CN111929226B

CN111929226B - Flow cytometer fluorescence collection lens and light path system thereof

Info

Publication number: CN111929226B
Application number: CN202010717346.9A
Authority: CN
Inventors: 杨波; 马永波; 秦晓琨; 李�根
Original assignee: Beijing Gaugene Biological Technology Co ltd
Current assignee: Beijing Gaugene Biological Technology Co ltd
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2022-03-25
Anticipated expiration: 2040-07-23
Also published as: CN111929226A

Abstract

The invention discloses a flow cytometer fluorescence collection lens and a light path system thereof, wherein the lens realizes larger numerical aperture through the combination of a plano-convex lens, a positive meniscus lens, a biconvex lens and two double cemented lenses, and can collect fluorescence scattered light within the range of half angle of 45 degrees; the lens meets the requirement that the working distance is more than 2mm, and can be compatible with a flow chamber used on a common flow cytometer; the lens has good chromatic aberration correction capability in the wavelength range of 400-820nm, can output parallel light, has the beam diameter within 5mm, meets the use requirement that the dichroic mirror and the color filter are sensitive to the incident angle of the light beam, simultaneously realizes light beam transmission by the color filter and the dichroic mirror with smaller size, greatly saves the coating cost and the cost of instruments, is simple and easy to obtain, and can be applied to common flow cytometry.

Description

Flow cytometer fluorescence collection lens and light path system thereof

Technical Field

The invention relates to the technical field of detection equipment adopting a flow cytometry, in particular to a flow cytometer fluorescence collection lens and a light path system thereof.

Background

Currently, in flow cytometers, cells are passed through the instrument at a rate of 10,000 cells (or more) per second and detected, which can provide a wide range of statistical functions for cell biology. Cells can generate specific fluorescent signals through laser beams of an instrument, then the specific fluorescent signals are collected through a collecting lens, the fluorescent light is further divided according to wave bands through a dichroic mirror and a color filter, and the fluorescent light is converged to a photoelectric detector to complete signal receiving. The existing flow cytometer fluorescence collection lens needs to meet several important technical indexes:

first, as the larger the Numerical Aperture (NA) of the collecting lens means the stronger the ability of collecting light, for the performance of the apparatus, the stronger the fluorescence signal, the better the detection performance, and the higher the sensitivity, the collecting lens of the flow cytometer generally needs to satisfy the technical index of large Numerical aperture.

Second, since the flow cell is placed in front of the flow cytometer lens, and the wall thickness of the flow cell is typically 2mm, the working distance of the lens is typically required to be greater than 2 mm.

Thirdly, since the flow cytometer generally has at least 4 fluorescence detection channels, and the related spectral range is 400-820nm, in order to ensure that the on-axis chromatic aberration and the size of the dispersed spot are small after the light beam collected by the lens is converged, so that the light beam is effectively received by the photodetector, the collecting lens is required to have good chromatic aberration correction capability.

Fourth, the collection lens is required to output a parallel beam, and there are two reasons, the first is that the dichroic mirror and the color filter are based on the principle of light interference and are therefore sensitive to the incident angle, which affects the optical path difference of light propagating in the medium. In general, the color filter is required to be incident at 0 degree in design, the allowed use angle range is 0-5 degrees, the dichroic mirror is more sensitive to the incident angle, the dichroic mirror is required to be incident at 45 degrees and can only be used within the range of +/-1 degrees, and if the dichroic mirror is larger than the incident angle range, a spectral spectrum can generate blue shift (or red shift) and deformation, so that the use effect and the performance are greatly influenced; secondly, the traditional flow cytometer divides channels for fluorescence wave bands by adopting a dichroic mirror and a color filter method, can realize arbitrary expansion of the number of the fluorescence channels in principle on the premise of outputting parallel light by a fluorescence collecting lens, and has strong expansibility.

Because the flow cytometer has strict requirements on the technical indexes of the collecting lens, the existing collecting lens generally needs a targeted design and is difficult to be directly obtained in the market, and the collecting lens has poor compatible use performance and higher cost.

Therefore, how to provide a flow cytometer fluorescence collection lens which is simple and easy to obtain and has low cost is a problem to be solved urgently by those skilled in the art.

