Discrimination system

Abstract

A discrimination system is provided. A discrimination system forms a liquid column and interrogates an object within the liquid column with an excitation source. The optical arrangement collects output electromagnetic radiation emanating from an excited object disposed within the liquid column and directs the output electromagnetic radiation to the detector. The analyzer reduces the positional correlation of the detected intensity by normalizing the value based on the position of each object.

Description

[0001] This application is a divisional application of the invention patent application with application number 202180019656.6 (international application number PCT / US2021 / 022381), application date March 15, 2021, entitled "Identification System, Identification Method and Detection System". Technical Field

[0002] This disclosure relates to identification systems, identification methods, and detection systems. Background Technology

[0003] Object identification devices and techniques distinguish between different types of objects, such as those with different properties. These devices and techniques are particularly useful for analyzing cells and even classifying cells based on specific properties of interest. Some cell classification methods rely on light emitted from cells or stained cells to determine their type. In some implementations, cells traveling in a liquid column are exposed to an excitation source to generate output electromagnetic radiation for detection. Cells of a first type or with special properties produce output electromagnetic radiation that differs from other cells in some property (e.g., wavelength and / or intensity). This difference serves as the basis for cell type identification and classification. Summary of the Invention

[0004] Some embodiments relate to a discrimination system for distinguishing between different types of objects based on electromagnetic radiation emitted from objects disposed within a liquid column. A liquid column forming structure generates a liquid column containing objects at different locations within it, and an excitation source generates excitation electromagnetic radiation directed toward the objects within the liquid column at a measurement region. The objects within the liquid column emit output electromagnetic radiation in response to this excitation electromagnetic radiation. An optical arrangement collects the output electromagnetic radiation from the objects, and a detector generates an electrical signal in response to the intensity of the output electromagnetic radiation. The analyzer includes instructions stored thereon for performing the following operations: i) normalizing the intensity of the output electromagnetic radiation, represented by the electrical signal, based on the position of the objects in the liquid column, and ii) distinguishing objects of a first type from other objects.

[0005] According to some embodiments of the detection system, an optical arrangement collects output electromagnetic radiation from an object in a liquid column, and a detector generates an electrical signal in response to the intensity of the output electromagnetic radiation collected by the optical arrangement. The analyzer has instructions stored thereon for performing the following operations: i) normalizing the intensity of the output electromagnetic radiation, represented by the electrical signal, based on the position of the object in the liquid column, and ii) distinguishing objects of a first type from other objects.

[0006] According to other embodiments, a method begins object identification by generating a liquid column containing objects at different locations within the column. Excitation electromagnetic radiation is directed toward the object in the liquid column at a measurement region. The object in the measurement region emits output electromagnetic radiation in response to the excitation electromagnetic radiation, which is collected and used to generate an electrical signal in response to the intensity of the output electromagnetic radiation. The intensity of the output electromagnetic radiation, represented by the electrical signal, is normalized based on the object's position in the liquid column and a first type of object distinguished from other objects. Attached Figure Description

[0007] Figure 1 This is a simplified diagram of a discrimination system according to certain embodiments.

[0008] Figure 2 It shows Figure 1 The xy plane cross section of the liquid column in the measurement area of ​​the system.

[0009] Figure 3 The image shows light emitted from an object located near the center of a liquid column, wherein there is substantially uniform light refraction at the liquid-gas interface of the liquid column relative to the optical device used as a collecting optics.

[0010] Figure 4 The image shows light emitted from an object located in the upper part of an elliptical core of a liquid column, where non-uniform light refraction is exhibited at the liquid-gas interface relative to the optical device used as a collecting optics.

[0011] Figure 5 The geometry used to develop an analytical formula for the angular dependence of in-plane light density as a function of position x is shown.

[0012] Figure 6 A series of graphs are provided showing the angular correlation of radiation at different object locations within a liquid column.

[0013] Figure 7 A series of graphs are provided showing the relationship between the relative intensity of light collected from the liquid column and the object's position along the x-axis for different numerical apertures of the collecting optics.

[0014] Figure 8 This is a flowchart of a method for identifying an object traveling in a liquid column by correcting for detected changes in the position of the output light, according to some embodiments.

[0015] Figure 9 A schematic diagram of an embodiment including position and intensity detection from a single detector is provided.

[0016] Figure 10 A schematic diagram of an embodiment including a first detector for detecting intensity and a second detector for determining the location of an object is provided.

[0017] Figure 11 The output of a simulation used to determine the location of an object using a split detector is shown.

[0018] Figure 12 The results of an experiment showcasing improvements in intensity measurement by correcting detection values ​​based on the position of the sperm nucleus are presented.

[0019] Figure 13 The results of an experiment showcasing improvements in intensity measurement by correcting detection values ​​based on the position of live sperm cells are presented.

[0020] Figure 14 The results of experiments seeking to improve the performance of misaligned instruments using a correction factor based on sperm nucleus location are presented.

[0021] Figure 15 A series of graphs showing the angular correlation of radiation at different object locations are provided, and exclusion areas are also shown.

[0022] Figure 16 The diagram shows the relationship between the relative intensity of light collected from a liquid column and the position of the object along the x-axis when the angle is not excluded, when rays with angles between -0.3 rad and +0.3 rad are excluded, and when rays with angles between -0.4 rad and +0.4 rad are excluded.

[0023] Figure 17 The results of experiments using both a correction factor and an element for optically reducing the positional dependence of intensity measurements are presented.

[0024] The accompanying drawings are not necessarily drawn to scale. Similar numbers used in the drawings refer to similar elements. However, it will be understood that the use of numbers to refer to parts in a given drawing is not intended to limit the parts to be labeled with the same numbers in another drawing. Detailed Implementation

[0025] The embodiments described herein relate to devices, systems, and methods for distinguishing between different types of objects. These objects emit output light in response to excitation light directed toward, for example, a column of liquid in a flowing stream. As used herein, the term "emit" refers to both reflected and fluorescent electromagnetic radiation, such as light. As used herein, the term "light" refers to both electromagnetic radiation at wavelengths in the visible spectrum and electromagnetic radiation at wavelengths in the infrared and ultraviolet spectra. Such output electromagnetic radiation may include light that is directly reflected and fluorescent from the object, as well as light that is reflected and fluorescent by a stain or dye associated with the object. In some embodiments, cell types are distinguished based on the intensity of the output electromagnetic radiation emitted from the object. The intensity may be determined as a peak intensity or even as a total intensity, for example, based on an integrated region of the intensity signal. Specific embodiments described herein relate to distinguishing X-chromosome-bearing sperm cells from Y-chromosome-bearing sperm cells. Further embodiments relate to distinguishing viable X-chromosome-bearing sperm cells from objects other than viable X-chromosome-bearing sperm cells, including Y-chromosome-bearing sperm cells and non-viable cells of both sexes.

