Systems and method for correction of positionally dependent electromagnetic radiation detected from objects within fluid column

Abstract

To provide favorable systems and method for correction of positionally dependent electromagnetic radiation detected from objects within a suitable fluid column.SOLUTION: A discrimination system forms a fluid column and interrogates objects within the fluid column with an excitation source. A fluid column forming a structure creates a fluid column containing objects at differing positions within the fluid column, and an excitation source generates excitation electromagnetic radiation directed toward objects in the fluid column at a measurement region. An optical arrangement collects output electromagnetic radiation emanating from the excited objects disposed within the fluid column and directs the output electromagnetic radiation to a detector. An analyzer reduces the positional dependency of the detected intensity by normalizing a value based on the position of each object.SELECTED DRAWING: Figure 1

Description

Background Art

[0001] Object discrimination devices and techniques distinguish different types of objects, such as objects having different characteristics. These devices and techniques are particularly useful for analyzing and further sorting cells according to a specified characteristic of interest. Some cell sorting techniques rely on light emitted from cells or stained cells to identify the cell type. In some embodiments, cells moving in a fluid column are exposed to an excitation source to generate output electromagnetic radiation for detection. A first type of cell or a cell having a particular characteristic generates output electromagnetic radiation having some characteristics, such as wavelength and / or intensity, different from other cells. Such differences function as a basis for discriminating and sorting cell types.

Summary of the Invention

Means for Solving the Problems

[0002] Some embodiments relate to a discrimination system that discriminates different types of objects based on electromagnetic radiation emitted from an object disposed within a fluid column. The fluid column forming the structure creates a fluid column containing objects at different positions within the fluid column, and an excitation source generates excitation electromagnetic radiation directed at the objects in the fluid column in a measurement region. The objects within the fluid column emit output electromagnetic radiation in response to the excitation electromagnetic radiation. An optical configuration collects the output electromagnetic radiation from the objects, and a detector generates an electrical signal responsive to the intensity of the output electromagnetic radiation. An analyzer includes stored instructions for i) normalizing the intensity of the output electromagnetic radiation represented in the electrical signal based on the position of the objects in the fluid column, and ii) discriminating a first type of object from other objects.

[0003] According to some embodiments of the detection system, an optical configuration collects output electromagnetic radiation from an object in a fluid column, and a detector generates an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical configuration. An analyzer includes stored instructions to i) normalize the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the object in the fluid column, and ii) discriminate a first type of object from other objects.

