to the angle of the vertex angle, the incident angle of the first incident light and the abscissa of the intersection point with the x-axis when the light first incident, determine the ordinate and the rotation angle of the intersection point between the light-receiving plane and the y-axis, and the rotation of the light-receiving plane The angle is the angle between the prism and the positive direction of the x-axis; the light-receiving plane is the receiving screen or the next prism to be injected by the light;

According to the angle of the vertex angle, the rotation angle of the light-receiving plane, the material parameters of the prism, the incident angle of the first incident light, the abscissa of the intersection point of the first incident light and the x-axis, the intersection point of the light-receiving plane and the y-axis The ordinate of determines the position of the final falling point of the ray on the receiving plane.

In some embodiments, in a flow cytometer, a bundle of parallel light rays obtained by collecting fluorescence through fluorescence detection (including light rays in a certain wavelength range, and assuming that each wavelength is uniformly distributed everywhere) passes through the prism from the environment of the reference refractive index One side of the vertex angle (between 0° and 360°, the internal relative refractive index n, n being the refractive index of conventional optical materials) enters the prism, and then completely shoots out from the other side of the prism’s vertex angle. This process After repeating 1 or more times, the image is formed on the receiving screen (or all light rays enter a convex lens and form an image on its focal plane). The establishment process of the prism dispersion model is shown in Figure 2, where Figure 2(a) The apex of the apex of the prism is taken as the origin, the apex side of the light incident for the first time is the x-axis, and the direction perpendicular to the x-axis is the y-axis, so that the direction in which the prism body is located in the first quadrant is the x-axis and the y-axis The plane Cartesian coordinate system constructed in the positive direction of .

In some embodiments, for the bundle of parallel rays, since each optical surface in the optical system of the flow cytometer is a plane, for the rays of the same wavelength, any one of the rays in the bundle of rays in the first When refraction occurs at two optical surfaces, since the complementary angle of the incident angle is the same angle formed by the intersection of parallel lines and straight lines, the incident angle is always the same, so the refraction angle is also the same. By analogy, for light rays of the same wavelength, the initially parallel rays are still parallel before hitting the plane or the lens, so ignore the width of this bundle of parallel rays, and first study the Condition;

As shown in Figure 2(b), the vertex angle and the incident angle of the first incident light are acute angles, and the first incident direction of the light is the negative direction of the x-axis and the positive direction of the y-axis from the bottom right to the top left. The position of the light receiving plane is calculated under the conditions, and the position of the light receiving plane includes the following three situations:

In the first case, the light-receiving plane intersects the positive semi-axis of the y-axis at a point;

In the second case, the light-receiving plane intersects the negative semi-axis of the y-axis at a point;

In the third case, the receiving plane is parallel to (disjoint) or completely coincident with the y-axis;

When the position of the light-receiving plane is the first case, let the vertical coordinate of the intersection point of the light-receiving plane and the y-axis be y ₁ , the initial orientation of the light-receiving plane is parallel to the x-axis, and the light-receiving side of the light-receiving plane is Toward the negative direction of the y-axis, the light-receiving plane rotates counterclockwise at this time, and the angle of counterclockwise rotation is a positive value, and this angle is recorded as θ, as shown in Figure 2(c).

The position of the light-receiving plane can be deduced through geometric optics under various circumstances. The formula F=f(α,θ,n,i ₁ ,x ₁ ,y ₁ ) of the final falling point position of the light and its influencing factors can be finally derived. ;

Among them, F is the final landing position of the light, α is the angle of the vertex of the prism, n is the material parameter of the prism, i ₁ is the incident angle of the first incident light, x ₁ is the distance between the first incident light and the x-axis The abscissa of the intersection point, y ₁ is the ordinate of the intersection point of the light-receiving plane and the y-axis.

