WO2017173896A1 - 流式细胞术检测装置和方法 - Google Patents

流式细胞术检测装置和方法 Download PDF

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WO2017173896A1
WO2017173896A1 PCT/CN2017/074626 CN2017074626W WO2017173896A1 WO 2017173896 A1 WO2017173896 A1 WO 2017173896A1 CN 2017074626 W CN2017074626 W CN 2017074626W WO 2017173896 A1 WO2017173896 A1 WO 2017173896A1
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spots
flow cytometry
light
spot
detection
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PCT/CN2017/074626
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English (en)
French (fr)
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尤政
赵精晶
李滨
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清华大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/09—Beam shaping, e.g. changing the cross-sectional area, not otherwise provided for
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/09—Beam shaping, e.g. changing the cross-sectional area, not otherwise provided for
    • G02B27/0938—Using specific optical elements

Definitions

  • the present invention relates to the field of micro-optical detection technology, in particular, the invention relates to a flow cytometry detection device, a flow cytometry detection method and a binary optical device for use in a flow cytometry detection or preparation flow cytometry detection device .
  • Flow cytometry can be used to detect biological particles such as fluorescently labeled cells, embryos, RNA, DNA, protein particles, microorganisms, viruses, and the like.
  • flow cytometry needs to be irradiated on the biological particles located at the center of the liquid stream by means of a shaped laser spot, and the biological particles emit scattered light and fluorescence under the excitation of the laser spot.
  • These scattered light and fluorescent signals are collected by photoelectric conversion devices such as photomultiplier tubes and photodiodes, and then analyzed by a detection system to obtain physical and chemical information of biological particles, such as cell size, surface particle size, and antigen information.
  • the shape and quality of the laser spot are the key factors affecting the sensitivity of detection and the resolution of individual particles.
  • commercial flow cytometry generally uses two cylindrical lenses to shape a Gaussian laser into an elliptical Gaussian spot.
  • the short axis of the elliptical spot is parallel to the flow direction of the biological particle, and the long axis is perpendicular to the flow direction of the biological particle to improve the single The resolution of biological particles, while preventing the detection of biological particles.
  • the stability of commercial flow cytometry detection and the resolution of individual biological particles are difficult to further improve, it is difficult to achieve high-speed and high-throughput detection, and the speed measurement requirements for biological particles cannot be met.
  • the inventors found through research that the key to the above problems lies in the uneven distribution of light energy in the elliptical spot of commercial flow cytometry. When the relative position of the biological particles changes through the spot, the optical signal of the output will change accordingly. And when the elliptical spot is Gaussian, the boundary of the spot is not clear, and at the same time, the single spot cannot meet the real-time speed measurement requirement for each biological particle.
  • the present invention aims to solve at least one of the technical problems in the related art described above to some extent. Therefore, the present invention proposes to realize spot shaping by using a binary optical device having a surface microstructure, thereby realizing a spot on the output plane with the following characteristics: the spot boundary is sharp, the light energy in the spot is uniformly distributed, and the biological particle can be realized.
  • High throughput assay The spot is composed of more than two patterns, which can realize the real-time measurement of the speed of each biological particle; the spot can be composed of a plurality of different size patterns, which can balance single particle resolution and weak signal detection.
  • the invention provides a flow cytometry detecting device.
  • a flow cytometry detecting device comprising: a spot forming assembly configured to form at least two spots on a flow line centerline.
  • the flow cytometry detecting device proposed by the embodiment of the invention can accurately measure the velocity of the biological particles, and the single particle resolution and the detection force for the weak signal are strong.
  • the above flow cytometry detecting apparatus may further include at least one of the following additional technical features:
  • the spot forming component is a binary optic.
  • the binary optical device further improves the detection stability, single particle resolution, and weak signal detection capability of the flow cytometry detecting apparatus of the embodiment of the present invention.
  • the at least two spots are each independently rectangular.
  • the rectangular spot further improves the detection stability, single particle resolution, and high speed and high throughput detection efficiency of the flow cytometry detecting apparatus of the embodiment of the present invention.
  • the at least two spots are spaced apart and the central axis of the rectangle is located on the centerline of the flow channel.
  • the arrangement of the above rectangular spot causes the detection sensitivity of the cytometry detecting device to be further improved.
  • the lengths of the long sides of the at least two spots are all 10 to 100 micrometers, and the lengths of the short sides of the spots of the at least two spots are determined based on the size of the sample to be tested.
  • the long side length of the rectangular spot is 10 to 100 microns to minimize the missed detection of biological particle samples.
  • the short side length of the spot at both ends is determined based on the size of the sample to be tested, which further improves the resolution of a single biological particle sample.
  • the two end spots of the at least two spots respectively have the same size, and the long sides of the at least two spots are of equal length. Furthermore, the flow cytometry apparatus of the embodiment of the present invention can improve the accuracy of the test by data processing methods such as weighted averaging in data processing.
  • the amplitude variance of the at least two spots is less than 10%, and the light intensity of the spots is equal. Furthermore, the flow cytometry apparatus of the embodiment of the present invention can improve the accuracy of the test by data processing methods such as weighted averaging in data processing.
  • the uniformity of the light intensity of at least two spots is higher than 80%, and the light intensity distribution is uniform, which further improves the accuracy and sensitivity of the flow cytometry detecting device.
  • the flow cytometry detecting device comprises: an optical path along the incident laser, sequentially provided with a laser, a beam expander, the spot forming assembly, the first aperture stop and the first focus lens Between the optical path of the forward scattered laser, a light blocking plate, a first filter and a first photoelectric sensor are sequentially disposed; and along the optical path of the side scattered laser, a second filter and a second photoelectric sensor are sequentially disposed a second focus is sequentially disposed along the optical path of the excited fluorescence a lens and a multiplex fluorescent detecting device, wherein the multiplex fluorescent detecting device comprises a dichroic mirror, a second small aperture diaphragm, a third filter, and a third photoelectric sensor; and sequentially arranged along the observation optical path An image light sensor, a fourth filter, and a third focus lens.
  • the flow cytometry detecting device of the embodiment of the invention is simple in operation, and the stability, accuracy, single particle resolution and high-speed and high-flux detection efficiency of
  • the invention proposes a flow cytometry detection method.
  • the detecting method comprises: forming at least two spots on the center line of the flow channel, wherein the at least two spots are as defined above.
  • the flow cytometry detection method proposed by the embodiment of the invention can accurately determine the speed of the biological particle sample to be tested, and the stability of the detection result is high, and the single particle resolution and the detection of the weak signal can be considered.
  • the above flow cytometry detecting method may further include at least one of the following additional technical features:
  • the method is carried out using the flow cytometry detection device described above.
  • the flow cytometry detecting apparatus of the embodiment of the present invention has the characteristics of high stability of detection results, strong resolution to single particles, and strong detection capability for weak signals, and the detection method of the embodiment of the present invention is adopted.
  • the flow cytometry detecting device of the embodiment of the invention detects the sample of the biological particle to be tested, and the stability of the detection result, the resolution of the single particle and the detection capability of the weak signal are further improved.
  • the invention provides the use of a binary optic device for flow cytometry detection or preparation of a flow cytometry detection device.
  • a binary optical device is used in flow cytometry detection or preparation of a flow cytometry detection device, which can accurately measure the velocity of biological particles and has high stability against detection results of biological particles. The resolution of single particles and the ability to detect weak signals are high.
  • the use of the above binary optical device in the flow cytometry detection or preparation of the flow cytometry detection device may further include at least one of the following additional technical features:
  • the binary optics modulate the phase of the incident laser light.
  • the accuracy of the velocity measurement of the biological particle is further improved, and the stability of the detection result of the biological particle, the resolution of the single particle, and the detection capability of the weak signal are further improved.
  • the material of the binary optical device is selected from at least one of quartz, glass, and polymethyl methacrylate.
  • the selection of binary optics by selecting at least one of the above materials further enhances the stability and sensitivity of the flow cytometry assay or flow cytometry assay device.
  • the laser light comprises at least one selected from the group consisting of Gaussian light, planar light, and multimode light.
  • the stability and sensitivity of the flow cytometry or flow cytometry detection device can be further significantly improved by selecting a suitable incident laser and its corresponding binary optics.
  • the structure of the binary optical device is a square having a side length of 3 mm and an inside having 128*128 steps.
  • the above embodiments of the present invention The structure of the binary optics further enhances the stability of the flow cytometry assay or flow cytometry assay device and the resolution of single particles and the ability to detect weak signals.
  • FIG. 1 is a schematic view of a double rectangular spot according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing the positional relationship between a double-rectangular spot, a biological particle, and a flow path when subjected to flow cytometry according to an embodiment of the present invention
  • FIG. 3 is a schematic diagram of an optical signal emitted by biological particles as they pass through a double rectangular spot, in accordance with an embodiment of the present invention
  • FIG. 4 is a phase diagram of a specific binary optical device in accordance with an embodiment of the present invention.