Disclosure of Invention

In view of this, the present invention provides a fluorescence collecting lens for a flow cytometer and a light path system thereof, where the lens realizes a larger numerical aperture by combining a plano-convex lens, a positive meniscus lens, a biconvex lens, and two double cemented lenses, has a good chromatic aberration correction capability, can output parallel light, is simple and easy to obtain, and is applicable to common flow cytometers.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the present invention provides a fluorescence collecting lens for a flow cytometer, comprising, along a propagation direction of an optical path: the lens comprises a planoconvex lens, a meniscus lens, a biconvex lens, a first biconvex lens and a second biconvex lens which are coaxially arranged in sequence;

the plane of plano-convex lens is the light incident plane, meniscus lens's concave surface is the light incident plane, biconvex lens goes up the curvature radius of light incident plane and is greater than the curvature radius of light exit face, the surface of first biconvex lens's positive lens is the light incident plane, the surface of first biconvex lens's negative lens is the light exit face, the surface of second biconvex lens's negative lens is the light incident plane, the surface of second biconvex lens's positive lens is the light exit face.

The present invention combines a plano-convex lens, a meniscus lens, a biconvex lens and two biconvex lenses. The requirements of larger numerical aperture, working distance larger than 2mm, chromatic aberration correction within the 400-820nm spectral range and the emergence requirement of small-size parallel beams are realized. The above requirements match well the application of flow cytometry to fluorescence collection lenses.

The plano-convex lens used in the present invention is one of convex lenses, and one surface of a light-passing surface thereof is a plane, and the other surface thereof is a convex surface, and may be a spherical surface or an aspherical surface. The plano-convex lens is made of H-K3 glass material, the refractive index of the H-K3 glass material is 1.50, and the dispersion rate of the H-K3 glass material is 64.78.

The meniscus lens used in the present invention has a concave light-passing surface and a convex light-passing surface, and both surfaces may be spherical or aspherical. The meniscus lens is made of ZF6 glass material, the refractive index of the ZF6 glass material is 1.76, and the dispersion ratio of the ZF6 glass material is 27.54.

The two light-passing surfaces of the biconvex lens used in the invention are convex surfaces, and the two surfaces can be spherical or aspherical lenses. The biconvex lens is made of an H-ZK3 glass material, the refractive index of the H-ZK3 glass material is 1.59, and the dispersion ratio of the H-ZK3 glass material is 61.25.

The two double cemented lenses used in the present invention are lenses cemented by a low dispersion positive lens (i.e., convex lens) and a high dispersion negative lens (i.e., concave lens), and the curvatures of the cemented surfaces of the positive and negative lenses are the same.

Specifically, the positive lens of the first cemented doublet is made of H-K9L material, the refractive index of the H-K9L material is 1.52, and the dispersion ratio is 64.21; the negative lens of the first cemented doublet is made of ZF2 glass material, the refractive index of the ZF2 glass material is 1.67, and the dispersion ratio is 32.22.

The negative lens of the second double cemented lens is made of H-ZF52A material, the refractive index of the H-ZF52A material is 1.85, and the dispersion ratio is 23.79; the positive lens of the second cemented doublet is made of H-ZBAF52A glass material, the refractive index of the H-ZBAF52A glass material is 1.67, and the dispersion ratio is 47.20.

Further, the air space between two adjacent lenses of the plano-convex lens, the meniscus lens, the double convex lens and the first double cemented lens is 0.5 mm.

Further, the air space between the first and second cemented doublets is 11.5 mm.

In another aspect, the present invention further provides a flow cytometer optical path system, which sequentially comprises, along an optical path propagation direction: the device comprises a laser light source, a flow chamber, the fluorescence collecting lens, a dichroic mirror, an optical filter and a focusing mirror;

the sample flow enters a detection area in the flow chamber, and is irradiated by a laser beam emitted by the laser light source in the detection area to generate fluorescence, the fluorescence enters the fluorescence collection lens, is effectively collected by the fluorescence collection lens and is emitted as parallel light, and the parallel light is subjected to spectrum selection through the dichroic mirror, is subjected to band selection through the optical filter and is converged into a fluorescence channel through the focusing mirror.