Furthermore, if the wavelength of this bundle of parallel rays changes, it will appear as a change in n, that is, F=f(α,θ,n(λ),i ₁ ,x ₁ ,y ₁ ), that is, If the bundle of parallel rays contains rays of different wavelengths, it will have a dispersion effect after passing through the optical system of the flow cytometer. The F of will also be distributed within a range, and each λ corresponds to an F;

If this bundle of parallel rays has a certain width (considering the case of monochromatic light first), then it is equivalent to the continuous value of x ₁ in a certain range, and the corresponding F will also be distributed in a range, each x ₁ corresponds to an F; if this beam of light is not parallel, it is also equivalent to a change in x ₁ ;

If this bundle of parallel rays has a certain width and contains rays of different wavelengths, then the result of F will be multiple continuously distributed light bands. Each (x ₁ ,λ) pair of ordered numbers corresponds to one F.

Further, according to the definition of dispersion rate, the line dispersion rate of a single light passing through the flow cytometer optical system is

Right now

Further, the resolution is affected and limited by various factors. There may be multiple factors limiting resolution in the entire flow cytometer optical system. The final resolution of the flow cytometer optical system depends on the most restrictive of these limitations. Since the resolution cannot be defined for a single ray, it is assumed that this bundle of parallel rays has a certain width △x. If two wavelengths of light separated by △λ are to be separated, the following two conditions need to be met:

The first condition is that the distance between the two wavelengths that are finally separated is not less than the smallest scale of the detector pixel;

The second condition, that the finally separated beams of these two wavelengths do not overlap in space (crosstalk);

For the first condition, if the minimum size of the detector pixel is D, then

For the second condition, f(α,θ,n(λ),i ₁ ,x ₁ +Δx,y ₁ ,)≤f(α,θ,n(λ+Δλ),i ₁ ,x ₁ ,y ₁ ,), in the case of small changes, it can be transformed into

Determine the minimum resolution wavelength according to the following formula:

Among them, △λ is the minimum resolution wavelength, and the max function means to take the maximum value;

According to the definition of resolution, the final resolution of the optical path

calculated

Further, for the light transmittance, first consider the loss of light energy caused by reflection, assuming that a beam of natural light passes through the optical path, and only consider the light transmittance lost by reflection, that is, the product of the transmittance of the two optical surfaces, determined by the following formula Transmittance of flow cytometer optical system:

Among them, the factors that affect the light transmittance also include the scattering of the interface and the scattering and absorption of the material inside the prism. For ordinary optical materials, the scattering and absorption of the material inside the prism are very weak and can be basically ignored, while the scattering of the interface It may be too large to be ignored, but considering that the scattering of the interface is diffuse reflection and has randomness, that is, isotropy, so its specific influence degree has nothing to do with the parameters required for this embodiment;

For the design of a single prism, there is no need to consider the factor of interface scattering;

For the situation where there is a lens in front of the position where the fluorescence is received, if the ideal lens model is used, for the dispersion part, the only definition difference is the resolution, and this situation still needs to satisfy the detector pixel condition, that is

In some embodiments, the second condition should be determined by the size of the diffraction spot combined with the Rayleigh criterion, but in fact, the various aberrations caused by the gap between the ideal optical system and the actual optical system are often more important factors , and the parallelism of the incident beam is often not good, so in fact, it is often more accurate to consider the imaging of the incident beam window on the image plane; but in any case, the lens always has a converging effect on the beam, so , under the conventional lens selection, the spot must be smaller than the beam width without the lens, therefore, for the case with the lens, this part considers directly referring to the program calculation or simulation results for evaluation and design;

For the case with a lens, it is also necessary to consider the loss of light energy caused by the addition of the lens. This part can also be calculated using the formula similar to the above formula for calculating the light transmittance of the optical path based on the results of the two incident angles and refraction angles obtained inside the algorithm. calculate.

In some embodiments, when light enters a prism and then reaches the receiving screen, the mathematical model structure of the single prism is as shown in Figure 3;

It should be noted that the prism dispersion, that is, the material dispersion method, essentially uses the different propagation speeds of light of different wavelengths in the material, that is, the difference in refractive index to achieve dispersion. spread in different directions.