  • FIG. 5 is a diagram showing an output light distribution obtained by using a binary optical device according to an embodiment of the present invention.
  • Figure 6 is a schematic illustration of a three rectangular spot in accordance with an embodiment of the present invention.
  • Figure 7 is a schematic illustration of the positional relationship between three rectangular spots, biological particles and flow channels in accordance with an embodiment of the present invention.
  • FIG. 8 is a schematic diagram of an optical signal emitted by a biological particle as it passes through a three-rectangular spot, in accordance with an embodiment of the present invention
  • FIG. 9 is a phase diagram of a specific binary optical device in accordance with an embodiment of the present invention.
  • FIG. 11 is a light path diagram of a flow cytometric device according to an embodiment of the present invention.
  • Figure 12 is a schematic diagram showing the structure of a flow cytometry detecting apparatus according to an embodiment of the present invention.
  • the invention provides a flow cytometry detection device.
  • a spot forming assembly configured to form at least two spots on a flow line centerline. Refer to Figure 1 for the location of the centerline of the runner.
  • the total time of irradiation of the biological particles by the at least two spots of the flow cytometry device in the embodiment of the present invention is increased, and the irradiation time of the single spot to the biological particles may be unchanged or even shortened compared with the prior art.
  • the at least two spots formed by the spot forming assembly are relatively fixed in position, and the flow cytometry detecting device proposed in the embodiment of the present invention can accurately measure the velocity of the biological particles.
  • the spot forming assembly is a binary optic.
  • the microstructure of the binary optics can change the optical path at each point of the incident laser, and a specific optical diffraction occurs, thereby forming at least two on the output plane. Spots.
  • the light intensity inside the spot formed by the binary optics is evenly distributed. Therefore, even if the biological particles are shifted by the relative position of the spot, the irradiation condition of the particles does not change significantly, and the optical signal of the output remains substantially unchanged.
  • the stability of the detection reduces the coefficient of variation of the detection signal; the boundary of the spot is sharp, so that the optical signal of the output has a clear boundary, which helps to identify the light signal emitted by the biological particle; the light intensity portion inside the spot is evenly distributed.
  • the amplitude of the output light signal when the biological particles pass through the rectangular spot is substantially constant, which facilitates calculation of the optical signal, such as calculating the height, width or area of the optical signal.
  • the intermediate spot is used to increase the irradiation time of the biological particles and prolong the duration of the optical signal, thereby facilitating collection, detection and analysis of the optical signals, improving the detection sensitivity, and particularly contributing to the realization of the weak Effective detection of optical signals.
  • the spot forming assembly is a binary optical device which further improves the detection stability, single particle resolution, and weak signal detection capability of the flow cytometry detecting apparatus of the embodiment of the present invention.
  • the at least two spots are each independently rectangular.
  • the energy distribution in the rectangular spot is uniform and the boundary is sharp, and thus the optical signal of the output of the biological particle does not change when the relative position of the spot changes, so that the rectangular spot makes the embodiment of the present invention
  • the detection stability, single particle resolution, and high-speed and high-throughput detection efficiency of the flow cytometry detection device are further improved.
  • the elliptical spot formed by the lens has a Gaussian light intensity, and the boundary continuously changes and is not sharp. Therefore, the excited fluorescent signal has a significant difference due to the position of the biological particles such as cells, and the signal Approximate Gaussian shape is not conducive to distinguishing the signals of biological particles such as single cells, reducing sensitivity and resolution.
  • At least two rectangular spots are spaced apart and the central axis of the rectangle is located on the center line of the flow channel.
  • the central axis of the rectangle is perpendicular to the long side of the rectangle and parallel to the short side of the rectangle.
  • the sample stream containing biological particles such as cells in the embodiment of the present invention is focused at the center of the flow channel, and the flow velocity of the liquid flow is highest at the center position of the flow channel, so that the central axis of the spot coincides with the center line of the flow channel to enable a single cell to pass through the spot.
  • the time is shortened, and the cell flux can be increased by passing more cells per unit time.
  • the long side of the rectangular spot is perpendicular to the central axis of the rectangle, that is, perpendicular to the center line of the flow channel, that is, perpendicular to the flow direction of the cell, so that when the cell deviates from the center line position of the flow channel, it can still be irradiated by the spot to achieve effective detection. Furthermore, the central axis of the rectangular spot of the embodiment of the present invention is located on the center line of the flow path, so that the detection flux and sensitivity of the flow cytometry detecting device are further improved.
  • At least two rectangular spots have a long side length of 10 to 100 micrometers, and a short side length of the two ends of the at least two rectangular spots is determined based on a size of the sample to be tested.
  • the two ends of the light spot means that when there are two light spots, the light spots at both ends are as shown in FIGS. 11 and 12 of the first embodiment, and when there are two or more light spots, such as three light spots, the light spots at both ends are as shown in FIG. 52 is shown. Meanwhile, when there are more than two rectangular spots, such as three spots, as shown in FIG. 6, 53 is called an intermediate spot.
  • the long sides of the rectangular spots at both ends refer to the sides having a slightly longer length
  • the long sides of the intermediate rectangular spots refer to the sides perpendicular to the center line of the flow path.
  • the long side length of the rectangular spot is 10 to 100 microns to minimize the missed detection of biological particle samples.
  • the length of the short side of the spot at both ends is determined based on the size of the sample to be tested, and the present invention
  • the short side length of the spot at both ends of the device of the example is close to the average of the size of the sample to be tested, thereby further improving the resolution of the single biological particle sample.
  • the two end spots of the at least two spots respectively have the same size, and the long sides of the at least two spots are of equal length.
  • the light intensity of the two ends of the light spot is equal, and the amplitude of the light signal generated by the biological particles passing through the at least two light spots is substantially equal (it is to be noted that the light intensity of the light spots described in the present specification is equal to mean equal, and Not exactly equal. Because binary optics can only be completely equal in theory, it does not exist in practice).
  • the accuracy of the test can be improved by a data processing method such as weighted averaging.
  • the amplitude mean deviation of at least two spots is less than 10%.
  • the amplitude of the at least two spots is less than 10%, and the light intensity of the spot is equal, the amplitude of the optical signal generated by the biological particle to be tested after passing at least two spots is substantially equal, in data processing.
  • the accuracy of the test can be improved by a data processing method such as weighted averaging.
  • the uniformity of the light intensity of at least two spots is higher than 80%, and the light intensity distribution is uniform, so that when the biological particles pass through at least two spots, the biological particles are excited by the laser even if the relative position changes. There is no significant difference, the optical signal of the output will not change, which reduces the signal difference caused by the position factor, and the rising and falling edges of the generated optical signal are more steep and the top is more flat, which is beneficial to the
  • the identification and processing of the optical signal further improves the stability, accuracy and sensitivity of the flow cytometry detection device.
  • the flow cytometry detecting apparatus includes: along the optical path of the incident laser, a laser 100, a beam expander 200, and the spot forming assembly 300 are sequentially disposed.
  • the first aperture lens 1400 and the first focusing lens 400, the laser light emitted by the laser 100 forms at least two spots on the center line of the flow channel 10 through the spot forming assembly 300, wherein the flow channel 10 may be a transparent micro flow channel. It may also be present in the air, wherein the aperture stop 1400 is used to remove the secondary of the spot forming assembly 300 beyond the main stage of the diffracted spot.
  • the sample When a sample (cell or other biological particle to be tested) passes through the spot along the centerline of the flow channel 10, the sample is excited to emit fluorescence, forward scattered light (forward scattered light) and side scattered light, along the optical path of the forward scattered laser.
  • the light blocking plate 1800, the first filter 500 and the first photoelectric sensor 600 are disposed in sequence, and the light blocking plate 1800 is located at the center of the optical path of the forward scattered light, and has a small area, which can block the signal directly from the laser and ensure the incident to the photoelectric
  • the signal of the sensor 600 is only a scattered light signal
  • the first filter 500 can make the signal incident on the photosensor 600 only a laser signal
  • the first photosensor 600 is used to convert the forward scattered light signal into an electrical signal for light.
  • the dichroic mirror 900 is used to separate the multi-color fluorescence into a monochromatic fluorescence, that is, the separation of the fluorescent signals of each color requires a dichroic mirror 900 to separate, and then the fluorescence of each color passes through the second.
  • a small aperture stop 1500, a third filter 1000 and a third photosensor 1100 where the dichroic mirror 900, the second aperture aperture 1500, the third filter 1000 and the third photosensor 1100 are series Dichroic mirror, series second small aperture diaphragm, series third filter and series third photoelectric sensor (refer to Figure 12), wherein the second small aperture stop 1500 is used to remove stray light, third
  • the filter 1000 is used to retain a fluorescent signal of a specific color
  • the third photosensor 1100 is used to convert a fluorescent signal of a specific color into an electrical signal, thereby performing optical signal analysis, thereby obtaining sample information.