[0004] According to other embodiments, a method of discriminating an object is initiated by creating a fluid column containing the object at different positions within the fluid column. Excitation electromagnetic radiation is directed at the object in the fluid column in 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 responsive 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 position of the object in the fluid column, and a first type of object is discriminated from other objects. This specification also provides, for example, the following items. (Item 1) A discrimination system, a fluid column forming structure that creates a fluid column containing an object at different internal positions, an excitation source that generates excitation electromagnetic radiation directed at the object in the fluid column in a measurement region, wherein the object in the fluid column emits output electromagnetic radiation in response to the excitation electromagnetic radiation, an optical configuration that collects output electromagnetic radiation from the object, a detector that generates an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical configuration, an analyzer that stores instructions to i) normalize the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the object in the fluid column and ii) discriminate a first type of object from other objects, and a discrimination system comprising the same. (Item 2) The detector includes a first detector, and the system further includes a second detector for detecting the position of an object in the fluid column, the discrimination system according to item 1. (Item 3) The second detector includes a position detector, the discrimination system according to item 2. (Item 4) The position detector is a detector selected from the group consisting of a camera, a CCD, a PSD, a SiPM segmented detector, and a photodiode array, the discrimination system according to item 3. (Item 5) The detector includes a first detector that i) generates an electrical signal in response to the intensity of the output electromagnetic radiation collected by the optical configuration, and ii) detects the position of an object in the fluid column, the discrimination system according to item 1. (Item 6) The first detector is not arranged on the image plane of the optical configuration, the discrimination system according to item 5. (Item 7) The first detector includes a detector selected from the group consisting of a PMT segmented detector, a SiPM segmented detector, a photodiode array, an array of PMTs, and an array of SiPMs, the discrimination system according to item 5. (Item 8) The fluid column includes an object in a core flow having a generally elliptical cross-section contained within a sheath fluid having a generally circular cross-section, and the object in the fluid column is arranged at different positions on the major axis of the generally elliptical cross-section of the core flow, the discrimination system according to item 1. (Item 9) The instructions stored in the analyzer for normalizing the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the object in the fluid column apply a correction to the intensity of the output electromagnetic radiation represented by the electrical signal, the discrimination system according to item 8. (Item 10) A correction factor is determined for each position on the major axis of the core flow, and the correction factor is applied to each event based on the position on the major axis of the core flow, the discrimination system according to item 9. (Item 11) The discrimination system according to item 1, wherein the object of the first type includes an object selected from the group consisting of viable sperm having an X chromosome and viable sperm having a Y chromosome. (Item 12) The discrimination system according to item 1, wherein the analyzer includes instructions for discriminating viable sperm having an X chromosome from viable sperm having a Y chromosome. (Item 13) The discrimination system according to item 1, wherein the object includes sperm disposed in an elliptical column of a sample fluid, the sample fluid is formed coaxially with an outer layer of a sheath fluid, and the sheath fluid generally has a cylindrical shape. (Item 14) The discrimination system according to item 1, further comprising an element for changing the output electromagnetic radiation so as to increase the uniformity of the output electromagnetic radiation collected by the optical configuration for objects at different positions. (Item 15) A detection system, an optical configuration for collecting output electromagnetic radiation from an object in a fluid column, a detector for generating an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical configuration, an analyzer storing i) instructions for normalizing the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the object in the fluid column and ii) instructions for discriminating an object of a first type from other objects, and comprising a detection system. (Item 16) The detection system according to item 15, wherein the detector includes a first detector and a second detector, and a mathematical operation on the electrical signal provided by the first detector is related to the position of the object in the fluid column based on a signal from the second detector. (Item 17) The detection system according to item 16, wherein the second detector includes a position detector. (Item 18) The detection system according to item 17, wherein the position detector is a detector selected from the group consisting of a camera, a CCD, a PSD, a SiPM segmented detector, and a photodiode array. (Item 19) The detector according to item 15, comprising: i) generating an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical configuration; and ii) a first detector for detecting the position of an object in the fluid column. (Item 20) The detection system according to item 19, wherein the first detector includes a detector selected from the group consisting of a PMT segmented detector, a SiPM segmented detector, a photodiode array, an array of PMTs, and an array of SiPMs. (Item 21) The detection system according to item 19, wherein the first detector is disposed outside the image plane of the optical configuration. (Item 22) The fluid column includes an object in a generally elliptical cross-section core flow contained within a generally circular cross-section sheath fluid, the object in the fluid column being located at different positions on the major axis of the generally elliptical cross-section of the core flow, and the instructions stored in the analyzer that normalize the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the object in the fluid column, apply a correction to the intensity of the output electromagnetic radiation represented by the electrical signal. The detection system according to item 15. (Item 23) The detection system according to item 22, wherein a correction factor is determined for each position on the major axis of the core flow, and the correction factor is applied to each event based on the position on the major axis of the core flow. (Item 24) The detection system according to item 15, wherein the first type of object includes an object selected from the group consisting of viable sperm having an X chromosome and viable sperm having a Y chromosome. (Item 25) Creating a fluid column containing objects at different internal positions; Generating excitation electromagnetic radiation; Directing the excitation electromagnetic radiation at an object in the fluid column in a measurement region, wherein the object in the fluid column emits output electromagnetic radiation in response to the excitation electromagnetic radiation. Collecting output electromagnetic radiation from the object; Generating an electrical signal responsive to the intensity of the collected output electromagnetic radiation; Normalizing the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the object in the fluid column; Discriminating a first type of object from other objects; A method comprising. (Item 26) The method according to item 25, further comprising detecting the position of the object in the fluid column. (Item 27) The fluid column contains an object in a generally elliptical cross-section core flow contained in a generally circular cross-section sheath fluid, and the object in the fluid column is arranged at different positions on the major axis of the generally elliptical cross-section of the core flow. The method according to item 25. (Item 28) The method according to item 27, further comprising applying a correction to the intensity of the output electromagnetic radiation represented by the electrical signal. (Item 29) The method according to item 28, further comprising determining a correction factor for each position on the major axis of the core flow and applying the correction factor to each event based on the position on the major axis of the core flow. (Item 30) The method according to item 25, wherein the first type of object includes an object selected from the group consisting of viable sperm having an X chromosome and viable sperm having a Y chromosome. (Item 31) The method according to item 25, wherein the analyzer includes instructions for discriminating viable sperm having an X chromosome from viable sperm having a Y chromosome. (Item 32) The method according to item 25, further comprising an element for changing the output electromagnetic radiation to increase the uniformity of the output electromagnetic radiation collected from objects at different positions.