In some embodiments, when light continuously enters at least two prisms and reaches the receiving screen, the essence of dispersion of multiple prisms (prism groups) is first analyzed, that is, when the interface of two different media is used, the light beam passes through the interface. , the propagation directions of different wavelengths of light are different, resulting in angular differences, and then using the distance between the interfaces to convert the angular differences into spatial distances to achieve the effect of dispersion.

In the embodiment of the present application, the prism dispersion model determines the shape of the optical path when the total dispersion rate value reaches the maximum value when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used, including:

When the light enters any prism and reaches the receiving plane, determine the relationship between the dispersion rate and the angle of the vertex angle of the prism and the angle of the first incident angle of the light according to the prism dispersion model;

According to the angle of the vertex angle of the prism at the maximum value of the dispersion rate and the angle of the incident angle of the light incident for the first time, determine the light path when the total dispersion rate value is maximum when the light enters a prism and reaches the light receiving plane.

In the embodiment of the present application, according to the prism dispersion model, the relationship between the dispersion rate and the angle of the vertex angle of the prism and the angle of the first incident angle of the light rays is determined, including: determining the final angle of the light rays on the light-receiving plane by the following formula: The location of the drop point:

F＝ _x2 + _dr2

Determine the dispersion rate according to the position of the final landing point of the light on the light-receiving plane, and determine the dispersion rate by the following formula:

in,

is the dispersion rate,

is the partial derivative of k with respect to n,

Among them, the partial derivative of k to n is determined by the following formula:

in,

where _the partial derivative of r with respect to n is determined by:

in,

Determine the abscissa of the intersection point with the x-axis when the light is incident for the first time and the distance from the position where the light exits the prism to the light-receiving plane according to the position of the final landing point of the light on the receiving screen, the intermediate variable k, and the refraction angle when the light exits the prism Scale value between distances.

In some embodiments, under normal circumstances, the light-receiving plane is perpendicular to the emitted light, and both x ₁ and d have nothing to do with n, that is, they have nothing to do with wavelength. Therefore, x ₁ and d are equivalent to being fixed, and only need to consider F and The relationship between the various angles;

in,

yes

and

The linear combination of , for the case with a lens, if the ideal lens model is used, it can be obtained

due to n and

It is related to the nature of the material and belongs to the fixed quantity, so this implementation mainly designs the angle of the vertex angle of the prism and the angle of the incident angle of the first incident light. The parameter scan of a single prism is shown in Figure 4, where 4(a) is a schematic diagram of the feasible range of the angle of the vertex angle of the prism and the angle of the angle of incidence of the first incident light, FIG. 4(b) is a schematic diagram of light transmittance, and FIG. 4(c) is a three-dimensional schematic diagram of light transmittance, Figure 4(d) is

Schematic diagram, Figure 4(e) is

Three-dimensional schematic diagram, Figure 4(f) is the case where the light transmittance is not lower than 80%.

Schematic diagram, Figure 4(g) is the case where the light transmittance is not lower than 80%.

3D schematic.

In some embodiments, in the feasible range shown in Fig. 4(a), the maximum value of the linear dispersion rate and the angle of the vertex angle of the prism and the angle of the light ray when reaching the maximum value are found under the lower limit of the same light transmittance. The angle of incidence of an incident is shown in Figure 5.

In some embodiments,

and

The distribution law of is similar, and the position where the optimal value is taken is also similar.

In some embodiments, when light enters a prism and then reaches the receiving screen, the optical path design method for a single prism includes the following steps:

Step 210, determining the lower limit of the light transmittance required by the optical path;

Step 220, for a determined lower limit of light transmittance, draw a short line across the angular region and connect the two sides of the angle by changing the ratio of _x1 and d in the angular region shown in Figure 5, this There is an intersection point between the short line and the value line conforming to the principle of minimum deflection angle;

Step 230 , determine the ratio of x ₁ to d (that is, the ratio of x ₁ to d at the intersection point in step 220 ), so that the value line of α and i ₁ under this ratio passes through the intersection point in step 220 .