[0026] It will be understood that the methods disclosed herein can be more generally applied to distinguish any different types of objects, provided that the output electromagnetic radiation emitted from one object type produces a identifiable difference in at least one characteristic when compared with electromagnetic radiation emitted from another object type. In some of the examples provided, the liquid column is a flow with curved boundaries or interfaces, where refraction of electromagnetic radiation may occur. For example, the curved boundary of the liquid column may be generally circular in cross-section. The liquid column may be confined by solid walls, such as within a cuvette or a microfluidic channel, or may be jetted into air, such as in an "air-jet" flow cytometer. The object may move along the liquid column through a central core formed by a sheath fluid at least partially surrounding a central core. In the case of sperm sorting applications, the central core may comprise a core flow containing a sample solution of sperm cells. For the purpose of orienting aspherical sperm cells, the core flow may be tuned to a generally band shape or may have a generally elliptical cross-section. The electromagnetic radiation emitted from the object encounters at least one optically refractive boundary between the object and other materials, such as at the interface between the liquid column and air.

[0027] At least in part, due to the different refractive properties of the sheath fluid and air, the light collection efficiency of light emitted from an object inside the column outside the liquid column depends on the position of the object in such a system. This position-dependent light collection efficiency is disadvantageous in applications where the light emitted from the object must be precisely quantified, and this precision is limited by random (not directly observable) positional fluctuations of the object. Specifically, in the case of sex differentiation of sperm, this system seeks to distinguish very bright and closely correlated fluorescence intensities. This distinction is typically achieved by staining sperm cells and sperm nuclei with Hoechst 33342. Hoechst 33342 is a bright, cell-permeable dye that selectively combines with AT base pairs in the minor groove of double-stranded nuclear DNA. Chemical staining of sperm cells with Hoechst 33342 distinguishes the X chromosome from the Y chromosome by slightly different amounts of nuclear DNA. For example, many domestic animals exhibit a difference of approximately 4%. When sperm cells are properly stained and oriented, these small differences can be distinguished by the fluorescence intensity of Hoechst 33342 associated with the sperm cell's nuclear DNA when the sperm cells are irradiated by an appropriate excitation source (such as a laser operating at or near a wavelength of 355 nm).

[0028] This 4% difference is difficult to detect for several reasons. First, sperm nuclear DNA resides within the sperm head and is amorphous or, in most species, paddle-shaped. This asymmetry causes sperm to fluoresce differently on the flatter and narrower sides. In fact, this fluctuation exceeding 4% in DNA content means that sperm must be oriented to distinguish them based on the amount of nuclear chromosomes. Oriented geometry often produces a core with a banded or elliptical cross-section. This elliptical cross-section provides sperm with a larger latitude than normal to align on an axis.

[0029] The method disclosed in this paper enhances the accuracy of systems that can be constrained by such fluctuations, such as "air-jet" flow cytometers. As described in more detail below, the positional variability of light intensity collected from an object in a liquid column can be addressed using algorithms that correct for the correlation between intensity and position.

[0030] The methods outlined in this paper are particularly applicable to flow cytometry. However, the methods can be applied to any system in which light emitted from an object is collected on one side of an interface and on the other side of the interface, where the interface causes a change in the path of the emitted light in a manner that depends on the position of the object relative to the detector. The method described in this paper for correcting for positional variations within the liquid column thus provides more accurate measurements to distinguish the types of objects.

[0031] exist Figure 1The “air-jet” flow cytometer system 100 schematically illustrated is a type of identification system that can be used to discuss the concepts of this disclosure. The “air-jet” flow cytometer system 100 includes a liquid column forming structure that generates a flow stream comprising a liquid column 150 ejected at a high velocity (e.g., approximately 20 m / s) from an outlet nozzle 160 of a chamber 110. The liquid column 150 exiting from the outlet nozzle 160 may be generally circular in cross-section and may have a diameter of approximately 10 μm to approximately 100 μm in some embodiments. In some embodiments, the interior of the chamber 110 and / or the outlet nozzle 160 is configured with an internal geometry that hydrodynamically orients sperm within the liquid column. As a non-limiting example, nozzles similar to those described in U.S. Patents 6,782,768 and 6,263,745 may be incorporated for the purpose of orienting sperm and generating a coaxial flow of liquid column. The liquid column 150 is constituted by a core flow 151 within a sheath fluid 152, wherein… Figure 1 The arrows indicate the flow directions of the core fluid 151 and the sheath fluid 152. The sheath fluid 152 may have a generally circular cross-section, while the core fluid has a generally elliptical cross-section with a major axis and a minor axis.

[0032] Within chamber 110, sample injection element 111 introduces a core flow 151 containing objects 171, 172, which may be of multiple types. The core flow 151 is confined by a sheath fluid 152 comprising a sheath fluid and is hydrodynamically shaped within chamber 110. The sheath fluid 152 at least partially surrounds the core flow 151, and the sheath fluid 152 and the core flow 151 are substantially non-mixed. A ramp or inclined wall 115 of chamber 110 applies a force that shapes the core flow 151 and accelerates the objects 171, 172 within it. As a liquid column 150 is ejected from chamber 110, the movement of the sheath fluid 152 constrains the objects 171, 172 in the core flow 151 toward the center of the liquid column 150. The liquid column 150 delivers the objects 171, 172 (e.g., sequentially) to a measurement region 175 of the liquid column 150.

[0033] As the object passes through the measurement region 175 of the liquid column 150, light from the excitation source 180 provides excitation light to the objects 171 and 172. The excitation source 180 can provide light in a wide or narrow wavelength band. For example, the excitation source 180 can be a laser. Any laser suitable for generating a response from the object or a dye associated with the object can be used. Pulsed lasers and continuous-wave lasers are each well-suited to produce a suitable response. In some configurations, the electromagnetic radiation generated by the excitation source (e.g., the excitation light) can be modified by optical elements 181. For example, the excitation light can be focused onto the measurement region 175 by one or more lenses 181. Lenses can be used to focus the excitation electromagnetic radiation into a suitable beam shape focused onto the measurement region. The object 172a in the measurement region 175 emits light in response to the excitation source 180, such as scattered light or fluorescence.

[0034] The first type of object 171 will emit output electromagnetic radiation that differs from the output electromagnetic radiation emitted from the second type of object 172 in at least one characteristic. For example, in some scenarios, the light emitted by the first type of object 171 will have a higher intensity than the light emitted from the second type of object 172.