Brief Description of the Drawings

[0005]

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Best Mode for Carrying Out the Invention

[0006] The figures are not necessarily to scale. Like numerals used in the figures refer to like components. However, it will be understood that the use of numerals to refer to a component in a given figure is not intended to limit the component in another figure denoted by the same numeral.

[0007] The embodiments described herein relate to devices, systems, and methods for discriminating between different types of objects. The objects emit output light in response to excitation light directed at the objects in a fluid column, such as a flow stream. As used herein, the term "emit" refers to both reflected electromagnetic radiation, such as light, and fluorescence-emitting electromagnetic radiation. 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 can include light directly reflected or fluorescence-emitted from the object and light reflected or fluorescence-emitted by dyes or pigments associated with the object. In some embodiments, cell types are distinguished based on the intensity of the output electromagnetic radiation emitted from the objects. The intensity can be specified as a total intensity, such as a peak intensity or an integrated area under the intensity signal. Certain embodiments described herein relate to the discrimination between X-chromosome sperm cells and Y-chromosome sperm cells. Further embodiments relate to discriminating germ cells having an X chromosome from objects other than germ cells having an X chromosome, including germ cells having a Y chromosome and non-viable cells of both sexes.

[0008] The techniques of the present disclosure can more generally be applied to the discrimination between any different types of objects, as long as the output electromagnetic radiation emitted from one object type creates a distinguishable difference in at least one characteristic when compared to the electromagnetic radiation emitted from another object type. In some of the examples provided, the fluid column is a flow stream having a curved boundary or interface where refraction of the electromagnetic radiation can occur. For example, the curved boundary of the fluid column can generally be a circular cross-section. The fluid column can be delimited by a solid wall such as within a cuvette or within a microfluidic channel, or can be injected into the air such as in a jet-in-air flow cytometer. The object can move through a central core that is shaped by a sheath fluid that at least partially surrounds the central core along the fluid column. In a sperm sorting application, the central core can include a core flow of a sample fluid containing sperm cells. The core flow can generally be shaped in a ribbon form, or can generally have an elliptical cross-section to orient aspherical sperm cells. The electromagnetic radiation emitted from the object faces at least one optical refraction boundary between the object and another material, such as an interface between the fluid column and the air.