In the embodiment of the present application, the prism dispersion model determines the optical path when the total dispersion rate reaches the maximum value when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used, including:

In the embodiment of the present application, the prism dispersion model determines the shape of the optical path when the total dispersion rate reaches the maximum value when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used, including: according to the shape of the optical path Determine the size, number and placement of the required prisms.

In the embodiment of this application, the size, number and placement of the required prisms are determined according to the shape of the optical path, including:

The dimensions of the prism are larger than the dimensions of the rays entering the prism, and the rays entering the prism are near but not at the top corners of the prism.

It should be noted that the light of two specific wavelengths passes through the entire flow cytometer optical system, which is equivalent to passing through several medium interfaces. After passing through the jth medium interface, the light of the two specific wavelengths is additionally separated The angle is △θj, and the distance between the jth interface and the (j+1)th interface is dj, then after the first interface and the interval between the first surface and the second surface, The distance between the two wavelengths of light is △θ ₁ d ₁ , after passing through the second interface and the interval between the second surface and the third surface, the distance between the two wavelengths of light is d=△ θ ₁ (d ₁ +d ₂ )+△θ ₂ d ₂ . By analogy, assuming that there are a total of p interfaces, the distance between the final two wavelengths of light is determined by the following formula:

d=Δθ ₁ (d ₁ +d ₂ +...+d _p )+Δθ ₂ (d ₂ +d ₃ +...+d _p )+...+Δθ _p d _p

＝d ₁ Δθ ₁ +d ₂ (Δθ ₁ +Δθ ₂ )+......+d _p (Δθ ₁ +Δθ ₂ +......+Δθ _p )

In some embodiments, in the optical system of the flow cytometer, the total path length that the light can pass through is certain, because one of the purposes of the optical path design is to realize the miniaturization requirement of the flow cytometer, in this case, for Maximizing the "distance separated by light" is essentially to allocate the distance d between each section of the interface. For different d, the coefficients multiplied in front of it are different. For a reasonable dispersion design, each △θ is non- Negative, therefore, the coefficient multiplied in front of dp is the largest. In order to maximize the "distance separated by light", dp should be maximized, that is, the values of d1, d2...dp are allocated in increasing form.

In the embodiment of this application, the size of the shape of the optical path is determined according to the total path length of the optical path, including:

The size of the shape of the optical path, that is, the value of d1, d2...dp, is determined according to the total path length of the optical path, and the values of d1, d2...dp are allocated in increments.

In some embodiments, the designed optical path structure is a planar structure, regardless of the three-dimensional layout. Therefore, the mechanical components of the optical path must not cross planes, so for the dispersion part, the design scheme can be, for example, to deflect the light by 180 degrees as a whole at most, It is also possible to make the incident light and the outgoing light form a structure close to 270 degrees perpendicular to the plane.

In some embodiments, when the light continuously enters at least two prisms and reaches the receiving screen, the parameter scanning of multiple prisms is as shown in Figure 7, Figure 7(a) is a schematic diagram of the light transmittance of the polygonal prism, Figure 7(b) It is a three-dimensional schematic diagram of the light transmittance of a polygonal prism.

Schematic diagram, Figure 4(g) is the polygonal mirror under the condition that the light transmittance is not lower than 80%.

3D schematic.

In some embodiments, the optical path design method of multiple prisms includes the following steps:

Step 310, determining the refractive index n of the prism used;

Step 320, repeating steps 210-230 to obtain the best vertex angle, incident angle, and number of prisms, the number of prisms determines the regular polygonal shape of the optical path direction;

Step 330, according to the size limitation of the optical path (or the size desired to be controlled), determine the size of the regular polygonal shape of the optical path, that is, the radius;

Step 340, according to the optimal vertex angle and incident angle value, configure a prism at each vertex of the regular polygon along the optical path, the size of the prism only needs to be larger than the incident size of the light, and the apex angle of the prism should be as close as possible to the optical path Just leave a certain margin.