  • photosensors include, but are not limited to, photodiodes, photomultiplier tubes, photodiode arrays, and multiplier arrays, wherein the scattered light multi-purpose photodiodes, fluorescent multi-purpose photomultiplier tubes or photodiode arrays or multiplier arrays.
  • an image sensor 1200, a fourth filter 1600 and a third focus lens 1300 are sequentially disposed, and the image sensor 1200 is used for real-time observation and monitoring of the sample in the flow path to facilitate the experimental operation, and the fourth filter
  • the mirror 1600 is used to remove interference from the laser signal.
  • the invention proposes a flow cytometry detection method.
  • the detection method comprises: forming at least two spots on the centerline of the flow channel, wherein the at least two spots are as defined above.
  • the flow cytometry detection method proposed by the embodiment of the invention can accurately determine the speed of the biological particle sample to be tested, and the stability of the detection result is high, and the single particle resolution and the detection of the weak signal can be considered.
  • the method is carried out using the flow cytometry detecting device described above.
  • the flow cytometry detecting apparatus of the embodiment of the present invention has the characteristics of high stability of detection results, strong resolution to single particles, and strong detection capability for weak signals, and the detection method of the embodiment of the present invention is adopted.
  • the flow cytometry detecting device of the embodiment of the invention detects the sample of the biological particle to be tested, and the stability of the detection result, the resolution of the single particle and the detection capability of the weak signal are further improved.
  • the invention provides the use of a binary optic device for flow cytometry detection or preparation of a flow cytometry detection device.
  • the binary optical device can design a corresponding binary optical device for different incident lasers according to the mode of the incident laser light, the wavelength of the laser light, and the diameter to be applied to the flow cytometry detection or preparation flow.
  • the cytometry detecting device at least two spots are formed on the center line of the flow channel, and the binary optical device is applied to the flow cytometry detection or the preparation of the flow cytometry detecting device, and the detecting method or device can accurately measure the biological particles.
  • the speed of the detection of biological particles is high, and the resolution of single particles and the detection of weak signals are high.
  • the binary optical device modulates the phase of the incident laser light, thereby forming at least two spots on the center line of the flow channel, the distance between the at least two spots is constant, the boundary is sharp, and the internal light intensity distribution Uniform, can take into account the single-particle resolution and detection of weak signals, and then use binary optics to modulate the phase of the incident laser, further improve the accuracy of the measurement of biological particles, and the stability of the detection results of biological particles. The resolution of single particles and the ability to detect weak signals are further improved.
  • the material of the binary optical device is at least one selected from the group consisting of quartz, glass, and polymethyl methacrylate.
  • the material selection of the binary optics is selected for different incident lasers.
  • the material of the binary optical device of the embodiment of the invention is selected from, but not limited to, quartz, glass, polymethyl methacrylate, as long as it can be in the flow channel. At least two spots are formed on the center line, and the at least two spots are again as described above.
  • the selection of the most suitable material for the preparation of binary optics further enhances the stability and sensitivity of the flow cytometry assay or flow cytometry assay device.
  • the laser of the embodiment of the present invention includes, but is not limited to, Gaussian light, planar light, and multimode light
  • the binary optical device according to the embodiment of the present invention is designed according to different incident lasers, and then flows. At least two spots required for the experiment are formed on the midline.
  • the stability and sensitivity of the flow cytometry or flow cytometry detection device can be further significantly improved by selecting a suitable incident laser and its corresponding binary optics.
  • the structure of the binary optical device when the laser light is Gaussian light having a diameter of 3 mm, the structure of the binary optical device is a square sheet having a side length of 3 mm and an internal 128*128 step. .
  • the structure of the above specific binary optical device of the embodiment of the present invention may form two or three rectangular spots on the center line of the flow channel, and the rectangular spot has the characteristics of at least one of the aforementioned spots, and the embodiment of the present invention
  • the structure of the above binary optical device further enhances the stability of the flow cytometry detection method or the flow cytometry detection device and the resolution of single particles and the ability to detect weak signals.
  • the inventors use binary optics to form two rectangular spots on the centerline of the flow channel.
  • the following is a description of the features and design of the rectangular spot:
  • the double rectangular spot 1 As shown in Fig. 1, the double rectangular spot 1, the two rectangular spots 11 and 12 are separated by a certain distance L, and the lengths of the long sides and the short sides of the two rectangular spots are l1 and w1, l2 and w2, respectively, and the light intensity in the spot is uniform. distributed.
  • the spot 1 is located at the center of the flow channel, that is, the center line of the long side of the rectangular spot and the center line 43 of the flow path coincide.
  • the flow center line 43 is located at the center of the flow path boundaries 41 and 42;
  • the flow direction 3 of the biological particles 2 is parallel to the flow path center line 43 and is as close as possible to the center line.
  • the biological particles are in the range of more than ten micrometers near the center line of the flow channel. Therefore, the inventors take two rectangular spots, and the long sides l1 and l2 are several tens of micrometers to prevent the leakage of biological particles. To accurately distinguish individual biological particles, the inventors adjusted the short sides w1 and w2 of the two rectangular spots to approximate the feature size of a single biological particle.
  • the optical signal emitted by the biological particles as they pass through the double rectangular spot is as shown in FIG. 3.
  • the optical signal herein may refer to a forward scattered light signal, a side scattered light signal or a fluorescent signal of a certain path, and the time interval of the two optical signals is T, amplitudes H1 and H2, durations T1 and T2, and signal versus time integrals are A1 and A2 (ie, area).
  • T time interval of the two optical signals
  • amplitudes H1 and H2 durations T1 and T2
  • signal versus time integrals are A1 and A2 (ie, area).
  • the rising and falling edges of the optical signal generated by the biological particles passing through the spot are more steep and the top is more flat, which is advantageous for identifying and processing the optical signal.
  • the amplitude and area of the optical signal are used to characterize the intensity of the optical signal, wherein the intensity of the fluorescent signal is positively correlated with the amount of surface antigen of the biological particle, and the intensity of the forward scattered light is positively correlated with the size of the biological particle, and the intensity of the side scattered light It is related to the self-factor of biological particles such as surface particle size, internal structure, and optical properties of biological particles.
  • the inventors chose two rectangular spots to be equal in size and equal in intensity. In this way, the optical signals generated by the two biological patches are substantially equal.
  • the accuracy of the test can be improved by using an equal data processing method, for example, the average value of the signals is (H1+H2)/2 , (T1+T2)/2 and (A1+A2)/2.
  • the inventor uses blood cells as biological particles to be examined, and specifically describes the parameters and design of the double rectangular spot:
  • the blood mainly contains red blood cells, white blood cells and platelets.
  • the red blood cells are concave dish-shaped, 7-8 micrometers in diameter, 2 to 2.5 micrometers thick, and the white blood cells are 10-15 micrometers in diameter.
  • the platelets are convex dish-shaped and 2 to 3 micrometers in diameter.
  • the inventors designed the short axis length of the rectangular spot to be 10 micrometers.
  • the biological particles are in the range of a few ten micrometers near the center line of the flow channel, and thus the inventors set the length of the long side of the rectangular spot to 50 ⁇ m to prevent missed detection of biological particles.
  • the flow rate of the biological particles is usually 3 to 10 m/s, and the inventor has designed the spacing of the two rectangular spots to be 100 ⁇ m to ensure that the time interval between the blood cells passing through the two rectangular spots is large, thereby improving the measurement of the blood flow rate. accuracy.
  • the time interval between two optical signals of the same channel from a blood cell through two spots is 2 microseconds, and the resulting flow rate is 5 m/s.
  • the inventor uses a binary optic device to modulate the phase of the incident laser light, that is, to change the optical path at each point of the incident laser light by using a micro-stepped structure on the surface of the binary optical device, and the modulated laser light passes through the lens and The desired spot is achieved on its focal plane.
  • the binary optics are made of transparent optical materials, such as quartz, glass, PMMA, etc., and different height steps are formed on the surface of the optical material by etching, focusing plasma processing or the like.
  • the incident laser can be Gaussian light, planar light or other mode laser, or even multimode light, laser wavelength, diameter and other parameters Variable, for different incident lasers, the corresponding binary optics can be designed.
  • the binary optical device in order to realize the above-mentioned spot formed by two rectangular spots, when a Gaussian light having a diameter of 3 mm is selected as the input laser, the binary optical device can be prepared into a square piece having a side length of 3 mm, and the inside thereof is divided into 128 ⁇ 128. Steps, the phase diagram of this binary optics is shown in Figure 4. The inventor further selected the focal length of the lens after the binary optics to be 10 mm, and the output light distribution on the focal plane is as shown in Fig. 5. The pure black area in the figure indicates that the light intensity is zero, and the pure white area indicates that the light intensity is the largest. The gray representation is somewhere in between. The inventors have found that the size of the output spot of the above binary optical device is consistent with the design, has a clear boundary, and the amplitude within the spot has a mean square error of less than 10% and a high degree of uniformity.