[0009] At least in part due to the different refractive properties of the sheath fluid and air, the light collection efficiency outside the fluid column of light emitted from an object within the column depends on the position of the object in such a system. The light collection efficiency that varies with position must precisely quantify the light emitted from the object, and such precision is detrimental in applications where it is limited by random (not directly observable) position fluctuations of the object. In particular, in the case of sex-differentiated sperm, such a system attempts to distinguish very bright and closely related fluorescence intensities. Sperm cells and sperm nuclei are generally stained with Hoechst33342 to make such a distinction. Hoechst33342 is a bright cell-permeable dye that selectively binds to A-T base pairs in the minor groove of double-stranded nuclear DNA. By stoichiometrically staining sperm cells with Hoechst33342, the X and Y chromosomes are distinguished as having slightly different amounts of nuclear DNA. For example, many livestock have a difference of about 4%. When sperm cells are appropriately stained and oriented, this small difference can be distinguished by the fluorescence intensity of Hoechst33342 associated with the nuclear DNA of the sperm cells when irradiated with an appropriate excitation source such as a laser operating at a wavelength of 355 nm or in the vicinity thereof.

[0010] This 4% difference is difficult to detect for several reasons. First, the sperm nuclear DNA is aspherical in most species or is present within the sperm head having a paddle-like shape. Due to this asymmetry, sperm fluoresce differently from the flat side and the narrower side. In fact, this variation exceeds the 4% difference in DNA content, meaning that the sperm must be oriented in order to distinguish based on the nuclear chromosomal content. The orientation geometry has a tendency to produce a core flow having a ribbon-shaped or elliptical cross-section. This elliptical cross-section provides sperm with a greater degree of freedom than the normal degree of freedom when arranged along a single axis.

[0011] The techniques disclosed herein enhance the precision of systems that can be limited by such variations, such as jet-in air flow cytometers. As described in more detail below, the positional variability of the light intensity collected from an object in a fluid column can be addressed using an algorithm that corrects the intensity's dependence on position.

[0012] The techniques outlined herein are particularly applicable to flow cytometry. However, the techniques are applicable to any system where light is emitted from an object on one side of an interface, collected from the other side of the interface, and the interface varies the path of the emitted light rays such that the position of the object relative to the detector is dependent. The techniques herein correct for positional variations within a fluid column and, therefore, provide more accurate measurements for distinguishing the type of object.

[0013] The "jet-in air" flow cytometer system 100, schematically shown in FIG. 1, is one type of discrimination system that can be utilized to consider the concepts of the present disclosure. The "jet-in air" flow cytometer system 100 forms a structure that creates a flow stream including a fluid column 150 that jets at high speed, e.g., about 20 m / s, from an outlet nozzle 160 of a chamber 110. The fluid column 150 discharged from the outlet nozzle 160 can have a generally circular cross-section and, in some embodiments, can have a diameter of from about 10 μm to about 100 μm. In some embodiments, an internal geometry is configured within the chamber 110 and / or the outlet nozzle 160 to hydrodynamically orient sperm within the fluid column. By way of non-limiting example, nozzles such as those described in U.S. Pat. Nos. 6,782,768 and 6,263,745 can be incorporated to orient sperm and generate a coaxial flow of the fluid column. The fluid column 150 is composed of a core flow 151 within a sheath flow 152, and the arrows in FIG. 1 indicate the direction of flow of the core flow 151 and the sheath flow 152. The sheath flow 152 can generally have a circular cross-section, while the core flow generally has an elliptical cross-section with a major axis and a minor axis.

[0014] Within chamber 110, sample injection element 111 introduces core stream 151 that includes objects 171, 172 of multiple possible types. Core stream 151 is delimited by sheath stream 152 that includes sheath fluid and is shaped by fluid forces in chamber 110. Sheath stream 152 at least partially surrounds core stream 151, and sheath stream 152 and core stream 151 do not substantially mix. The inclined or slanted wall 115 of chamber 110 shapes core stream 151 and imparts a force that accelerates objects 171, 172 within core stream 151. The movement of sheath stream 152 confines objects 171, 172 to core stream 151 and causes them to move toward the center of fluid column 150 when fluid column 150 is ejected from chamber 110. Fluid column 150 carries objects 171, 172 to measurement region 175 of fluid column 150, for example, in a single row.