As a scenario implementation, according to the method proposed in this embodiment, considering the actual requirements of the dispersion part of the flow cytometer, in the case of the glass material of the dispersion prism, according to the material parameters of the dispersion prism. For example, F2 glass has a refractive index of 1.63 at 500nm, and the optimal solution is three dispersion prisms, which can guarantee a light transmittance of 60%. In this case, the 32-channel dispersion optical path design in the 480-700nm spectral band range, The optical path design results of discrete devices are shown in Figure 7, where Figure 7(a) is the optical path design diagram, Figure 7(b) is the physical diagram of the optical path, and Figure 7(c) is the specific output light wavelength position verified using a monochromator Figure 7(d) is the average spectrum result graph of the fluorescent signal of the green fluorescent microspheres actually measured in the flow cytometer system.

As a scenario realization, according to the method proposed in this embodiment, with the on-chip dispersion as the design target, when adopting the design scheme that makes the light deflect at most 180 degrees as a whole, according to the material parameters of the PDMS material used in micromachining, its D light (589.3 nm) refractive index is 1.414, and the optimal design scheme contains 5 prisms. In this case, the mold can be processed on the silicon wafer and formed at one time by inverting the mold, as shown in Figure 8, where Figure 8(a) is the first An on-chip optical path design diagram, Figure 8(b) is a schematic diagram of a silicon wafer mold.

In some embodiments, when adopting a design scheme in which the incident light and the outgoing light form a structure close to 270 degrees perpendicular to the plane, the optimal design scheme includes 8 prisms, as shown in FIG. 9 , wherein no mechanical elements appear plane intersection.

To sum up, the method proposed in the embodiment of this application determines the required total light transmittance and the material parameters of the prism used; establishes the prism dispersion model, and the prism dispersion model determines the continuous light according to the total light transmittance and the material parameters of the prism used The shape of the optical path when the total dispersion rate value reaches the maximum after entering at least one prism and reaching the receiving screen; determine the total path length of the optical path, and determine the size of the optical path shape according to the total path length of the optical path. This application avoids the shading slit required when the light of the grating dispersion system enters, improves the utilization rate, signal-to-noise ratio and sensitivity of the collected fluorescent energy, and effectively realizes the miniaturization requirements of the flow cytometer while having the most Good dispersion and best light transmittance.

In order to realize the above embodiments, the present application also proposes an optical path design device for fluorescence dispersion of a flow cytometer.

As shown in Figure 10, an optical path design device for fluorescence dispersion of a flow cytometer includes:

The input module 101 is used to determine the required total light transmittance and the material parameters of the prism used;

The prism dispersion model module 102 is used to establish the prism dispersion model. The prism dispersion model determines the optical path shape when the total dispersion rate value is maximum when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used. .

The determination module 103 is configured to determine the total path length of the optical path, and determine the size of the shape of the optical path according to the total path length of the optical path.

To sum up, the device proposed in the embodiment of the present application determines the required total light transmittance and the material parameters of the prism used through the input module; The material parameters determine the shape of the optical path when the total dispersion rate value is the largest when the light rays continuously enter at least one prism and reach the receiving screen; the determination module determines the total path length of the optical path, and determines the size of the optical path shape according to the total path length of the optical path. The optical path obtained by this application has strong stability, and it can work only with general mechanical positioning and fixing. It can be used to realize an on-chip optical system. It has a processing approach for one-time molding of the optical path, which is conducive to system integration, and is more suitable for flow cytometers. miniaturization needs.

It should be noted that, in the description of the present application, terms such as "first" and "second" are used for description purposes only, and should not be understood as indicating or implying relative importance. In addition, in the description of the present application, unless otherwise specified, "plurality" means two or more.

Any process or method descriptions in flowcharts or otherwise described herein may be understood to represent modules, segments or portions of code comprising one or more executable instructions for implementing specific logical functions or steps of the process , and the scope of preferred embodiments of the present application includes additional implementations in which functions may be performed out of the order shown or discussed, including in substantially simultaneous fashion or in reverse order depending on the functions involved, which shall It should be understood by those skilled in the art to which the embodiments of the present application belong.