[0035] An optical collection arrangement 190 is positioned to collect output electromagnetic radiation 161 emitted from an object 172a within a measurement region 175, which crosses the optical refractive boundary of a liquid column 150 at a liquid-gas interface 153. In some embodiments, the optical arrangement 190 may be configured to modify the output electromagnetic radiation 161 to provide modified output electromagnetic radiation 162, which focuses the output electromagnetic radiation emitted from the object 172a in the measurement region 175 onto a detector 185. In some embodiments, the optical collection arrangement 190 may include elements to reduce the positional dependence of the output electromagnetic radiation 161. The detector 185 receives the modified output electromagnetic radiation 162 and, in response, generates an electrical signal representing the characteristics of the modified output electromagnetic radiation. As an example only, the detector 185 may be a forward fluorescence detector. Of course, other detectors may be incorporated to detect characteristics of interest, such as scattering, attenuation, phase shift, or other characteristics of interest. As a non-limiting example only, the detector may be a photomultiplier tube (PMT), a silicon photomultiplier tube (SiPM), a photodiode array, or a split detector. In some embodiments, detector 185 may represent more than one detector. In some embodiments, a second position detector may be utilized. In other embodiments, a side detector may be employed to detect side scattering or side fluorescence. In addition to detector 185, other embodiments may incorporate both a position detector and a side detector.

[0036] In some scenarios, the amplitude of the electrical signal can differ for different object types. Analyzer 187 uses the electrical signal to distinguish between different types of objects 171, 172. For example, analyzer 187 can be configured to compare the amplitude of the electrical signal with a threshold to distinguish between a first type of object 171 and a second type of object 172. Analyzer 187 may include one or more analog circuits and / or digital processors for manipulating one or more signals from one or more detectors. Just as an example, a side detector can be employed at 90 degrees relative to detector 185 to detect side scattering or side fluorescence. In the case of sperm sorting, side fluorescence allows analyzer 187 to distinguish properly oriented sperm from unoriented sperm.

[0037] The analyzer 187 may include a processor 188 having executable instructions stored thereon. In addition to those instructions 198 known for collecting, comparing, and manipulating information from the detector signal, the processor may also include instructions 192 for normalizing the intensity value of the output electromagnetic radiation from the detector, expressed as an electrical signal, based on the position of the object 172a in the liquid column 150 at the measurement region 175. The intensity value can be normalized in any number of ways. As just one example, a user may input a hand-drawn line or curve into the graphical user interface based on an initial sample of data including fluorescence intensity and position information.

[0038] The processor 188 may also include instructions 182 for identifying objects. Figure 2 It shows in Figure 1 The image depicts a cross-section of the liquid column 150 in the measurement region 175 in the xy plane. In the xy cross-section of the measurement region 175, the core stream 151 is elliptical in shape, and the liquid in the core stream 151 includes at least one object 172a suspended in a buffer solution, which may also be referred to as a sample. Sheath fluid 152 substantially surrounds the core stream 151. In the particular example used for this discussion in this disclosure, objects 171, 172 are sperm cells, and system 100 is implemented to distinguish X-chromosome sperm from Y-chromosome sperm.

[0039] Sperm cells 172a within measurement region 175 are irradiated by a focused laser beam generated by excitation source 180. Cells 171 and 172 are stained with fluorescent dye, and electromagnetic radiation is excited to cause cells 172a within measurement region 175 to emit fluorescent output electromagnetic radiation. The purpose of the generally elliptical core 151 is to orient sperm cells 172a so that their flattened sides face upwards. Figure 2The left and right sides are shown. In this orientation, the flattened side 172a of the sperm cell faces the laser 180 and the optical collection arrangement 190, respectively. When each cell 171, 172 is present in a similar orientation at the measurement region 175, the random variability based on orientation can be greatly reduced. However, the elliptical cross-section, which plays an auxiliary role in this orientation, also provides a significant latitude relative to the position of the cell within the liquid column 150.

[0040] To achieve the desired orientation, the elliptic core flow 151 exists and... Figure 2 The x-axis depicted is parallel to the long axis. Sperm cells 172a can take any number of positions along the x-axis within the core 151. Figure 2 Three representative possible locations of sperm cells 172a within the elliptical core 151 are shown, although it is understood that sperm can be located anywhere between the depicted locations. Figure 2 In the orientation shown, the first possible position of the sperm cells 172a in the core 151 is approximately at the center of the elliptical core 151 (on the optical axis 199 of the optical collection arrangement 190), the second possible position is at the top of the core 151 (above the optical axis 199), and the third possible position is at the bottom of the core 151 (below the optical axis 199). Position-related refraction of the output light rays emitted from the sperm cells 172a occurs at the liquid-gas interface 153 at different locations within the core 151. As used herein, relative positional terms such as “top,” “bottom,” “upper,” and “lower” should be understood as descriptive in relation to the relationships between the features depicted in the figures and not limiting of the claims, particularly the position of the sperm in the core 151.

[0041] When sperm cell 172a is in the first position and the liquid column 150 has the following characteristics: Figure 2 In the circular cross-section shown, in-plane rays of light emitted from sperm cell 172a are incident approximately normally on the liquid-gas interface 153. Rays emanating from points away from the center of sperm cell 172a, or from the plane shown in the figure, are not incident perfectly normally on interface 153; these rays are not considered in this simplified discussion, but those skilled in the art will know how the discussion can be extended to include them. Therefore, any light refraction occurring at the liquid-gas interface 153 occurs in a more uniform manner relative to detector 185.

[0042] Figure 3 The simplified diagram shows when sperm cell 172a is in Figure 2 The uniform refraction of output electromagnetic radiation 298 emitted from sperm cell 172a at the first position within the elliptical core 151 shown is due to electromagnetic radiation crossing interface 153. Correspondingly, from... Figure 3The in-plane density of the ray 298 emitted from the liquid column 150 is uniform with respect to the ray angle. The uniform angular density of the ray corresponds to the uniform radiation that varies with the ray angle.

[0043] In contrast, when sperm cell 172a deviates from the optical axis 199 and is closer to the top or bottom of the elliptical core 151, for example, in Figure 2 At the second and third positions of the elliptical core 151 shown, at least some of the output rays emitted from the sperm cells 172a encounter the liquid-gas interface 153 at an oblique angle. These output rays are refracted at the liquid-gas interface 153 in a non-uniform manner compared to the normal incident scenario described above. The most oblique rays are the most severely refracted. The refraction of the light causes the radiation distribution of the fluorescence emitted from the liquid column 150 to become non-uniform across the liquid-gas interface 153 and varies with the position of the cell 172a along the x-axis. That is, this refraction alters the radiation distribution of the output electromagnetic radiation emitted from the sperm cells 172a outside the liquid column 150.