  • the inventors use binary optics to form three rectangular spots on the centerline of the flow channel.
  • the following is a description of the features and design of the rectangular spot:
  • the spot 5 formed by three rectangles is composed of two rectangular spots 51 and 52 spaced apart by a distance L, and a rectangular spot 53 located therebetween, and the intermediate rectangular spot is in the flow direction of the biological particles.
  • the size is larger, and the dimensions of the three rectangles are w1 ⁇ l1, w2 ⁇ l2, and w3 ⁇ l3, respectively, and the light intensity in the spot is evenly distributed.
  • the spot is located at the center of the flow, and the common line of the three rectangles coincides with the center line 43 of the flow path, and the flow
  • the track center line 43 is located at the center of the flow path boundaries 41 and 42; the flow direction 3 of the biological particles 2 is parallel to the flow path center line 43 and is as close as possible to the center line.
  • the rectangular spots 51 and 52 at both ends are used to distinguish and measure the individual biological particles, and the wide rectangle 53 in the middle is used to increase the irradiation time of the biological particles, prolong the duration of the optical signal, and facilitate the collection of the optical signals.
  • the biological particles are in the range of more than ten micrometers near the center line of the flow channel, and the inventors have adjusted the lengths of the long sides l1, l2, and l3 of the rectangular spot to several tens of micrometers to prevent missed detection of biological particles.
  • the inventors adjusted the short sides w1 and w2 of the rectangular spots 51 and 52 to approximate the feature size of a single biological particle.
  • the optical signal emitted by the biological particles as they pass through the three rectangular spots is as shown in Fig. 8.
  • the optical signals may refer to forward scattered light signals, side scattered light signals or fluorescent signals of a certain path, through rectangular spots 51 and 52 at both ends.
  • the time interval is T
  • the optical signal amplitudes are H1, H2, and H3
  • the durations are T1, T2, and T3
  • the integration of the signals with time is A1, A2, and A3 (ie, area).
  • the rising and falling edges of the optical signal generated by the biological particle passing through the spot are more steep and the top is more flat, which is advantageous for identifying and processing the optical signal.
  • the flow velocity V L / T of the biological particles is obtained by calculating the time interval T of the biological particles passing through the rectangular spots 51 and 52 at both ends.
  • the area of the optical signal i.e., the time integral of the optical signal
  • the amplitude and area of the optical signal are used to characterize the intensity of the optical signal, where the intensity of the fluorescent signal is positively correlated with the amount of surface antigen of the biological particle,
  • the intensity of the scattered light is positively correlated with the size of the biological particles, and the intensity of the side scattered light is related to the self-factor of the biological particles such as the surface particle size, internal structure, and optical characteristics of the biological particles.
  • the inventors designed the light intensity of the three spots to be equal.
  • the three rectangular spots are equal to the side lengths l1, l2 and l3 perpendicular to the flow direction, and the short sides w1 and w2 of the rectangular spots 51 and 52 are equal.
  • the amplitude of the optical signals passing through the three rectangular spots of the biological particles is substantially equal, so that the accuracy of the test can be improved by data processing such as weighted averaging in data processing.
  • the weighted average of the amplitude of the optical signal is (H1 ⁇ w1 + H2 ⁇ w2 + H3 ⁇ w3) / (w1 + w2 + w3) or (H1 ⁇ T1 + H2 ⁇ T2 + H3 ⁇ T3) / (T1 + T2+T3), and signal mean (H1+H2)/2, (H1+H2+H3)/3, (T1+T2)/2 and (A1+A2)/2.
  • the inventor uses blood cells as biological particles to be examined, and specifically describes the parameters and design of the three rectangular spots:
  • the blood mainly contains red blood cells, white blood cells and platelets.
  • the red blood cells are concave dish-shaped, 7-8 micrometers in diameter, 2 to 2.5 micrometers thick, and the white blood cells are 10-15 micrometers in diameter.
  • the platelets are convex discs and the diameter is 2-3 micrometers. .
  • the inventors adjusted the lengths of the short sides w1 and w2 of the rectangular spots 51 and 52 at both ends to 10 ⁇ m.
  • the inventors adjusted the side length w3 of the intermediate rectangular spot 53 in the flow direction of the biological particles to 50 ⁇ m.
  • the biological particles are in the region of a few micrometers near the center line of the flow channel, and the inventors adjusted the lengths of the three rectangular spots perpendicular to the flow direction side lengths l1, l2, and l3 to 50 ⁇ m to prevent missed detection of biological particles.
  • the flow rate of the biological particle flow is usually 3 to 10 m/s, and the inventor adjusts the spacing of the two narrow rectangular spots at both ends to 100 micrometers to ensure that the time interval of the two rectangular spots of the blood cell passer is sufficiently large, thereby improving the pair.
  • the accuracy of blood cell flow rate measurement For example, the time interval of the two optical signals of the same channel emitted by the biological particles through the two spots 51 and 52 at both ends is 2 microseconds, and the flow rate is 5 m/s.
  • the inventor uses a binary optic device to modulate the phase of the incident laser light, that is, to change the optical path at each point of the incident laser light by using a micro-stepped structure on the surface of the binary optical device, and the modulated laser light passes through the lens and The desired spot is achieved on its focal plane.
  • the binary optics can be made of transparent optical materials, such as quartz, glass, PMMA, etc., and different height steps are formed on the surface of the optical material by etching, focusing plasma processing or the like.
  • the incident laser can be Gaussian light, planar light or other mode laser, or even multimode light. The wavelength and diameter of the laser can be varied. For different incident lasers, corresponding binary optics can be designed.
  • the binary optical device in order to realize the above-mentioned spot formed by three rectangular spots, when Gaussian light having a diameter of 3 mm is selected as the input laser, the binary optical device can be made into a square piece having a side length of 3 mm, and the inside thereof is divided into 128. ⁇ 128 steps, the phase diagram of the corresponding binary optics is shown in Figure 9.
  • the inventor chose the focal length of the lens after the binary optics to be 10 mm, and the output light distribution on the focal plane is as shown in Fig. 10.
  • the pure black area in the figure indicates that the light intensity is zero, and the pure white area indicates the maximum light intensity, gray.
  • the representation is somewhere in between; the inventors found that the size of the output spot of the above binary optics Consistent with the design, with clear boundaries, the amplitude within the spot is less than 15%, with a high degree of uniformity.
  • the inventors have described the optical path of the flow cytometry device in detail:
  • the laser beam 61 is expanded by the beam expander 200 to reach the design aperture of the binary optical device.
  • the laser beam is shaped by the spot forming assembly 300 (this embodiment is a binary optical device) and then passed through the aperture 1400.
  • the 400 is focused at the center of the flow path 10, and the biological particles at the center of the flow path are irradiated, and the above constitutes a spot forming optical path.
  • the biological particles in the flow channel generate fluorescent and scattered light signals as they pass through the spot, and the optical signals are detected by the detection optical path.
  • the fluorescent and scattered light signals are separated one by one by the detection optical path, such as forward scattered light 63, side scattered light 62, fluorescent light generated by excitation, and fluorescent light 161 to 16N obtained by color separation, and each optical signal is photomultiplied.
  • Photoelectric detection devices such as tubes and photodiodes are converted into electrical signals, and the electrical signals are processed on subsequent processing platforms.
  • the image sensor 1200 and the second focus lens 1300 and the filter 1600 may be added to the detection optical path to realize the bright field 21 observation.
  • first and second are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated.
  • features defining “first” and “second” may include one or more of the features either explicitly or implicitly.
  • the meaning of "a plurality” is two or more unless specifically and specifically defined otherwise.
  • the terms “installation”, “connected”, “connected”, “fixed” and the like shall be understood broadly, and may be either a fixed connection or a detachable connection, unless explicitly stated and defined otherwise. , or integrated; can be mechanical connection, or can be electrical connection; can be directly connected, or can be indirectly connected through an intermediate medium, can be the internal communication of two elements or the interaction of two elements.
  • installation can be understood on a case-by-case basis.