[0015] As the object passes through measurement 175 of fluid column 150, light from excitation source 180 provides excitation light to objects 171, 172. Excitation source 180 can provide light in a broad or narrow wavelength band. For example, excitation source 180 can be a laser. Any laser suitable for producing a response from the object or a dye associated with the object may be employed. Both pulsed lasers and continuous wave lasers are each well-suited for generating an appropriate response. In some configurations, the electromagnetic radiation generated by a source such as excitation light can be modified by optical element 181. For example, the excitation light can be focused onto measurement region 175 by one or more lenses 181. The lens can be used to focus the excitation electromagnetic radiation into a suitable beam shape for the measurement region. Object 172a in measurement region 175 emits light, such as scattered light or fluorescence, in response to excitation source 180.

[0016] The first type of object 171 emits output electromagnetic radiation that differs in at least one characteristic compared to the output electromagnetic radiation emitted by the second type of object 172. For example, in some situations, the first type of object 171 emits light having a higher intensity than the light emitted from the objects of the second type of object 172.

[0017] The optical collection configuration 190 is positioned to collect output electromagnetic radiation 161 emitted from an object 172a within a measurement region 175 that traverses the optical refractive boundary of the fluid column 150 at the fluid-air interface 153. In some embodiments, the optical configuration 190 may be configured to modify the output electromagnetic radiation 161 to provide a modified output electromagnetic radiation 162 that focuses the output electromagnetic radiation emitted from the object 172a in the measurement region 175 onto the detector 185. In some embodiments, the optical collection configuration 190 may include elements that reduce the position dependence of the output electromagnetic radiation 161. The detector 185 receives the modified output electromagnetic radiation 162 and, in response, generates an electrical signal representative of the characteristics of the modified output electromagnetic radiation. By way of example only, the detector 185 may be a forward fluorescence detector. Of course, other detectors may be incorporated to detect the target characteristics such as the target scattering, attenuation, phase shift, or other characteristics. By way of non-limiting example only, the detector may be a photomultiplier tube (PMT), a silicon photomultiplier (SiPM), a photodiode array, or a split detector. In some embodiments, the detector 185 may represent two or more detectors. In some embodiments, a second position detector may be utilized. In other embodiments, a lateral detector may be employed to detect lateral scattering or lateral fluorescence. Still other embodiments may incorporate both a position detector and a lateral detector in addition to the detector 185.

[0018] In some situations, the amplitude of the electrical signal may vary for different object types. The electrical signal is used by an analyzer 187 to distinguish between different types of objects 171, 172. For example, the analyzer 187 may be configured to compare the amplitude of the electrical signal to a threshold to distinguish between a first type of object 171 and a second type of object 172. The analyzer 187 may include one or more analog circuits and / or digital processors to manipulate one or more signals from one or more detectors. By way of example only, a lateral detector may be employed at 90 degrees with respect to the detector 185 to detect lateral scattering or lateral fluorescence. In the case of sperm sorting, the lateral fluorescence allows the analyzer 187 to distinguish between non-oriented sperm and appropriately oriented sperm.

[0019] The parser 187 may include a processor 188 in which executable instructions are stored. In addition to known instructions 198 for collecting, comparing, and manipulating information from the detector signal, the processor may include an instruction 192 for normalizing the intensity value of the output electromagnetic radiation represented by the electrical signal from the detector based on the position of the object 172a in the fluid column 150 in the measurement region 175. The intensity value may be normalized in any way. As a mere example, based on an initial sampling of data including fluorescence intensity and position information, a handwritten line or curve may be input by a user into the graphical user interface.