[0044] For example, when cell 172a is located off the optical axis by 199 degrees, for example, in Figure 2 At the second or third position shown, the light density on the air side of interface 153 is higher when it is at an angle relative to the optical axis 199 (either a positive or negative ray angle). Therefore, the radiation on the air side of interface 153 is higher when it is at a positive or negative ray angle relative to the optical axis 199. (Positive and negative refer to...) Figure 5 Mid-ray angle The plus or minus sign. Figure 4 This is a simplified diagram illustrating light ray 299 emitted from cell 172a and exiting the liquid column 150 through the liquid-gas interface 153 when cell 172a is located in the second position of the elliptical core 151. In this scenario, the density of light rays or radiation at a positive ray angle is greater than the density of light rays parallel to the optical axis 199 or at a negative ray angle. For an optical system with a predetermined numerical aperture (NA), the amount collected by the system from cells of the same type (e.g., collection efficiency) can vary depending on whether the cell is in the first or second position. The positional dependence of the system's collection efficiency leads to inaccuracies in determining the cell type.

[0045] refer to Figure 5 Using Snell's law to determine the angle of the ray and sperm position x The analytical formula for the changing light density, where, It is the angle of the light ray emitted from the object relative to the optical axis after refraction at the liquid-gas interface. This analysis only considers rays within or tangential to the two-dimensional cross-section of the flow.

[0046] We want to solve for the relationship between light rays and angles. We can use the density to determine the location of each sperm. x The density of rays at the incident pupil of the optical collection system. This can be written as:

[0047] For our purposes, we can assume that sperm cells emit light uniformly in all directions, so the emitted light rays are relative to the angle. The density is:

[0048] In other words, from arrive Uniformly distributed. Through geometric analysis:

[0049] Among them, angle and distance x exist Figure 5 As shown in the figure. Because the flow has a refractive index... n Snell's law yields another relationship between angles:

[0050] interface The density of external light is expressed by the following formula and interface. The density of light inside is related, among which, This represents the average value of the transmissivity across the interface across two polarizations:

[0051] The following formula relates transmittance to the Fresnel reflection coefficients of s-polarization and p-polarization. and :

[0052] Use equation (7) together with the above and the following additional relation:

[0053] We have ray relative to The expression for density:

[0054] Now, the optical collection array NA is determined by the maximum ray angle. Given the sine of NA, we can solve for this angle using NA:

[0055] Ultimately, depending on the sperm location x The relative intensity of the collected light changes from... arrive Integrating equation (15) and by using x Integer values ​​equal to 0 are given after normalization:

[0056] Using the formula for ray density distribution from equation (15), the angular correlation of ray density (radiation) with different sperm locations can be expressed as follows: Figure 6 The drawing is shown in [the image]. Figure 6 In the diagram, each line represents a specific sperm location. x Rays with angle And the density changes, where the angle It is in the form of an arc. The curve graph and its location relative to... x = 0 corresponds to a series of positions within a symmetrical range, (and) Figure 6 (Corresponding to Figure 404 in the image), this is where the ray density (radiation) varies uniformly with angle. When x When positive (e.g., Figure 2 The second position in the diagram (corresponding to figure 402) represents the relative radiation angle of the orthogonal ray. It is high and for negative ray angle It is low, and when x When it is negative, the situation is exactly the opposite (for example, Figure 2 The third position in the diagram, corresponding to figure 403.

[0057] If optical devices are collected ( Figure 1 and Figure 2 In an optical collecting arrangement (190), a large numerical aperture, for example, close to 1, results in a relatively small variation in the collected optical intensity relative to position for light emitted from an object within the elliptical core. This is because virtually all light emitted from the object and directed to the right will be collected by the collecting optics regardless of the precise ray direction, and the total amount of emitted light remains constant with respect to the object position (given uniform excitation). In contrast, a small numerical aperture leads to a relatively large variation in collected intensity relative to the object position, as changes in object position affect the radiation distribution, and a small numerical aperture means that only a portion of this changed radiation distribution is collected. Practical systems can have NAs significantly smaller than 1, for example, less than 0.5, or less than 0.3. Figure 7 The series of graphs provided in the image illustrate the light collected from an object by collecting optics with different NAs as the object's position changes. x And the relative intensity changes. Figure 6 Demonstrated through Figure 7The angle captured by different numerical apertures The range.

[0058] exist Figure 7 In a series of figures, figure 412 illustrates a collection optics with a numerical aperture (NA) of 0.2 (e.g., in...). Figure 1 and Figure 2 The relative intensity of the optical collecting arrangement 190 shown is along the x-axis relative to the position; Figure 414 shows the relative intensity along the x-axis relative to the position for a collecting optics with an NA of 0.4; Figure 416 shows the relative intensity along the x-axis relative to the position for a collecting optics with an NA of 0.6; Figure 418 shows the relative intensity along the x-axis relative to the position for a collecting optics with an NA of 0.8; and Figure 419 shows the relative intensity along the x-axis relative to the position for a collecting optics with an NA of 0.9. Figure 6 and Figure 7 It is clear that, compared to collecting optics with a larger NA, collecting optics with a smaller NA produce a larger variation in the intensity of the collected light relative to the object's position. Furthermore, collecting optics with a larger NA collect light with a wider range of refraction angles compared to those with a smaller NA, and therefore have a higher overall collection efficiency.

[0059] Especially regarding applications of sperm identification or classification, it is understandable that the major axis of the ellipse of Core 151 ( Figure 1 and Figure 2 The length can be approximately 50 µm, thus providing approximately 25 µm of sperm in latitude to move in any direction. (Return to reference) Figure 7 It can be seen that when the object is approximately 17 µm off-center, a NA of 0.2 captures only about 90% of the object's relative intensity. Similarly, for an object approximately 17 µm off-center, a NA of 0.4 captures only 92% of the relative intensity, and at the same location, a NA of 0.6 captures slightly more than 94% of the relative intensity. It can be further understood that the NA for collecting optics for sperm classifiers can be between approximately 0.3 and 0.6. Although... Figure 7 While the benefits of increasingly larger numerical apertures have been demonstrated, these apertures are becoming increasingly expensive and have shallower depth of field, meaning that larger apertures must be placed closer to the nozzle. However, there are limitations on how close the collection optics can be placed in sperm sorting applications. In typical sperm sorting instruments, the aperture can be between 0.5 and 0.6. The embodiment described herein, which corrects for the positional correlation of the intensity of the measurement, allows smaller numerical aperture collection optics to perform functions similar to those of higher numerical aperture collection optics.