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Abstract

一种流式细胞术检测装置及方法和二元光学器件在流式细胞术检测或制备流式细胞术检测装置中的用途。所述流式细胞术检测装置包括：光斑形成组件（300），所述光斑形成组件（300）被配置为适于在流道（10）中线上形成至少两个光斑。

Description

流式细胞术检测装置和方法
优先权信息
本申请请求2016年04月08日向中国国家知识产权局提交的、专利申请号为201610218106.8的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及微光学检测技术领域，具体地，本发明涉及流式细胞术检测装置、流式细胞术检测方法以及二元光学器件在流式细胞术检测或制备流式细胞术检测装置中的用途。
背景技术
流式细胞术可用于检测经过荧光标记的细胞、胚胎、RNA、DNA、蛋白质颗粒、微生物、病毒等生物微粒。检测中，流式细胞术需要借助于经过整形的激光光斑照射在位于液流中心的生物微粒上，生物微粒在激光光斑的激发下发出散射光和荧光。这些散射光和荧光信号被光电倍增管、光电二极管等光电转换器件收集，再经过检测系统分析，从而得出生物微粒的物理和化学信息，如细胞尺寸、表面粒度、抗原信息等。基于流式细胞术的检测原理，激光光斑的形状和质量是影响检测灵敏度、单个微粒分辨力的关键因素。
目前，商用流式细胞仪一般利用两个柱透镜把高斯激光整形为椭圆形的高斯光斑，椭圆形光斑的短轴平行于生物微粒的流向、长轴垂直于生物微粒的流向，以提高对单个生物微粒的分辨力、同时防止对生物微粒的漏检。
然而，流式细胞术检测装置仍有进一步开发和改进。
发明内容
本申请是基于发明人对以下问题和事实的发现而提出的：
目前商用流式细胞仪检测的稳定性和对单个生物微粒的分辨力难以进一步提高，难以实现高速和高通量检测，并且无法满足对生物微粒的测速要求。发明人通过研究发现，造成上述问题的关键在于目前商用流式细胞仪椭圆形光斑内光能分布不均匀，生物微粒通过光斑的相对位置发生改变时，其输出的光学信号会随之发生改变，并且椭圆光斑为高斯分布时，光斑的边界不清晰，同时，单个光斑无法满足对每个生物微粒的实时测速要求。
本发明旨在至少在一定程度上解决上述相关技术中的技术问题之一。为此，本发明提出了利用具备表面微结构的二元光学器件来实现光斑整形，进而在输出平面上实现具有如下特点的光斑：光斑边界锐利，光斑内光能均匀分布，能够实现对生物微粒的高通量测定； 光斑由二个以上的图形组成，能够实现对每个生物微粒的速度实时测定；光斑可由多个不同尺寸的图形组成，能够兼顾单微粒分辨力和微弱信号检测。
进而，在本发明的第一方面，本发明提出了一种流式细胞术检测装置。根据本发明的实施例，包括：光斑形成组件，所述光斑形成组件被配置为适于在流道中线上形成至少两个光斑。本发明实施例所提出的流式细胞术检测装置能够实现对生物微粒速度的准确测定，单微粒分辨力和对微弱信号的检测力强。
根据本发明的实施例，上述流式细胞术检测装置还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述光斑形成组件为二元光学器件。二元光学器件使得本发明实施例的流式细胞术检测装置的检测稳定性、单微粒分辨力和对微弱信号检测能力进一步提高。
根据本发明的实施例，所述至少两个光斑分别独立地为矩形。矩形光斑使得本发明实施例的流式细胞术检测装置的检测稳定性、单微粒分辨力和高速和高通量检测效率进一步提高。
根据本发明的实施例，所述至少两个光斑间隔设置，并且所述矩形的中轴线位于所述流道中线上。上述矩形光斑的设置使得细胞术检测装置的检测灵敏度进一步提高。
根据本发明的实施例，所述至少两个光斑的长边长度均为10～100微米，所述至少两个光斑的两端光斑的短边长度是基于待测样品的尺寸确定的。矩形光斑的长边长度为10～100微米可最大限度的避免对生物微粒样品的漏检。两端光斑的短边长度是基于待测样品的尺寸确定的，可进一步提高对单个生物微粒样品的分辨力。
根据本发明的实施例，所述至少两个光斑的的两端光斑分别具有相同的尺寸，并且所述至少两个光斑的长边长度相等。进而，本发明实施例的流式细胞检测装置在数据处理中可通过加权平均等数据处理方法提高测试的准确度。
根据本发明的实施例，所述至少两个光斑的幅值方差小于10％，光斑的光强相等。进而，本发明实施例的流式细胞检测装置在数据处理中可通过加权平均等数据处理方法提高测试的准确度。
根据本发明的实施例，至少两个光斑的光强的均匀性高于80％，光强分布均匀，进一步提高了流式细胞术检测装置的准确度和灵敏度。
根据本发明的实施例，所述流式细胞术检测装置包括：沿着入射激光的光路，依次设置有激光器、扩束镜、所述光斑形成组件、第一小孔光阑和第一聚焦透镜；沿着前向散射激光的光路，依次设置有挡光片、第一滤光镜和第一光电传感器；沿着侧向散射激光的光路，依次设置有第二滤光镜和第二光电传感器；沿着被激发荧光的光路，依次设置有第二聚焦 透镜和多路荧光检测装置，其中，所述多路荧光检测装置包括二向色镜、第二小孔光阑、第三滤光镜和第三光电传感器；以及沿着观察光路，依次设置有图像光感器、第四滤光镜和第三聚焦透镜。本发明实施例的流式细胞术检测装置在上述组件组成下，操作简便，其检测的稳定性、准确性、单微粒分辨力和高速和高通量检测效率进一步提高。
在本发明的第二方面，本发明提出了一种流式细胞术检测方法。根据本发明的实施例，所述检测方法包括：在流道中线上形成至少两个光斑，其中，所述至少两个光斑是如前所定义的。本发明实施例所提出的流式细胞术检测方法可准确测定生物微粒待测样品的速度，检测结果的稳定性高，可兼顾单微粒分辨力和对微弱信号的检测。
根据本发明的实施例，上述流式细胞术检测方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述方法是利用前面所述的流式细胞术检测装置进行的。如前所述，本发明实施例的流式细胞术检测装置具有检测结果稳定性高、对单微粒的分辨力强以及对微弱信号的检测能力强的特点，进而本发明实施例的检测方法采用本发明实施例的流式细胞术检测装置对生物微粒待测样品进行检测，其检测结果的稳定性、对单微粒的分辨力和对微弱信号的检测能力进一步提高。
在本发明的第三方面，本发明提出了二元光学器件在流式细胞术检测或制备流式细胞术检测装置中的用途。根据本发明的实施例，二元光学器件应用在流式细胞术检测或制备流式细胞术检测装置，该检测方法或装置可准确测定生物微粒的速度，对生物微粒的检测结果的稳定性高，对单微粒的分辨力和对微弱信号的检测能力高。
根据本发明的实施例，上述二元光学器件在流式细胞术检测或制备流式细胞术检测装置中的用途还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述二元光学器件对入射激光的相位进行调制。利用二元光学器件对入射激光的相位进行调制，对生物微粒的速度测定准确性进一步提高，对生物微粒的检测结果的稳定性和对单微粒的分辨力和对微弱信号的检测能力进一步提高。
根据本发明的实施例，所述二元光学器件的材料选自石英、玻璃、聚甲基丙烯酸甲酯的至少之一。选择上述的材料的至少之一制备二元光学器件，可进一步提高所述流式细胞术检测或流式细胞术检测装置的稳定性和灵敏性。
根据本发明的实施例，所述激光包括选自高斯光、平面光和多模光的至少之一。根据本发明的实施例，选择合适的入射激光及其相应的二元光学器件，可进一步显著提高所述流式细胞术检测或流式细胞术检测装置的稳定性和灵敏性。
根据本发明的实施例，当所述激光为直径为3mm的高斯光时，所述二元光学器件的结构为正方形，所述正方形的边长为3mm且内部具有128*128个台阶。本发明实施例的上述 二元光学器件的结构进一步提高了所述流式细胞术检测方法或流式细胞术检测装置的稳定性和对单微粒的分辨力和对微弱信号的检测能力。
附图说明
图1是根据本发明实施例的双矩形光斑的示意图；
图2是根据本发明实施例的流式细胞术检测时，双矩形光斑、生物微粒和流道的之间的位置关系示意图；
图3是根据本发明实施例的生物微粒通过双矩形光斑时发出的光信号的示意图；
图4是根据本发明实施例的一个具体的二元光学器件的相位图；
图5是根据本发明实施例的利用二元光学器件得出的输出光分布图；
图6是根据本发明实施例的三矩形光斑的示意图；