[0020] The processor 188 may also include an instruction 182 for discriminating objects. FIG. 2 shows an x-y plane cross section of the fluid column 150 in the measurement region 175 shown in FIG. 1. In the x-y cross section in the measurement region 175, the core flow 151 has an elliptical shape, and the fluid of the core flow 151 contains at least one object 172a suspended in a buffer solution, which may also be called a sample. The sheath flow 152 substantially surrounds the core flow 151. In the specific example used in this discussion of the present disclosure, the objects 171, 172 are sperm cells, and the system 100 is implemented to discriminate X-chromosome sperm from Y-chromosome sperm.

[0021] The focused laser beam generated by the excitation source 180 irradiates the sperm cells 172a within the measurement region 175. The cells 171, 172 are stained with a fluorescent dye, and the excitation electromagnetic radiation causes the cells 172a within the measurement region 175 to emit fluorescent output electromagnetic radiation. Generally, the purpose of the elliptical core flow 151 is to orient the sperm cells 172a such that the flat sides of the sperm cells face left and right as shown in FIG. 2. In this orientation, the flat sides of the sperm cells 172a face the laser 180 and the optical collection configuration 190, respectively. If each cell 171, 172 is present in the same orientation in the measurement region 175, the random variability based on the orientation can be greatly reduced. However, the elliptical cross section aimed at this orientation also provides a large degree of freedom regarding the position of the cells within the fluid column 150.

[0022] To obtain the desired orientation, the elliptical core stream 151 has a major axis parallel to the x-axis shown in FIG. 2. The spermatid 172a can take any number of positions along the x-axis within the core stream 151. FIG. 2 shows three representative positions of the spermatid 172a possible in the elliptical core 151, but it can be understood that the sperm may be placed anywhere between the illustrated positions. In the orientation shown in FIG. 2, the first possible position of the spermatid 172a in the core stream 151 is generally at the center of the elliptical core 151 (on the optical axis 199 of the optical collection configuration 190), the second possible position is above the core stream 151 (above the optical axis 199), and the third possible position is below the core stream 151 (below the optical axis 199). The position-dependent refraction of the output light rays emitted from the spermatid 172a occurs at different positions at the fluid-air interface 153 within the core stream 151. As used herein, relative position terms such as "upper", "lower", "upper part", and "lower part" should be understood as being described in relation to the relationship between the illustrated features and do not limit the claims, particularly the position of the sperm in the core stream 151.

[0023] When the spermatid 172a is placed in the first position and the fluid column 150 has a circular cross-section as shown in FIG. 2, the in-plane light rays emitted from the spermatid 172a are incident on the fluid-air interface 153 approximately perpendicularly. Light rays emitted from points of the spermatid 172a away from the center or from out of the plane of the figure are not incident exactly perpendicularly on the interface 153, and these light rays are not considered in this simplified consideration, but one skilled in the art will recognize that the considerations here can be generalized to include those light rays. Therefore, as long as any refraction of light occurs at the fluid-air interface 153, the refraction occurs more uniformly with respect to the detector 185.

[0024] The figure of FIG. 3 shows the uniform optical refraction of the output electromagnetic radiation 298 emitted from the spermatid 172a as the electromagnetic radiation passes through the interface 153 when the spermatid 172a is in the first position within the elliptical core 151 shown in FIG. 2. Correspondingly, the in-plane density of the light rays 298 exiting the fluid column 150 in FIG. 3 is uniform with respect to the light ray angle. The uniform angular density of the light rays corresponds to a uniform radiance as a function of the light ray angle.

[0025] In contrast, when the sperm cell 172a is offset from the optical axis 199 and is closer to above or below the elliptical core 151, for example, at the second and third positions of the elliptical core 151 shown in FIG. 2, at least some of the output light rays emitted from the sperm cell 172a face the fluid-air interface 153 at an oblique angle. These output light rays refract non-uniformly at the fluid-air interface 153, in contrast to the normal incidence situation described above. The most oblique rays refract the most. The refraction of the light rays makes the emission luminance distribution of the fluorescence exiting the fluid column 150 across the fluid-air interface 153 non-uniform and vary with the position of the cell 172a along the x-axis. That is, this refraction changes the radiation luminance distribution of the output electromagnetic radiation emitted from the sperm cell 172a to the outside of the fluid column 150.