[0060] Sperm are located in core 151 near Figure 2 At the second and third positions, the electromagnetic radiation emitted is significantly lower in total intensity, which is ultimately detected for analysis and differentiation. In fact, at high event rates (60,000 events per second and greater), Core 151 can have an elliptical major axis of approximately 50 µm in length. Some sperm will deviate from the center by up to 20 µm or even as much as approximately 25 µm to either side of the first position. Against a background of extremely bright and closely correlated fluorescent signals, this variation can mask approximately 4% of the difference in stained nuclear DNA that distinguishes sperm carrying the X chromosome from those carrying the Y chromosome.

[0061] Furthermore, increasing the number of events at a given sperm concentration in the sample within the buffer requires increasing the sample volume per unit time that traverses the measurement region in the column. Increasing the number of events detected per second in this manner also increases the elliptical cross-section of the core flow within the column, including the length of its major axis. As a natural consequence, and as those skilled in the art will know, generally, increasing the sorting speed by increasing the sample flow rate reduces the sensitivity of the sperm sorting equipment. Therefore, the embodiments described herein not only improve the accuracy of sperm sorting at conventional speeds but also provide sperm sorting at increased total speeds based on throughput without suffering a loss of accuracy.

[0062] exist Figure 8 The flowchart illustrates a method for identifying an object traveling in a liquid column in the presence of positional changes. The process includes generating a 510-column liquid stream containing the object at different locations within it. The liquid stream can be a coaxial flow produced by an "air-jet" flow cytometer. Such a liquid stream may include a core stream with an elliptical cross-section having a major axis along which the object can be positioned. The core stream may be coaxially contained within a sheath fluid. In some embodiments, the liquid stream may have a gas-liquid interface, thereby allowing refraction to occur. In other embodiments, the liquid stream may be formed within a cuvette or microfluidic channel. In this case, a liquid-glass interface and possibly a glass-gas interface may be present, and the emitted light may be refracted twice. This double-refracted light is expected to greatly benefit from the angular correlation corrections of some embodiments.

[0063] The process continues by generating 520 excitation electromagnetic radiation and directing 530 excitation electromagnetic radiation toward an object in the liquid column at the measurement region. The object within the liquid column emits output electromagnetic radiation in response to the excitation electromagnetic radiation at the measurement region. 540 output electromagnetic radiation is collected from the object in the liquid column (including objects at different positions within the liquid column at the measurement region), and a detector generates an electrical signal 550 in response to the intensity of the output electromagnetic radiation collected by the optical arrangement.

[0064] Next, the analyzer or other suitable device normalizes the intensity represented by the output signal of 560 based on the position of the object in the liquid column. Normalization can be performed using correction, which deviates from the central axis (towards and including) Figure 2 The generated signal (based on the second and third positions) is amplified by an appropriate correction factor based on their positions. Figure 7 The appropriate correction factor value is determined. Once normalization is achieved through correction, the method continues by identifying 570 objects of the first type from other objects. Identification can occur within the flow cytometer analyzer and can include one or more additional operations. For example, a univariate histogram showing the distribution of fluorescence intensity can be generated. A bivariate histogram can also be generated using the corrected signal and further calculated values. These corrected and calculated values ​​can be compared to a gating region in the flow cytometer analyzer or to a lookup table to distinguish the first type of object from other types of objects.

[0065] As an example, sperm can be identified as sperm carrying the X chromosome or sperm carrying the Y chromosome. Furthermore, in addition to secondary quenching dyes, sperm can be stained with DNA-selective dyes. Quenching dyes typically penetrate damaged sperm cells, such as dead or dying sperm cells, and significantly reduce the fluorescence produced by DNA-selective dyes associated with those damaged cells. These quenched cells are effectively removed from the closely related population undergoing identification / classification. In this way, the system can distinguish live or viable sperm cells from dying or damaged sperm cells. The system can also distinguish viable X-chromosome-carrying sperm cells from all remaining cells, Y-chromosome-carrying sperm cells from all remaining cells, or even simultaneously distinguish viable X-chromosome-carrying and Y-chromosome-carrying sperm cells from all other sperm cells.

[0066] Figure 9 It shows basically with Figure 1 and Figure 2 In a first embodiment of a discrimination system similar to the one depicted, output electromagnetic radiation 161 emitted from an object 172a located in the measurement area is collected by an optical collection arrangement 190. The optical collection arrangement 190 may include a collection lens that focuses the modified output electromagnetic radiation onto a detector 185. In the depicted embodiment, this detector functions both as a measurement of the characteristics of the modified output electromagnetic radiation and as a position detector 186 for determining the position of the object 172a within the spool 151 of the liquid column 150.

[0067] The detector 185, suitable for determining both the characteristics of the modified output electromagnetic radiation 162 and the position of the object 172a in the measurement region, may include a split detector or a PMT, SiPM, pin-type photodiode detector array, or the like. These detectors may be located in the image plane or the Fourier plane of the object to determine its position. In the image plane, the detector directly measures the object's position, while in the Fourier plane, position information is extracted from the lateral intensity distribution (e.g., left-right asymmetry).

[0068] Flow cytometry applications typically require highly sensitive (down to single-photon counting) and fast (objects moving 10 µm at approximately 20 m / s) detectors. Detectors with the necessary speed and sensitivity are typically those that provide internal gain. In photomultiplier tubes (PMTs), also known as pixelated avalanche photodiodes or silicon photomultipliers (SiPMs), single photon generation can reach up to approximately 10-10. 6 The cascade of electrons. Both detector types are commercially available as detector arrays. SiPMs may be better suited for detector arrays designed to determine the location of objects because they are fabricated on a silicon wafer using standard techniques. Some detectors, such as SiPMs, are particularly well-suited for placement in the Fourier plane to distribute light over a larger area of ​​the detector.

[0069] Figure 10 An alternative embodiment is shown in which a portion of the power of the modified output electromagnetic radiation 162 is redirected by a beam splitter 191 or other suitable optics. Most of the modified output electromagnetic radiation 162 is redirected and focused onto detector 185. In this embodiment, detector 185 includes a first detector 176 for detecting the characteristic of interest. The first detector 176 can be any detector generally suitable for quantifying a specific characteristic of interest. In typical flow cytometry applications, photodiodes, photomultiplier tubes (PMTs), and silicon photomultipliers can be particularly well-suited for detecting the intensity of scattered or fluorescent electromagnetic radiation.