图7是根据本发明实施的三矩形光斑、生物微粒和流道的之间的位置关系示意图；
图8是根据本发明实施例的生物微粒通过三矩形光斑时发出的光信号的示意图；
图9是根据本发明实施例的一个具体的二元光学器件的相位图；
图10是根据本发明实施例的利用二元光学器件得出的输出光分布图；
图11是根据本发明实施例的流式细胞检测装置的光路图；以及
图12是根据本发明实施例的流式细胞术检测装置的结构示意图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
流式细胞术检测装置
在本发明的第一方面，本发明提出了一种流式细胞术检测装置。根据本发明的实施例，包括：光斑形成组件，所述光斑形成组件被配置为适于在流道中线上形成至少两个光斑。流道中线的位置参考图1。本发明实施例中的流式细胞检测装置的至少两个光斑对生物微粒的照射的时间总和增加了，而单个光斑对生物微粒的照射时间可能不变甚至较现有技术有所缩短，因此，在流道中线上形成至少两个光斑兼顾了对微弱信号的检测能力和对单个生物微粒的分辨力，本发明实施例所提出的流式细胞术检测装置单微粒分辨力和对微弱信号的检测力强。光斑形成组件所形成的至少两个光斑相对位置固定，进而本发明实施例所提出的流式细胞术检测装置能够实现对生物微粒速度的准确测定。
具体地，根据本发明的实施例，该光斑形成组件为二元光学器件。二元光学器件的微结构可改变入射激光各点处的光程，发生特定的光学衍射，进而可在输出平面上形成至少两 个光斑。二元光学器件所形成的光斑内部光强均匀分布，因而即使生物微粒通过光斑的相对位置发生一定偏移时，微粒受到的照射条件无明显变化、其输出的光学信号基本保持不变，从而提升了检测的稳定性、降低了检测信号的变异系数；光斑的边界锐利，使得输出的光学信号具有清晰的边界，有助于识别生物微粒发出的光信号；光斑内部的光强部分布均匀，这使得生物微粒通过矩形光斑时的输出光信号幅值基本保持恒定，便于对光信号进行计算，如计算光信号的高度、宽度或面积。当形成两个以上光斑时，中间光斑用于增长对生物微粒的照射时间、延长光信号的持续时间，便于对光信号进行收集、检测和分析，提高了检测灵敏度，特别有助于实现对微弱光信号的有效检测。光斑形成组件为二元光学器件使得本发明实施例的流式细胞术检测装置的检测稳定性、单微粒分辨力和对微弱信号检测能力进一步提高。
具体地，根据本发明的实施例，该至少两个光斑分别独立地为矩形。根据本发明的实施例，矩形光斑内的能量分布均匀并且边界锐利，进而生物微粒通过光斑的相对位置发生改变时，其输出的光学信号不会随之发生改变，从而矩形光斑使得本发明实施例的流式细胞术检测装置的检测稳定性、单微粒分辨力和高速和高通量检测效率进一步提高。而现有技术中，通过透镜形成的椭圆形光斑，其光强成高斯分布，边界连续变化、不锐利，因而其激发的荧光信号因细胞等生物微粒通过位置的不同而具有显著差异，且信号呈近似的高斯形，不利于分辨单个细胞等生物微粒的信号，降低了灵敏度和分辨力。
根据本发明的实施例，至少两个矩形光斑间隔设置，并且所述矩形的中轴线位于所述流道中线上。其中，矩形的中轴线与矩形的长边垂直，与矩形的短边平行。本发明实施例的包含细胞等生物微粒的样本流被聚焦在流道的中心位置，并且流道的中心位置上液流的流速最高，因而光斑中轴线与流道中线重合能够使单个细胞通过光斑的时间缩短，既单位时间内能够通过更多的细胞，提高了细胞通量。矩形光斑的长边与矩形的中轴线垂直，也即与流道中线垂直，也即与细胞流向垂直，这样当细胞偏离流道中线位置时依然能够被光斑照射，实现有效的检测。进而，本发明实施例的矩形光斑的中轴线位于所述流道中线上，使得流式细胞术检测装置的检测通量和灵敏度进一步提高。
根据本发明的实施例，至少两个矩形光斑的长边长度均为10～100微米，所述至少两个矩形光斑的两端光斑的短边长度是基于待测样品的尺寸确定的。其中，两端光斑是指，当具有两个光斑时，两端光斑如图1的11、12所示，当具有两个以上的光斑，如三个光斑，两端光斑如图6的51、52所示。同时，当有两个以上的矩形光斑，如三个光斑，如图6所示，53被称为中间光斑。在本发明中，两端矩形光斑的长边是指长度稍长的边，中间矩形光斑的长边是指与流道中线垂直的边。矩形光斑的长边长度为10～100微米可最大限度的避免对生物微粒样品的漏检。两端光斑的短边长度是基于待测样品的尺寸确定的，本发明实 施例的装置的两端光斑的短边长度接近待测样品尺寸的平均值，进而可进一步提高对单个生物微粒样品的分辨力。
根据本发明的实施例，至少两个光斑的的两端光斑分别具有相同的尺寸，并且所述至少两个光斑的长边长度相等。进而，两端光斑的光强相等，并且生物微粒通过至少两个光斑后产生光信号的幅值基本相等(需要说明的是，本说明书中所述的光斑的光强相等是指近似相等，而不是完全相等。因为二元光学器件只能在理论上实现完全相等，实际中并不存在)。进而在数据处理中可通过加权平均等数据处理方法提高测试的准确度。
同时，根据本发明的实施例，至少两个光斑的幅值均方差小于10％。本发明实施例中，至少两个光斑的幅值均方差小于10％，光斑的光强相等，则生物微粒待测样品通过至少两个光斑后产生光信号的幅值基本相等，在数据处理中可通过加权平均等数据处理方法提高测试的准确度。
根据本发明的实施例，至少两个光斑的光强的均匀性高于80％，光强分布均匀，因而当生物微粒通过至少两个光斑时，即使相对位置发生改变，生物微粒所受激发激光并无显著差异，其输出的光学信号不会随之发生改变，从而减弱了位置因素造成的信号差异，并且产生的光信号的上升沿和下降沿更加陡直、顶部更加平整，进而有利于对光信号进行识别和处理，进一步提高了流式细胞术检测装置的稳定性、准确度和灵敏度。
更具体地，根据本发明的实施例，所述流式细胞术检测装置，参考图12，包括：沿着入射激光的光路，依次设置有激光器100、扩束镜200、所述光斑形成组件300、第一小孔光阑1400和第一聚焦透镜400，激光器100发射出的激光通过光斑形成组件300在流道10中线上形成至少两个光斑,其中流道10可以是透明的微流道，也可是存在于空气中，其中小孔光阑1400用于去除光斑形成组件300输衍射光斑主级之外的各次级。当样品(细胞或其它待测生物微粒)沿流道10中线通过光斑时，样品被激发出荧光、前向散射光(正向散射光)和侧向散射光，沿着前向散射激光的光路，依次设置有挡光片1800、第一滤光镜500和第一光电传感器600，挡光片1800位于前向散射光的光路中心，面积小，能够遮挡激光直射过来的信号，保证入射到光电传感器600的信号仅为散射光信号，第一滤光镜500可使入射到光电传感器600的信号仅为激光信号，第一光电传感器600用于将前向散射光信号转化为电信号，进行光信号分析，进而分析样品信息；沿着侧向散射激光的光路，依次设置有第二滤光镜700和第二光电传感器800，第二滤光镜700可使入射到光电传感器800的信号仅为激光信号，第二光电传感器800用于将侧向散射光信号转化为电信号，进行光信号分析，进而分析样品信息；沿着被激发荧光的光路，依次设置有第二聚焦透镜1700和多路荧光检测装置2000，第二聚焦透镜1700用于收集空间中的荧光，以便增强荧光信号，其中所述多路荧光检测装置2000包括二向色镜900、第二小孔光阑1500、第三滤光镜1000和 第三光电传感器1100，二向色镜900用于将多色荧光分离成单色荧光，即每色荧光信号的分离均需一个二向色镜900进行分离，进而每色荧光均依次通过第二小孔光阑1500、第三滤光镜1000和第三光电传感器1100，此处的二向色镜900、第二小孔光阑1500、第三滤光镜1000和第三光电传感器1100为系列二向色镜、系列第二小孔光阑、系列第三滤光镜和系列第三光电传感器的统称(参考图12)，其中，第二小孔光阑1500用于去除杂散光，第三滤光镜1000用于保留特定颜色的荧光信号，第三光电传感器1100用于将特定颜色的荧光信号转化为电信号，进而进行光信号分析，从而获得样品信息。其中，荧光信号的强度与生物微粒的表面抗原数量正相关，前向散射光的强度与生物微粒的大小正相关，侧向散射光的强度与生物微粒的表面粒度、内部结构、光学特性等生物微粒的自身因素相关。根据本发明的实施例，光电传感器包括但不限于光电二极管、光电倍增管、光电二极管阵列和倍增管阵列，其中，散射光多用光电二极管，荧光多用光电倍增管或光电二极管阵列或倍增管阵列。以及沿着观察光路，依次设置有图像传感器1200、第四滤光镜1600和第三聚焦透镜1300，图像传感器1200用于流道中的样品进行实时观测和监控，以方便实验操作，第四滤光镜1600用于去除激光信号的干扰。本发明实施例的流式细胞术检测装置在上述组件组成下，操作简便，其检测的稳定性、准确性、单微粒分辨力和高速和高通量检测效率显著提高。