In the present invention, the working distance, i.e. the distance of the sample from the plane of the plano-convex lens, is set to 2.10 mm. The flow chambers used on the conventional flow cytometer can be compatible with each other.

Further, since there may be a plurality of fluorescent channels in the optical path, there may be a plurality of dichroic mirrors, color filters, and focusing mirrors, and the number of color filters and focusing mirrors used in the optical path is the same as that of the dichroic mirrors, and the plurality of dichroic mirrors, color filters, and focusing mirrors are correspondingly disposed. The spectrum of a certain wavelength range is reflected, then passes through a color filter and a focusing mirror, and a fluorescence channel is formed. The residual spectrum penetrates through the dichroic mirror, and other fluorescence channels can be formed under the selective action of other dichroic mirrors, so that the number of the fluorescence channels can be increased, and strong expansibility is realized.

According to the technical scheme, compared with the prior art, the invention discloses and provides the fluorescence collecting lens of the flow cytometer and the optical path system thereof, wherein the lens is formed by combining a plano-convex lens, a positive meniscus lens, a biconvex lens and two double cemented lenses, so that the larger numerical aperture is realized, and the fluorescence scattered light in the half-angle range of 45 degrees can be collected; the lens meets the requirement that the working distance is more than 2mm, and can be compatible with a flow chamber used on a common flow cytometer; the lens has good chromatic aberration correction capability in the wavelength range of 400-820nm, can output parallel light, has the beam diameter within 5mm, meets the use requirement that the dichroic mirror and the color filter are sensitive to the incident angle of the light beam, simultaneously realizes light beam transmission by the color filter and the dichroic mirror with smaller size, greatly saves the coating cost and the cost of instruments, is simple and easy to obtain, and can be applied to common flow cytometry.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic structural diagram of a fluorescence collecting lens of a flow cytometer according to the present invention;

fig. 2 is a schematic structural diagram of an optical path system of a flow cytometer provided in the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In one aspect, as shown in fig. 1, an embodiment of the present invention discloses a fluorescence collecting lens for a flow cytometer, where the lens includes, along a propagation direction of an optical path: a planoconvex lens 11, a meniscus lens 12, a biconvex lens 13, a first doublet cemented lens 14 and a second doublet cemented lens 15, and the planoconvex lens 11, the meniscus lens 12, the biconvex lens 13, the first doublet cemented lens 14 and the second doublet cemented lens 15 are coaxially arranged in sequence;

the plane of the plano-convex lens 11 is a light incident plane, the concave surface of the meniscus lens 12 is a light incident plane, the surface with a large curvature radius on the biconvex lens 13 is a light incident plane, the outer surface of the positive lens of the first cemented doublet 14 is a light incident plane, the outer surface of the negative lens of the first cemented doublet 14 is a light exit plane, the outer surface of the negative lens of the second cemented doublet 15 is a light incident plane, and the outer surface of the positive lens of the second cemented doublet 15 is a light exit plane.

In more detail, referring to fig. 1, the plano-convex lens 11 comprises a surface 111 and a surface 112; meniscus lens 12 comprises surface 121 and surface 122; the lenticular lens 13 comprises a surface 131 and a surface 132; first cemented doublet 14 comprises surface 141 and surface 142; the second doublet lens 15 includes a surface 151 and a surface 152.

In this embodiment, the plano-convex lens is made of H-K3 glass material (n is 1.50, V)_d64.78 where n represents the refractive index of the glass material and V_dRepresents the dispersion ratio of glass materials, and is two most basic parameters common to optical glass materials); the meniscus lens adopts ZF6 glass material (n is 1.76, V)_d27.54); the biconvex lens adopts H-ZK3 glass material (n is 1.59, V)_d61.25); the first cemented doublet uses two glass materials, of which the positive lens uses H-K9L material (n is 1.52, V)_d64.21), the negative lens adopts ZF2 glass material (n is 1.67, V)_d32.22); the second doublet lens uses two glass materials, wherein the negative lens uses H-ZF52A material (n is 1.85, V)_d23.79), the positive lens adopts H-ZBAF52A glass material (n is 1.67, V)_d＝47.20)。

Details of the lens parameters included in the fluorescence collection lens are described in detail below by way of specific examples, as shown in table 1 below:

TABLE 1 parameters of the lenses in the fluorescence Collection lens

As can be seen from the above table, the fluorescence collecting lens consists of a plano-convex lens, a positive meniscus lens, a biconvex lens and two biconvex lenses. In the planar lens 11, the curvature radius R1 of the surface 111 is ∞, the curvature radius R2 of the surface 112 is-4.049 mm, the thickness of the planar lens 11 is 4.25mm, the caliber is 8mm, and the material is H-K3 glass material.