It should be understood that each part of the present application may be realized by hardware, software, firmware or a combination thereof. In the embodiments described above, various steps or methods may be implemented by software or firmware stored in memory and executed by a suitable instruction execution system. For example, if implemented in hardware, as in another embodiment, it can be implemented by any one or combination of the following techniques known in the art: Discrete logic circuits, ASICs with suitable combinational logic gates, programmable gate arrays (PGAs), field programmable gate arrays (FPGAs), etc.

Those of ordinary skill in the art can understand that all or part of the steps carried by the methods of the above embodiments can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. During execution, one or a combination of the steps of the method embodiments is included.

In addition, each functional unit in each embodiment of the present application may be integrated into one processing module, each unit may exist separately physically, or two or more units may be integrated into one module. The above-mentioned integrated modules can be implemented in the form of hardware or in the form of software function modules. If the integrated modules are realized in the form of software function modules and sold or used as independent products, they can also be stored in a computer-readable storage medium.

The storage medium mentioned above may be a read-only memory, a magnetic disk or an optical disk, and the like.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations on the present application, and those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims

A method for designing an optical path for fluorescence dispersion of a flow cytometer, wherein the method includes:

Determine the required total light transmittance and the material parameters of the prism used;

Establishing a prism dispersion model, the prism dispersion model determines the shape of the optical path when the total dispersion rate value is maximum when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used;

The total path length of the light path is determined, and the size of the shape of the light path is determined according to the total path length of the light path.
The method according to claim 1, wherein the material parameter of the prism is the refractive index of the prism; and the wavelength of the light is determined according to the material parameter of the prism.
The method according to claim 2, wherein said establishment of a prism dispersion model comprises:

On a plane, when light enters any prism, take the vertex of the prism as the origin, the side of the vertex where the light first enters is the x-axis, and the direction perpendicular to the x-axis is the y-axis to ensure that the prism The direction in which the subject is located in the first quadrant is the x-axis and the positive direction of the y-axis to construct a plane Cartesian coordinate system, the vertex angle and the angle of incidence of the first incident light are acute angles, and the direction of the first incident light is the positive direction of the x-axis , the negative direction of the y-axis;

According to the angle of the vertex angle, the angle of incidence of the light for the first time and the abscissa of the intersection with the x-axis when the light is incident for the first time, the ordinate and the rotation angle of the intersection of the light-receiving plane and the y-axis are determined. The rotation angle of the light plane is the angle between the prism and the positive direction of the x-axis; the light-receiving plane is the receiving screen or the next prism to be injected by the light;

According to the angle of the vertex angle, the rotation angle of the light-receiving plane, the material parameters of the prism, the incident angle of the first incident light, the abscissa of the intersection point of the first incident light and the x-axis, the light-receiving plane and the y-axis The ordinate of the intersection point determines the position of the final falling point of the ray on the receiving plane.
The method according to claim 3, wherein the prism dispersion model determines the maximum value of the total dispersion rate when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used. Light path shape, including:

When the light enters any prism and reaches the light-receiving plane, determine the relationship between the dispersion rate and the angle of the vertex angle of the prism and the angle of the first incident angle of the light according to the prism dispersion model;

Use the relationship between the dispersion rate and the angle of the prism vertex angle and the angle of the incident angle of the light for the first time to determine the abscissa of the intersection point with the x-axis when the light is incident for the first time and the distance from the position where the light exits the prism to the receiving screen The proportional value between the distances;

Determine the maximum value of the dispersion rate and the angle of the vertex angle of the prism at this time and the angle of the incident angle of the light incident for the first time according to the ratio value;

According to the angle of the vertex angle of the prism at the maximum value of the dispersion rate and the angle of the incident angle of the light incident for the first time, determine the light path when the dispersion rate value is the maximum when the light enters a prism and reaches the light receiving plane.
The method according to claim 4, wherein said determining the relationship between the dispersion rate and the angle of the vertex angle of the prism and the angle of the first incident angle of light according to the prism dispersion model comprises: by the following formula Determine where the ray ends up falling on the receiving plane:

F＝ x2 + dr2

Among them, F is the final landing position of the light, x2 is the abscissa of the light on the light-receiving plane, r2 is the refraction angle when the light is emitted from the prism, and d is the distance from the position where the light is emitted from the prism to the light-receiving plane, α is the angle of the apex angle of the prism, n is the material parameter of the prism, i 1 is the incident angle of the light incident for the first time, x 1 is the abscissa of the intersection point with the x-axis when the light is incident for the first time, and k is an intermediate variable;

Determine the distance from the position where the light exits the prism to the light-receiving plane according to the rotation angle of the light-receiving plane and the ordinate of the intersection point of the light-receiving plane and the y-axis;

Determine the dispersion rate according to the position of the final falling point of the light on the light-receiving plane, and determine the dispersion rate by the following formula:

in,
is the dispersion rate,
is the partial derivative of k with respect to n,
is the partial derivative of r 2 to n, F is the final landing position of the light, λ is the wavelength of the light, n is the material parameter of the prism, k is the intermediate variable, r 2 is the refraction angle when the light exits the prism, and x 1 is The abscissa of the intersection point of the ray and the x-axis when the ray is incident for the first time, d is the distance from the position where the ray exits the prism to the receiving plane;

Wherein, the partial derivative of k to n is determined by the following formula:

in,
is the partial derivative of k to n, k is an intermediate variable, α is the angle of the apex angle of the prism, n is the material parameter of the prism, and i 1 is the incident angle of the light incident for the first time;

Wherein, the partial derivative of said r to n is determined by the following formula:

in,
is the partial derivative of r 2 to n, r 2 is the refraction angle when the light exits the prism, α is the angle of the apex angle of the prism, n is the material parameter of the prism, and i 1 is the incident angle of the first incident light;

Determine the abscissa of the point of intersection with the x-axis when the light is incident for the first time and the position from the position of the light exiting the prism to the light collection according to the position of the final landing point of the light on the receiving screen, the intermediate variable k, and the refraction angle when the light exits the prism Scale value between the distances of the planes.
The method according to claim 4, wherein the prism dispersion model determines the maximum value of the total dispersion rate when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used. Light path, including:

According to the optical path when the total dispersion rate value is the largest when the light enters at least one prism and reaches the receiving screen after the light enters each prism continuously, determine the total dispersion when the light continuously enters at least one prism and reaches the receiving screen The shape of the light path when the power value is maximum.
The method according to claim 1, wherein the prism dispersion model determines the maximum value of the total dispersion rate when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used The shape of the optical path includes: determining the size, number and placement of the required prisms according to the shape of the optical path.
The method according to claim 7, wherein said determining the size, number and placement of the required prisms according to the shape of the optical path comprises:

The size of the prism is larger than the size of the incident light rays, and the light rays incident on the prisms are close to but not located at the vertex corners of the prisms.
The method according to claim 1, wherein said determining the size of the shape of the optical path according to the total path length of the optical path comprises:

When light continuously enters at least one prism and reaches the receiving screen, the distance between light with different wavelengths in the light is Δθ 1 (d 1 +d 2 +…+d p )+Δθ 2 (d 2 +d 3 +… ...+d p )+...+Δθ p d p ;

Among them, △θp is the angle at which light with different wavelengths in the light is separated after the light passes through the pth prism; dp is the medium interface between the medium interface where the light exits the pth prism and the light receiving plane that the light reaches after exiting the pth prism the distance between the interfaces;

The size of the shape of the optical path, that is, the values of d1, d2...dp is determined according to the total path length of the optical path, and the values of d1, d2...dp are allocated in increments.
An optical path design device for fluorescence dispersion of a flow cytometer, wherein the device includes:

Input module for determining the required total light transmittance and the material parameters of the prisms used;

The prism dispersion model module is used to establish a prism dispersion model, and the prism dispersion model determines when the total dispersion rate value is maximum when the light continuously enters at least one prism and reaches the receiving screen according to the total light transmittance and the material parameters of the prisms used The shape of the optical path;

The determining module is configured to determine the total path length of the optical path, and determine the size of the shape of the optical path according to the total path length of the optical path.