流式细胞术检测方法
在本发明的第二方面，本发明提出了一种流式细胞术检测方法。根据本发明的实施例，该检测方法包括：在流道中线上形成至少两个光斑，其中，所述至少两个光斑是如前所定义的。本发明实施例所提出的流式细胞术检测方法可准确测定生物微粒待测样品的速度，检测结果的稳定性高，可兼顾单微粒分辨力和对微弱信号的检测。
具体地，根据本发明的实施例，该方法是利用前面所述的流式细胞术检测装置进行的。如前所述，本发明实施例的流式细胞术检测装置具有检测结果稳定性高、对单微粒的分辨力强以及对微弱信号的检测能力强的特点，进而本发明实施例的检测方法采用本发明实施例的流式细胞术检测装置对生物微粒待测样品进行检测，其检测结果的稳定性、对单微粒的分辨力和对微弱信号的检测能力进一步提高。
二元光学器件在流式细胞术检测或制备流式细胞术检测装置中的用途
在本发明的第三方面，本发明提出了二元光学器件在流式细胞术检测或制备流式细胞术检测装置中的用途。根据本发明的实施例，二元光学器件可根据入射激光的模式、激光的波长、直径，针对不同的入射激光，设计出相应的二元光学器件，以应用在流式细胞术检测或制备流式细胞术检测装置中，在流道中线上形成至少两个光斑，进而二元光学器件应用在流式细胞术检测或制备流式细胞术检测装置，该检测方法或装置可准确测定生物微粒 的速度，对生物微粒的检测结果的稳定性高，对单微粒的分辨力和对微弱信号的检测能力高。
根据本发明的具体实施例，所述二元光学器件对入射激光的相位进行调制，进而在在流道中线上形成至少两个光斑，至少两个光斑的距离恒定，边界锐利，内部光强分布均匀，能兼顾单微粒分辨力和对微弱信号的检测，进而利用二元光学器件对入射激光的相位进行调制，对生物微粒的速度测定准确性进一步提高，对生物微粒的检测结果的稳定性和对单微粒的分辨力和对微弱信号的检测能力进一步提高。
另外，根据本发明的实施例，所述二元光学器件的材料选自石英、玻璃、聚甲基丙烯酸甲酯的至少之一。二元光学器件的材料的选择是针对不同的入射激光进行选择的，本发明实施例的二元光学器件的材料选自但不限于石英、玻璃、聚甲基丙烯酸甲酯，只要能在流道中线上形成至少两个光斑，所述至少两个光斑又如前面所描述的即可。选择最适的材料制备二元光学器件，可进一步提高所述流式细胞术检测或流式细胞术检测装置的稳定性和灵敏性。
另外，根据本发明的实施例，本发明实施例的激光包括但不限于高斯光、平面光和多模光，本发明实施例的二元光学器件会根据不同的入射激光进行设计，进而在流道中线上形成实验所需要的至少两个光斑。选择合适的入射激光及其相应的二元光学器件，可进一步显著提高所述流式细胞术检测或流式细胞术检测装置的稳定性和灵敏性。
根据本发明的具体实施例，当所述激光为直径为3mm的高斯光时，所述二元光学器件的结构为正方形的薄片，所述正方形的边长为3mm且内部具有128*128个台阶。本发明实施例的上述具体的二元光学器件的结构可在流道中线上形成两个或三个矩形光斑，所述矩形光斑具有前面所述的光斑的至少之一的特点，本发明实施例的上述二元光学器件的结构进一步提高了所述流式细胞术检测方法或流式细胞术检测装置的稳定性和对单微粒的分辨力和对微弱信号的检测能力。
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
在本实施例中，发明人采用二元光学器件在流道中线上形成两个矩形光斑，以下是对矩形光斑的特征和设计的描述：
如图1所示，双矩形光斑1，两个矩形光斑11和12相距一定距离L，两个矩形光斑的长边和短边长度分别为l1和w1、l2和w2，光斑内的光强均匀分布。
流式细胞术检测时，光斑、生物微粒和流道的之间的位置关系情况如图2所示：光斑1位于流道的中心位置，即矩形光斑长边的中线和流道中线43重合，流道中线43位于流道边界41和42的中心；生物微粒2的流动方向3平行于流道中线43，并尽量靠近中线。一般生物微粒在流道中线附近十几微米的范围内，因而发明人取两个矩形光斑长边l1和l2均为几十微米，以防止对生物微粒的漏检。为精确分辨出单个生物微粒，发明人将两个矩形光斑的短边w1和w2调整为接近单个生物微粒的特征尺寸。
生物微粒通过双矩形光斑时发出的光信号如图3所示，这里的光信号可指代前向散射光信号、侧向散射光信号或某一路的荧光信号，两次光信号的时间间隔为T、幅值为H1和H2、持续时间为T1和T2、信号对时间的积分为A1和A2(即面积)。发明人发现光斑边界清晰、内部光强均匀分布，生物微粒通过光斑时产生的光信号的上升沿和下降沿更加陡直、顶部更加平整，这有利于对光信号进行识别和处理。通过生物微粒通过两个光斑的时间间隔T，可得出生物微粒的流速V＝L/T。光信号的幅值和面积用于表征光信号的强度，其中荧光信号的强度与生物微粒的表面抗原数量正相关，正向散射光的强度与生物微粒的大小正相关，侧向散射光的强度与生物微粒的表面粒度、内部结构、光学特性等生物微粒的自身因素相关。为便于信号处理，发明人选取两个矩形光斑的尺寸相等和光强相等。这样生物微粒通过两个矩形光斑的产生的光信号基本相等，在数据处理中可以通过取平均等数据处理方法提高测试的准确度，例如，得出信号的平均值为(H1+H2)/2、(T1+T2)/2与(A1+A2)/2。
发明人以血细胞作为待检的生物微粒，对双矩形光斑的参数和设计作具体描述：
血液中主要包含红细胞、白细胞和血小板，红细胞为凹碟形、直径为7～8微米、厚2～2.5微米，白细胞直径多为10～15微米，血小板为凸碟形、直径2～3微米。为能够准确分辨出一个血细胞的光信号，发明人将矩形光斑的短轴长度设计为10微米。一般，生物微粒在流道中线附近十几微米的范围内，因而发明人将矩形光斑的长边长度设为50微米，以防止对生物微粒的漏检。此外，生物微粒的流速多为3～10m/s，进而发明人将两个矩形光斑的间距设计为100微米，以保证血细胞通过两个矩形光斑的时间间隔较大，从而提高对血细胞流速测量的准确性。例如，血细胞通过两个光斑所发出的同一通道的两个光信号的时间间隔为2微秒，则得出的其流速为5m/s。
为实现这种光斑，发明人采用二元光学器件对入射激光的相位进行调制，即利用二元光学器件表面的微台阶结构改变入射激光各点处的光程，调制后的激光经过透镜并在其焦面上实现期望光斑。二元光学器件采用透明的光学材料制成，如石英、玻璃、PMMA等，通过刻蚀、聚焦等离子术加工等方法在光学材料的表面制作出不同高度的台阶。入射激光可为高斯光、平面光或其他模式的激光、乃至多模光，激光的波长、直径等参数 可变，针对不同的入射激光，可设计出相应的二元光学器件。例如：为实现上述的由两个矩形光斑所形成的光斑，当选取直径3mm的高斯光作为输入激光时，可将二元光学器件制备成边长3mm的正方形薄片，其内部分为128×128个台阶，此二元光学器件的相位图如图4所示。发明人进一步选取二元光学器件后的透镜焦距为10mm，得出焦面上的输出光分布如图5所示，图中的纯黑区域表示光强为零，纯白区域表示光强最大，灰色表示介于两者之间。发明人发现，上述二元光学器件的输出光斑的尺寸与设计相一致、具有清晰的边界，光斑内的幅值均方差小于10％、具有高度的均匀性。
实施例2
在本实施例中，发明人采用二元光学器件在流道中线上形成三个矩形光斑，以下是对矩形光斑的特征和设计的描述：
如图6所示，由三个矩形形成的光斑5由两个相距距离L的矩形光斑51和52、以及位于两者中间的矩形光斑53共同组成，中间的矩形光斑在生物微粒流动方向上的尺寸更大，三个矩形的尺寸分别为w1×l1、w2×l2和w3×l3，光斑内的光强均匀分布。
流式细胞术检测时，光斑、生物微粒和流道的之间的位置关系情况如图7所示：光斑位于液流的中心位置，既三个矩形共同的中线与流道中线43重合，流道中线43位于流道边界41和42的中心；生物微粒2的流动方向3平行于流道中线43，并尽量靠近中线。两端的矩形光斑51和52用于分辨单个生物微粒、并对其进行测速，中间的宽矩形53用于增加对生物微粒的照射时间、既延长光信号的持续时间，便于对光信号进行收集、检测和分析，提高了检测灵敏度，特别有助于实现对微弱光信号的有效检测。一般生物微粒在流道中线附近十几微米的范围内，进而发明人将矩形光斑的长边l1、l2和l3的长度调整为几十微米，以防止对生物微粒的漏检。为精确分辨出单个生物微粒，发明人将矩形光斑51和52的短边w1和w2调整为接近单个生物微粒的特征尺寸。