The meniscus lens 12 is a positive meniscus lens, wherein the radius of curvature R1 of the surface 121 is-13.3 mm, the radius of curvature R2 of the surface 122 is-10.00 mm, the thickness of the positive meniscus lens is 2.5mm, the caliber is 13mm, and the material is ZF6 glass material.

In the lenticular lens 13, the curvature radius R1 of the surface 131 is 440.00mm, the curvature radius R2 of the surface 132 is-15.43 mm, the thickness of the lenticular lens 13 is 3mm, the caliber is 13mm, and the material is H-ZK3 glass material.

In the first cemented doublet 14, the radius of curvature R1 of the surface 141 is 10.47, the radius of curvature R2 of the cemented surface is-9.37 mm, the radius of curvature R3 of the surface 142 is-57.43 mm, the thickness is 8mm, the caliber is 13mm, and the positive lens and the negative lens are made of glass materials of H-K9L and ZF2, respectively.

In the second doublet 15, the curvature radius R1 of the surface 151 is-24.40 mm, the curvature radius R2 of the cemented surface is-5.20 mm, the curvature radius R3 of the surface 152 is-22.30, the thickness is 5mm, the caliber is 9mm, and the negative lens and the positive lens are made of H-ZF52A glass material and H-ZBAF52A glass material, respectively.

Specifically, the air space between two adjacent lenses, i.e., the plano-convex lens 11, the meniscus lens 12, the double convex lens 13, and the first cemented doublet 14, is 0.5mm in this embodiment. The air space between the first doublet lens 14 and the second doublet lens 15 was 11.5 mm.

On the other hand, referring to fig. 2, the embodiment of the present invention further discloses a flow cytometer optical path system, which sequentially includes, along the optical path propagation direction: a laser light source (not marked in the figure), a flow chamber 2, the fluorescence collecting lens 1, a dichroic mirror 3, a filter 4 and a focusing mirror 5, wherein the flow chamber 2 has a section of detection area;

when the sample flow passes through the detection area of the flow chamber 2, the laser beam emitted by the laser light source irradiates the detection area to generate fluorescence, the fluorescence enters the fluorescence collection lens 1, is effectively collected by the fluorescence collection lens 1 and is emitted as parallel light, the parallel light is subjected to spectrum selection through the dichroic mirror 3, and is converged into a fluorescence channel through the focusing mirror 5 after being subjected to band selection through the optical filter 4, so that the fluorescence can be received by the detector.

In summary, the fluorescence collecting lens disclosed in the embodiment of the present invention is simpler and more easily available, and the following effects are achieved by combining and optimizing the lenses:

1. a larger numerical aperture is achieved. This embodiment can collect fluorescence scattered light in the half angle 45 deg.. Since the environment in which fluorescence is generated by the flow cytometer is in a liquid environment (in general, the liquid contains water as a main component, and the refractive index n is 1.333), the calculation is performed according to the following numerical aperture calculation formula (1):

NA＝n×sinθ.........................(1)

in the formula, NA represents a numerical aperture, n represents a refractive index, and θ represents a collection angle (half angle).

The final calculation gave a numerical aperture of 0.94 in this example.

2. The requirement that the working distance is more than 2mm is met. In this embodiment, the distance (i.e. the working distance) between the sample and the plane of the plano-convex lens is 2.10mm, and all the flow chambers used in the conventional flow cytometer can be compatible with each other.