[0026] For example, when the cell 172a is offset from the optical axis 199 and is disposed at the second or third position shown in FIG. 2, the density of the light rays, and thus the distribution of the emission luminance on the air side of the interface 153, is higher at each of the positive or negative ray angles with respect to the optical axis 199 compared to the emission luminance on the air side of the interface 153 at an angle parallel to the optical axis 199. Positive and negative refer to the signs of the ray angle γ in FIG. 5. FIG. 4 is a diagram showing the light rays 299 emitted from the cell 172a and exiting the fluid column 150 through the fluid-air interface 153 when the cell 172a is disposed at the second position of the elliptical core 151. In this situation, the density of the light rays or the radiation luminance at a positive ray angle is higher than the density of the light rays at an angle parallel to the optical axis 199 or at a negative ray angle. For an optical system having a given numerical aperture (NA), the amount of light (e.g., collection efficiency) collected by the system from the same type of cell can vary depending on whether the cell is in the first position or the second position. The position dependence of the system collection efficiency introduces inaccuracies in the identification of the cell type.

[0027] Referring to FIG. 5, the analytical expression of the light ray density as a function of the light ray angle γ and the sperm position x is determined using Snell's law, where γ is the angle of the light ray emitted from the object with respect to the optical axis after refraction at the fluid-air interface. This analysis only considers the light rays in the two-dimensional cross-section of the flow stream or the light rays in the tangential direction.

[0028] It is desired to solve for the light ray density with respect to the angle γ, which can be used to specify the light ray density at the entrance pupil of the optical collection system at each sperm position x. This is I γ (γ) (1) can be written as.

[0029] For the purposes of the present invention, it can be assumed that the spermatids emit light uniformly in all directions, whereby the density of the light rays generated with respect to the angle θ is I θ (θ)=1 / π (2) i.e.,

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[0030] The density I of the light rays outside the interface β(β) is the density I of the light rays inside the interface surface, given by the following formula α related to (α), where T(α) represents the average over the bipolarity of the transmission through the interface surface.

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[0031] The transmission is given by the following formula: T(∝)=1 - R(∝) (8)

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[0032] Using the above and the following additional relationships in Equation (7),

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[0033] Here, the NA of the optical collection configuration is given by the sine of the maximum ray angle γ0, and thus this angle can be solved for in terms of NA. γ0 = sin -1 (NA) (16)

[0034] Finally, the relative collected light intensity as a function of the sperm position x is given by integrating Equation (15) from - γ0 to γ0 and normalizing by the value of that integral at x = 0.

Number

[0035] Using the formula for the optical density distribution of Equation (15), the angular dependence of the optical density (radiance) at different sperm positions can be plotted as shown in FIG. 6. In FIG. 6, each line represents the optical density as a function of the angle γ at a given sperm position x, where the angle γ is in radians. The plot corresponds to a series of positions centered on x = 0 that are symmetric in the range where the optical density (radiance) is uniform as a function of the angle (corresponding to graph 404 in FIG. 6). When x is positive (e.g., the second position in FIG. 2 corresponding to graph 402), the relative radiance is higher at positive optical angles γ and lower at negative optical angles γ, and vice versa when x is negative (e.g., the third position in FIG. 2 corresponding to graph 403).