生物微粒通过三矩形光斑时发出的光信号如图8所示，这里的光信号可指代前向散射光信号、侧向散射光信号或某一路的荧光信号，通过两端矩形光斑51和52的时间间隔为T，光信号幅值为H1、H2和H3，持续时间为T1、T2和T3，信号对时间的积分为A1、A2和A3(即面积)。发明人发现，光斑边界清晰、内部光强均匀分布，生物微粒通过光斑时产生的光信号的上升沿和下降沿更加陡直、顶部更加平整，这有利于对光信号进行识别和处理。通过计算生物微粒通过两端矩形光斑51和52的时间间隔T得出生物微粒的流速V＝L/T。通过计算生物微粒通过中间矩形光斑53时光信号的面积(即光信号的时间积分)，能够有效增强对微弱光信号的检测能力。光信号的幅值和面积用于表征光信号的强度，其中荧光信号的强度与生物微粒的表面抗原数量正相关，正 向散射光的强度与生物微粒的大小正相关，侧向散射光的强度与生物微粒的表面粒度、内部结构、光学特性等生物微粒的自身因素相关。为便于信号处理，发明人设计三个光斑的光强相等，三个矩形光斑垂直于流向的边长l1、l2和l3相等，矩形光斑51和52的短边w1和w2相等。这样生物微粒通过三个矩形光斑的光信号的幅值基本相等，这样在数据处理中可以通过加权平均等数据处理方法提高测试的准确度。例如：光强相等时H1:H2:H3≈1:1:1，流速稳定时有T1:T2:T3≈w1:w2:w3、A1:A2:A3≈T1:T2:T3≈w1:w2:w3，光信号幅值的加权平均值为(H1×w1+H2×w2+H3×w3)/(w1+w2+w3)或(H1×T1+H2×T2+H3×T3)/(T1+T2+T3)，以及信号均值(H1+H2)/2、(H1+H2+H3)/3、(T1+T2)/2与(A1+A2)/2。
发明人以血细胞作为待检的生物微粒，对三矩形光斑的参数和设计作具体描述：
血液中主要包含红细胞、白细胞和血小板，红细胞为凹碟形、直径为7～8微米、厚2～2.5微米，白细胞直径多为10～15微米，血小板为凸碟形、直径微2～3微米。为能够准确分辨出一个血细胞的光信号，发明人将两端矩形光斑51和52的短边w1和w2的长度调整为10微米。为增加血细胞通过中间矩形光斑的时间，发明人将中间矩形光斑53在生物微粒流动方向上的边长w3调整为50微米。一般，生物微粒在流道中线附近十几微米的区域，发明人将三个矩形光斑垂直于流向的边长l1、l2和l3的长度调整为50微米，以防止对生物微粒的漏检。此外，生物微粒流的流速多为3～10m/s，发明人将两端的两个窄矩形光斑的间距调整为100微米，以保证血细胞通过者两个矩形光斑的时间间隔足够大，从而提高对血细胞流速测量的准确性。例如，生物微粒通过两端的两个光斑51和52所发出的同一通道的两个光信号的时间间隔为2微秒，则得出其流速为5m/s。
为实现这种光斑，发明人采用二元光学器件对入射激光的相位进行调制，即利用二元光学器件表面的微台阶结构改变入射激光各点处的光程，调制后的激光经过透镜并在其焦面上实现期望光斑。二元光学器件可采用透明的光学材料制成，如石英、玻璃、PMMA等，通过刻蚀、聚焦等离子术加工等方法在光学材料的表面制作出不同高度的台阶。入射激光可为高斯光、平面光或其他模式的激光、乃至多模光，激光的波长、直径等参数可变，针对不同的入射激光，可设计出相应的二元光学器件。例如：为实现上述的由三个矩形光斑所形成的光斑，当选取直径为3mm的高斯光作为输入激光时，可将二元光学器件为制作成边长3mm的正方形薄片，其内部分为128×128个台阶，相应二元光学器件的相位图如图9所示。发明人选取二元光学器件后的透镜焦距为10mm，得出焦面上的输出光分布如图10所示，图中的纯黑区域表示光强为零，纯白区域表示光强最大，灰色表示介于两者之间；发明人发现，上述二元光学器件的输出光斑的尺寸 与设计相一致、具有清晰的边界，光斑内的幅值均方差小于15％、具有高度的均匀性。
实施例3
在本实施例中，发明人对流式细胞检测装置的光路进行了详细描述：
参考图11，激光61经过扩束器200扩束后达到二元光学器件的设计口径，激光经过光斑形成组件300(此实施例为二元光学器件)整形后再经过小孔光阑1400被透镜400聚焦在流道10的中心处，流道中心处的生物微粒被照射，以上构成了光斑成型光路。流道中的生物微粒通过光斑时产生荧光和散射光信号，光信号被检测光路检测。荧光和散射光信号通过检测光路被逐一分离，如前向散射光63、侧向散射光62、激发产生的荧光131以及被分色后得到的各色荧光161～16N，各路光信号被光电倍增管、光电二极管等光电检测器件转换为电信号，电信号在后续的处理平台上完成信号处理。为调试方便，检测光路中还可加入图像传感器1200和第二聚焦透镜1300和滤光镜1600实现明场21观测。
在本发明的描述中，需要理解的是，术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”、“顺时针”、“逆时针”、“轴向”、“径向”、“周向”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任 一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (16)

  1. 一种流式细胞术检测装置，其特征在于，包括：
    光斑形成组件，所述光斑形成组件被配置为适于在流道中线上形成至少两个光斑。
  2. 根据权利要求1所述的流式细胞术检测装置，其特征在于，所述光斑形成组件为二元光学器件。
  3. 根据权利要求1所述的流式细胞术检测装置，其特征在于，所述至少两个光斑分别独立地为矩形。
  4. 根据权利要求3所述的流式细胞术检测装置，其特征在于，所述至少两个光斑间隔设置，并且所述矩形的中轴线位于所述流道中线上。
  5. 根据权利要求4所述的流式细胞术检测装置，其特征在于，所述至少两个光斑的长边长度均为10～100微米，所述至少两个光斑的两端光斑的短边长度是基于待测样品的尺寸确定的。
  6. 根据权利要求1所述的流式细胞术检测装置，其特征在于，所述至少两个光斑的两端光斑分别具有相同的尺寸，并且所述至少两个光斑的长边长度相等。
  7. 根据权利要求1所述的流式细胞术检测装置，其特征在于，所述至少两个光斑的幅值均方差小于10％。
  8. 根据权利要求1所述的流式细胞术检测装置，其特征在于，所述至少两个光斑的光强的均匀性高于80％。
  9. 根据权利要求1所述的流式细胞术检测装置，其特征在于，
    沿着入射激光的光路，依次设置有激光器、扩束镜、所述光斑形成组件、第一小孔光阑和第一聚焦透镜；
    沿着前向散射激光的光路，依次设置有挡光片、第一滤光镜和第一光电传感器；
    沿着侧向散射激光的光路，依次设置有第二滤光镜和第二光电传感器；
    沿着被激发荧光的光路，依次设置有第二聚焦透镜和多路荧光检测装置，其中所述多路荧光检测装置包括二向色镜、第二小孔光阑、第三滤光镜和第三光电传感器；以及
    沿着观察光路，依次设置有图像光感器、第四滤光镜和第三聚焦透镜。
  10. 一种流式细胞术检测方法，其特征在于，包括：
    在流道中线上形成至少两个光斑，
    其中，所述至少两个光斑是如权利要求1～9任一项中所定义的。
  11. 根据权利要求10所述的方法，其特征在于，所述流式细胞术检测方法是利用权利要求1～9任一项所述的流式细胞术检测装置进行的。
  12. 二元光学器件在流式细胞术检测或制备流式细胞术检测装置中的用途。
  13. 根据权利要求12所述的用途，其特征在于，所述二元光学器件对入射激光的相位进行调制。
  14. 根据权利要求11所述的用途，其特征在于，所述二元光学器件的材料选自石英、玻璃、聚甲基丙烯酸甲酯的至少之一。
  15. 根据权利要求13所述的用途，其特征在于，所述激光包括选自高斯光、平面光和多模光的至少之一。
  16. 根据权利要求13所述的用途，其特征在于，当所述激光为直径为3mm的高斯光时，所述二元光学器件的结构为正方形，所述正方形的边长为3mm且内部具有128*128个台阶。
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