3. Has good chromatic aberration correction capability for the wavelength range of 400-820 nm. For five set wavelengths (0.488nm, 0.530nm, 0.585nm, 0.67nm and 0.780nm), a conventional N-BK7 material is adopted, a lens with an effective focal length of 12.7mm is used for focusing, and the radius of the obtained dispersed light spot can be controlled within 100 um;

4. in this embodiment, the lens outputs parallel light, and the beam diameter is within 5 mm. This satisfies the use requirements of dichroic mirrors and color filters (which are sensitive to the angle of incidence of the light beam). Meanwhile, the color filter and the dichroic mirror realize light beam light transmission by using smaller size, and the film coating cost and the instrument cost are greatly saved.

5. The objective lens of the microscope on the market is most similar to the lens of the invention, but the objective lens of the microscope with large numerical aperture can not be applied to the flow cytometer, and the main reasons are two points: firstly, the microscope objective is characterized in that the working distance and the numerical aperture have an inverse relation, the numerical aperture can be 0.9 or more, the working distance is basically within 0.5mm, and part of the microscope objective needs to be immersed in oil during use, is complicated to use and is not suitable for being applied to a flow cytometer; secondly, the chromatic aberration correction range of the microscope objective is 400-700 nm, and the wavelength range of the flow cytometer cannot be covered. Therefore, it should be emphasized that the lens described in the embodiments of the present invention is not directly improved from the lens on the market.

6. All the lens materials used in the embodiment of the invention are common optical glass and are very easy to obtain. Meanwhile, the lenses are all common spherical lenses and are easy to process.

In summary, the lens disclosed in the embodiments of the present invention has the advantages of simplicity, easy availability, and low price, and also has the advantages of being widely used in a flow cytometer and being unable to be replaced.

Meanwhile, the fluorescence collecting lens disclosed by the embodiment of the invention is used on a flow cytometer in a targeted manner, and is possibly applicable to other application fields with large numerical apertures.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A flow cytometer fluorescence collection lens, comprising, in the direction of propagation of an optical path: the lens comprises a planoconvex lens, a meniscus lens, a biconvex lens, a first biconvex lens and a second biconvex lens which are coaxially arranged in sequence;

2. The fluorescence collecting lens of flow cytometer as described in claim 1, wherein said plano-convex lens is made of H-K3 glass material, said H-K3 glass material has a refractive index of 1.50, and said H-K3 glass material has an abbe number of 64.78.

3. The fluorescence collection lens of the flow cytometer as described in claim 1, wherein said meniscus lens is made of ZF6 glass material, said ZF6 glass material has refractive index of 1.76, and said ZF6 glass material has dispersion ratio of 27.54.

4. A flow cytometer fluorescence collection lens as described in claim 1, wherein said lenticular lens is made of H-ZK3 glass material, said H-ZK3 glass material has a refractive index of 1.59, and said H-ZK3 glass material has an abbe number of 61.25.

5. The fluorescence collecting lens for flow cytometer as described in claim 1, wherein said positive lens of said first cemented doublet is made of H-K9L material, said H-K9L material has refractive index of 1.52 and dispersion ratio of 64.21;

the negative lens of the first cemented doublet is made of ZF2 glass material, the refractive index of the ZF2 glass material is 1.67, and the dispersion ratio is 32.22.

6. A flow cytometer fluorescence collection lens as described in claim 1, wherein the negative lens of said second doublet is made of H-ZF52A material, said H-ZF52A material has refractive index of 1.85 and dispersion ratio of 23.79;

the positive lens of the second cemented doublet is made of H-ZBAF52A glass material, the refractive index of the H-ZBAF52A glass material is 1.67, and the dispersion ratio is 47.20.

7. The fluorescence collection lens of flow cytometer as described in claim 1, wherein the air gap between two adjacent lenses of said plano-convex lens, meniscus lens, biconvex lens and first biconvex lens is 0.5 mm.

8. A flow cytometer fluorescence collection lens as described in claim 1, wherein the air space between said first cemented doublet and said second cemented doublet is 11.5 mm.

9. A flow cytometer optical path system comprising, in order along an optical path propagation direction: a laser light source, a flow cell, the fluorescence collection lens of any of claims 1-8, a dichroic mirror, a filter, and a focusing mirror;

10. A flow cytometer optical path system as described in claim 9, wherein said dichroic mirror, color filter and focusing mirror are provided in plural and same number, and said dichroic mirror, color filter and focusing mirror are correspondingly provided.