[0070] Beam splitter 191 may include dielectric mirror 197; however, those skilled in the art will appreciate that other suitable optical components, such as cubic beam splitters, prism beam splitters, and the like, may be used to redirect a portion of the modified output electromagnetic radiation 162 power. Regardless of how the output power is split, the first beam portion 164 is guided along a first path to the detector, and the second beam portion 165 is guided along a different path to a second detector 173 in the form of a position detector 177. The position detector may be a camera, a position-sensitive device (“PSD”) (such as an isotropic sensor or charge-coupled device (CCD)), a split detector, a detector array of a PMT, a SiPM, a pin photodiode, or the like.

[0071] Turn Figure 11 A simulation was performed to demonstrate the feasibility of using split-type detectors to determine positional information in a flow cytometry system. The simulation employed a split-type SiPM detector comprising 3 mm SiPM detectors mounted side-by-side. The intersection of the detectors was calibrated to a central x-coordinate position, simulating the interrogation of the laser beam axis and the center of symmetry of the liquid column. A 1.5 mm spot size was swept across the split-type detectors at x-positions ranging from approximately -12 mm to 12 mm, and the relative intensity measured by each detector was recorded. First graph 601 shows the relative intensity recorded for a beam spot from one of the detectors at x-positions ranging from approximately -12 mm to approximately 12 mm, where the x-position corresponds to the plane of the SiPM detector. Graph 602 shows the corresponding relative intensity of beam spots detected by the other detectors in the range of x-positions from approximately -12 mm to approximately 12 mm. As can be seen, positional differences between the two detectors, based on the x-position of the 1.5 mm spot, result in different measured intensities. These differences are position-dependent and can be converted into approximate positional information by processing devices. Noise was included in the simulation, but it is independent of the intensity. At maximum intensity, the noise corresponds to a coefficient of variation of 0.8%. Simulations demonstrate that the x-position in a split detector arrangement can be determined based on the relative intensity detected by each SiPM in the split detector arrangement. Those skilled in the art will understand that embodiments of the invention are not limited to this configuration, and other detector configurations suitable for determining the position of particles within a liquid column are contemplated herein. This is merely an example; other detectors can be employed in a split detector arrangement. Those skilled in the art will understand that the detectors should have low noise because the combined signal must have a sufficiently low coefficient of variation.

[0072] Figure 12This demonstrates how incorporating positional correction for sperm nuclei in a liquid column leads to significantly improved experimental results in distinguishing between sperm nuclei carrying the X chromosome and those carrying the Y chromosome. Sperm nuclei stained with Hoechst 33342 were processed using a Genesis III sperm sorting instrument manufactured by Cytonome. This instrument is equipped with a SiPM splitting detector. Sample and sheath pressure were adjusted to establish an event rate of 35,000 events per second. Nuclei were probed using a Coherent Genesis CW-355 laser at an average power of 150 mW. Graph 610 depicts a bivariate histogram showing the sum of fluorescence intensities from each detector in the splitting detector, plotted at the position Δ of the nuclei in the control liquid column. As previously described, the range of position Δ represents the major axis of the elliptical core along which the nucleus can enter the measurement region. The population 612 of X-chromosome-carrying nuclei is considered crescent-shaped. As expected, the measured intensity is maximum near position Δ, with the curve contracting downwards as the nucleus moves away from the central position. Population 614, which carries the nucleus of the Y chromosome, is considered the second crescent below the X population, and similarly, the highest intensity occurs near position 0 Δ, where the relative intensity is significantly reduced when the nucleus moves away from the central position.

[0073] Curve graph 620 presents a univariate histogram of summed fluorescence intensity corresponding to the intensity plotted in curve graph 610. Although different populations 612 carrying X chromosome nuclei and 614 carrying Y chromosome nuclei are visible, the contrast between curve graph 610 and curve graph 620 clearly shows the increasing overlap between off-center X chromosome-carrying sperm nuclei and center-aligned Y chromosome-carrying sperm nuclei. In fact, the peak-to-trough ratio was calculated to be 76.8%.

[0074] According to an embodiment of the invention, the correction factor 616 is represented as a curved line in graph 610. The correction factor 616 represents the degree of correction required to remove variations in the detected fluorescence intensity introduced by the random location of the event. Corresponding corrections are applied to the fluorescence values ​​depicted in graph 630 to produce a corrected population 632 of nuclei carrying the X chromosome and a corrected population 634 of nuclei carrying the Y chromosome. The corrected population 632 of nuclei carrying the X chromosome forms a generally rectangular shape and no longer demonstrates fluctuations based on the position of the nuclei in the liquid column. A more pronounced difference between the corrected population 632 of nuclei carrying the X chromosome and the corrected population 634 of nuclei carrying the Y chromosome can be seen in graph 630. Graph 640 shows a corresponding univariate histogram with a peak-to-valley ratio of 94% between the corrected population 632 of nuclei carrying the X chromosome and the corrected population 634 of nuclei carrying the Y chromosome. The stark contrast between graphs 620 and 640 is visually apparent. Furthermore, this difference is quantifiable, exceeding the previous difference by 17.2 percentage points.

[0075] Figure 13 Results of an example of incorporation correction according to the embodiments described herein are shown. Live sperm stained with Hoechst 33342 were processed using a Genesis III sperm sorting instrument manufactured by Cytonome. Sample and sheath pressure were adjusted to achieve an event rate of 43,000 events per second, and sperm were probed using a Coherent Genesis CW-355 laser operating at an average power of 100 mW. Graph 710 shows a bivariate histogram of summed fluorescence intensity and the relative position of live sperm in the core stream. Similarly, the population 712 carrying the X chromosome can be considered as the first population above the population 714 carrying the Y chromosome. A correction factor 716 for normalizing the summed intensity values ​​is also depicted in graph 710. Graph 720 shows a univariate histogram of the uncorrected summed intensity and demonstrates a peak-to-trough ratio of 75.3% between the population 712 carrying the X chromosome and the population 714 carrying the Y chromosome.

[0076] Curve graph 730 provides a type of bivariate histogram commonly used in sperm classification applications. In this case, the corrected positive fluorescence intensity is plotted against the control side fluorescence. Positive fluorescence versus lateral fluorescence histograms are useful for classifying live sperm because the lateral fluorescence provides information about the orientation of each cell. In contrast, sperm nuclei are sonicated and removed from the aspherical sperm head. Therefore, orientation is not an issue when classifying sperm nuclei. For this reason, nuclei are easier to classify and are often used to calibrate sperm classification flow cytometers. Curve graph 730 depicts the corrected population of sperm carrying the X chromosome 732 and the corrected population of sperm carrying the Y chromosome 734.

[0077] Similar to the previous example, curve 740 still correlates with the corrected positive fluorescence of curve 730 on the Y-axis. In the univariate plot of curve 740, the corrected X-chromosome-carrying sperm population 732 and the corrected Y-chromosome-carrying sperm population 734 can be viewed as having more pronounced peaks with a machine-calculated peak-to-trough ratio of 81.0%. And similarly, the corrected histogram shows a significant improvement over curve 720, which demonstrates the corrected position for live sperm.

[0078] On the other hand, the embodiments described herein can provide systems and methods that are substantially easy to perform alignment processes in flow cytometry. For example, in the case of sperm, the measurement area, detector, and even the structures forming the sheath fluid must be properly and precisely aligned to generate and collect sufficiently clear signals to distinguish very bright and closely related populations of X-chromosome-carrying sperm from those carrying Y-chromosome-carrying sperm. Even in precise and proper alignment, oriented sperm in the liquid column can have any number of positions along the long axis of the core flow. As stated above regarding… Figures 3 to 7 This means that even when the components of the flow cytometer are perfectly aligned, there is an angular correlation with the detected output electromagnetic radiation. This angular correlation introduces noise-like variations because cells can be randomly positioned within the core.

[0079] In commercial sperm sorting applications, technicians typically perform several process adjustments, followed by several fine adjustments to multiple components across multiple axes to align the instrument. Due to the instrument's sensitivity to each adjustment, the extremely close correlation of the detected signals, and the number of adjustments that may be made, this alignment can be a time-consuming task for technicians operating sperm sorting instruments. Machine alignment for commercially sorted sperm can take several minutes, even up to 5 minutes, when switching between samples. After cleaning or otherwise removing, replacing, or adjusting other components that require calibration, technicians may need 5 minutes, 15 minutes, and in rare cases, up to 30 minutes, to properly align the instrument for commercially sex-sorted sperm.

[0080] Figure 14 Results are shown for an example of a process that significantly reduces alignment in identifying sperm nuclei. Sperm nuclei stained with Hoechst 33342 were processed using a Genesis III sperm classifier manufactured by Cytonome. This instrument is equipped with a SiPM splitting detector. Positive fluorescence detection resulted in alignment in less than one minute, leading to coarse alignment. Sperm nuclei were probing at an event rate of 33,000 nuclei per second and with a Coherent Genesis CW-355 laser operating at an average power of 150 mW. Curve graph 810 shows a bivariate histogram plotting the summed positive fluorescence of the control at each event, plotted against the position detected by SiPM. Misalignment was evident in each of the populations 812 carrying X chromosome nuclei and 814 carrying Y chromosome nuclei. In the misalignment, the crescent shape was asymmetrical, and the fluorescence intensity values ​​decreased significantly in the positive x-direction compared to the negative x-direction. The same skewness was demonstrated in the population 814 carrying Y chromosome nuclei.

[0081] The correction factor 816 is represented as a line between the two populations. This correction factor 816 indicates the degree of correction that will be performed on the summed fluorescence values ​​at each x-direction. In other words, the correction factor 816 represents a curved line that will be normalized to a flat line by the correction. Each summed fluorescence value at the corresponding x-position along the line receives the same amount of increase or decrease as the correction factor 816.

[0082] The distortion caused by coarse alignment is more evident in the fluorescence intensity histogram of curve 820, where the increased overlap between population 812 carrying the nucleus of the X chromosome and population 814 carrying the nucleus of the Y chromosome results in a peak-to-valley ratio of 72.3%.

[0083] In graph 830, the corrected sum of positive fluorescence values ​​is plotted as a bivariate histogram against the detected location for each event. It can be seen that, similarly, by normalizing the fluorescence intensity values ​​using a correction factor 816 based on cell location, two clean cell populations emerge. The corrected population 832 carrying X chromosome nuclei and the corrected population 834 carrying Y chromosome nuclei are more clearly and distinctly grouped in graph 830. Importantly, the orthogonality of these populations is converted into a univariate fluorescence intensity histogram as seen in graph 840, where the two distinct univariate peaks have a calculated peak ratio of 94.4%.

[0084] In addition to using corrections, some embodiments disclosed herein also include elements to reduce variations in the intensity of the collected light relative to the position of an object in the flow stream. Some embodiments described herein can provide modified output light with measured intensity variations of less than about 3%, or less than about 2%, or even less than about 1%, when the positional deviation of the object is less than 60% of the radius of the flow stream away from its center along an axis perpendicular to the optical axis. Many applications are sensitive to intensity measurement errors, which can be caused by various root causes. Since reducing intensity fluctuations by precisely controlling the position of the object within the flow stream is difficult, it is more useful to reduce variations in the intensity of the collected light relative to the object's position by carefully designing the optical collection arrangement. For applications such as X / Y sperm classification, the situation is typically that two or more cell populations are separated based on the differences in measured fluorescence intensity between these populations. If the fluctuation in collected light intensity caused by random positional variations is greater than the nominal difference in fluorescence intensity between the two populations, it is impossible to distinguish them simultaneously with high yield and high purity. The fluorescence intensity difference between X and Y sperm cells is typically only a few percentage points (e.g., about 4% for bovine sperm). Current sperm classifier systems can theoretically achieve high throughput by increasing the core flow rate, but this has the effect of increasing the core width. Therefore, the sperm position within the core of the flow stream becomes highly uncertain. This positional uncertainty, and the resulting fluctuations in the collected fluorescence intensity, limit the maximum throughput of current sperm classifier systems to a level that does not mask the small fluorescence intensity differences between X and Y sperm.

[0085] You can refer to this. Figure 6 and Figure 7 Let's understand a method for intensity-position correction. Figure 6 The brackets highlight the integration region corresponding to a fluorescence collecting optics with a given NA. Figure 7 China provides targeted Figure 6 A graph showing the collected intensity variation of NA relative to the object's location. Figure 7In this process, for a given NA, an integral is performed over the fluorescence collection region, allowing the intensity of the collected light to be plotted based on each sperm location. Figure 7 It is evident that increasing the NA of the collecting optics helps reduce the influence of the object location on the fluorescence intensity collected by the collecting optics.

[0086] In some embodiments, the collecting optics can be modified with elements that reduce the variation in the intensity of the collected light relative to the object location (e.g., Figure 1 and Figure 2 The optical collecting arrangement 190 is as described above. This embodiment is described in more detail in U.S. Patent Application No. 16 / 133,531, which is incorporated herein by reference. According to some of these embodiments, the collecting optics operate by masking certain rays in "angle space," that is, the collecting optics selectively collect, attenuate, and / or block rays from different angles. The rays are used to achieve the desired intensity contrast positional variation curve. In practice, an "angle-space" masking function can be applied at the pupil of an optical system (e.g., entrance pupil, exit pupil, or aperture stop), where the position and angle of the rays intersecting the pupil plane are determined. Correspondingly, in some embodiments, the collecting optical arrangement achieves a desired (e.g., a flatter) intensity contrast position curve by preferably collecting light rays at higher angles (pointing away from the optical axis) and excluding some lower-angle light rays.

[0087] Figure 15 and Figure 16 This demonstrates how excluding low-angle reflected rays at a given NA flattens the intensity-to-position curve. Excluding low-angle rays removes the rays that produce the most variation in the intensity-to-position curve, while angular changes in radiation at high positive angles tend to eliminate the corresponding changes at high negative angles. Figure 15 This shows the relative radiation angle to the ray for different positions of the object along the x-axis. The curve, where the angle It takes the form of an arc. Figure 15 In the middle, each figure is related to... Figure 5 The position x of the object within the core of the flow indicated in the middle corresponds to the flow direction. Figure 15 The brackets indicate that rays with an angular value less than 0.3 rads are excluded. Figure 15 (The bottom brackets in the text) and when rays with an angle value less than 0.4 rads are excluded ( Figure 15 The portion of light excluded by the collecting optics for each position x (in the top brackets) will be the portion of light excluded by the collecting optics for each position x.

[0088] Figure 16The figure shows the relative collected light intensity of the object along the x-axis to the position when the angle is not excluded (Figure 900), when the angle between -0.3 rad and +0.3 rad is excluded (Figure 903), and when the angle between -0.4 rad and +0.4 rad is excluded (Figure 904). Figure 16 The diagram shows that when lower-angle rays are excluded, the relative intensity versus location plot exhibits a smaller intensity variation relative to location.

[0089] Figure 17 Experimental results are demonstrated for incorporating both software-based position correction and hardware-based components into the light collection path to reduce the variation in collected light intensity relative to the object position, as described. Sperm nuclei stained with Hoechst 33342 were processed using a Genesis III sperm classifier manufactured by Cytonome. The sperm classifier is equipped with a SiPM splitting detector having wires placed in the light collection path to exclude low-collection-angle electromagnetic radiation generated from the sperm nuclei. Suitable wires and other components for blocking low-collection-angle electromagnetic radiation are described in U.S. Patent Application No. 16 / 133,531.

[0090] The sample and sheath pressure were adjusted to achieve an event rate of 60,000 events per second, and nuclei were probed using a Coherent Genesis CW-355 laser operating at an average power of 90 mW. As can be seen in graph 1010, the guide wire mitigates some of the influence of intensity correlation on nucleus location within the liquid column. However, a significant reduction in relative intensity still exists as the nucleus moves further along the x-axis in the positive direction. Populations 1012 of X-chromosome-carrying sperm nuclei and 1014 of Y-chromosome-carrying nuclei are considered to droop significantly in the positive direction along the x-axis. The corresponding peak-to-trough ratio calculated from the fluorescence intensity histogram of graph 1020 is 81.5%. Similarly, X-chromosome-carrying nuclei located towards one end of the liquid column are not sufficiently detected. Therefore, the summed fluorescence intensity of nuclei at this end has a similar intensity value to that of the centrally located Y-chromosome-carrying nuclei within population 1014. This skewness is evident in the univariate histogram of curve graph 1020, manifested as a downward shift of the shoulders and an exaggerated peak in population 1014 carrying the Y chromosome nucleus.

[0091] The correction factor 1016 is shown on graph 1010. For each location, a correction value is added to the corresponding correction factor for the detected fluorescence intensity. Graph 1030 shows a bivariate histogram of the populations 1032 with corrected nuclei carrying the X chromosome and 1034 with corrected nuclei carrying the Y chromosome; these two populations are more clearly rectangular. Graph 1040 provides a corresponding univariate histogram of the corrected summed intensity values, independent of the location of each event. The populations 1032 with corrected nuclei carrying the X chromosome and 1034 with corrected nuclei carrying the Y chromosome show significantly more equal peak heights and a peak-to-valley ratio of 92.6%.

[0092] The foregoing description of various embodiments has been presented for illustrative and descriptive purposes and for non-limiting purposes. The disclosed embodiments are not intended to exaggerate or limit possible implementations to the disclosed embodiments. Many modifications and variations are possible based on the above teachings.

Claims

1. A discrimination system, the discrimination system comprising: A collecting optical device is used to collect the output electromagnetic radiation of sperm carrying the X chromosome and sperm carrying the Y chromosome within the measurement area. A split detector that generates an electrical signal in response to the intensity of the output electromagnetic radiation collected by the collecting optics, wherein the split detector detects the position of sperm cells within the measurement area; as well as An analyzer that stores instructions for distinguishing between sperm carrying the X chromosome and sperm carrying the Y chromosome.

2. The identification system according to claim 1, wherein, The split detector is not located on the image plane of the collecting optics.

3. The identification system according to claim 1, wherein, The split detector is selected from the group consisting of: PMT split detector and SiPM split detector.

4. The identification system according to claim 1, wherein, The analyzer includes further instructions stored thereon for normalizing the intensity of the output electromagnetic radiation represented in the electrical signal and correcting the intensity of the output electromagnetic radiation represented in the electrical signal based on the location of the sperm cell in the measurement area.

5. The identification system according to claim 4, wherein, The amount of correction is determined for each position on the main axis of the core flow in the measurement area, and wherein the correction is based on the detection position of each sperm cell on the main axis of the core flow applied to the electrical signal representing each sperm cell.

6. The identification system according to claim 1, wherein, The sperm cells are located within an elliptical sample liquid column, in which the sample liquid and the outer layer of the sheath fluid are coaxially formed, and the sheath fluid is generally cylindrical.

7. The discrimination system of claim 1, further comprising an element for modifying the output electromagnetic radiation to improve the uniformity of the output electromagnetic radiation collected by the collecting optics from the sperm cells at different locations.

8. The identification system according to claim 1, wherein, The split detector is located on the Fourier plane.

9. The discrimination system of claim 1, further comprising a mask for attenuating low-angle refracted light rays of the output electromagnetic radiation.

10. The identification system according to claim 1, wherein, The cover includes wires.

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