[0036] When the numerical aperture of the collection optical system (optical collection configuration 190 in FIGS. 1 and 2) is large, e.g., approaching 1, the variation in the collection optical intensity with respect to the position of light emitted from an object within the elliptical core is relatively small. This is especially so because all the light emitted from the object and directed to the right is collected by the collection optical system regardless of the exact ray direction, and the total amount of light emitted is invariant with respect to the object position (given uniform excitation). In contrast, when the numerical aperture is small, the collection intensity variation with respect to the object position becomes relatively large, because the change in the object position affects the radiance distribution and the small numerical aperture implies that only a portion of this varying radiance distribution is collected. Practical systems may have a numerical aperture much smaller than 1, e.g., less than 0.5 or less than 0.3. The family of graphs provided in FIG. 7 shows the relative intensity of light collected from an object as a function of the object position x through collection optical systems using different numerical apertures. FIG. 6 shows the range of angles γ captured by the different numerical apertures of FIG. 7.

[0037] In the family of graphs of FIG. 7, graph 412 shows the relative intensity with respect to the position along the x-axis in a collection optical system having a numerical aperture (NA) of 0.2 (optical collection configuration 190 shown in FIGS. 1 and 2), graph 414 shows the relative intensity with respect to the position along the x-axis in a collection optical system having an NA of 0.4, graph 416 shows the relative intensity with respect to the position along the x-axis in a collection optical system having an NA of 0.6, graph 418 shows the relative intensity with respect to the position along the x-axis in a collection optical system having an NA of 0.8, and graph 419 shows the relative intensity with respect to the position along the x-axis in a collection optical system having an NA of 0.9. From FIGS. 6 and 7, it is clear that the smaller the NA of the collection optical system, the greater the variation in the collected light intensity with respect to the object position, as compared to a collection optical system with a larger NA. Furthermore, the larger the NA of the collection optical system, the more it collects rays having a wider range of refractive angles than a collection optical system with a smaller NA, and thus has a higher overall collection efficiency.

[0038] Particularly with regard to the use of sperm discrimination and sorting, it can be understood that the elliptical major axis of the core stream 151 (FIGS. 1 and 2) can be about 50 μm in length, providing sperm with a degree of freedom of movement of about 25 μm in any direction. Referring again to FIG. 7, it can be seen that when the object is displaced about 17 μm from the center, an NA of 0.2 captures only about 90% of the object relative intensity. Similarly, an NA of 0.4 captures only 92% of the relative intensity of an object displaced about 17 μm from the center, and an NA of 0.6 captures just slightly above 94% of the relative intensity at the same position. It can be further understood that the NA of the collection optical system of the sperm sorter can be from about 0.3 to about 0.6. FIG. 7 shows the advantages of an increasingly large numerical aperture, but such an aperture becomes increasingly expensive, has a shallower depth of field, and means that a larger aperture must be placed closer to the nozzle. However, in the sperm sorting application, there is a limit to how close the collection optical system can be placed. In a typical sperm sorting device, the aperture can be from about 0.5 to 0.6. The embodiments described herein correct the position dependence of the measured intensity, enabling a collection optical system with a smaller numerical aperture to perform as well as a collection optical system with a higher numerical aperture.

[0039] Thus, sperm located at positions approaching the second and third positions of FIG. 2 in the core stream 151 emit electromagnetic radiation with a much lower overall intensity that is ultimately detected for analysis and discrimination. In fact, the core stream 151 can have an elliptical major axis that is approximately 50 μm in length at a high event rate (on the order of 60,000 events per second or more). Some sperm deviate to either side of the first position from the center by up to 20 μm and even about 25 μm. In the context of a very bright and closely related fluorescence signal, this variation can obscure a roughly 4% difference in the stained nuclear DNA that distinguishes sperm with a Y chromosome from sperm with an X chromosome.

[0040] Furthermore, to increase the number of events at a given sperm concentration within a buffer sample, it is necessary to increase the sample volume per unit time in the fluid column passing through the measurement region. Increasing the number of detected events per second in this way increases the elliptical cross-sectional area of the core stream within the fluid column, including the length of the major axis. As a natural consequence, and as would be recognized by one of ordinary skill in the art, increasing the sort speed by increasing the sample flow rate generally decreases the sensitivity of the sperm sorting device. Thus, the embodiments described herein can provide a sperm sort that not only improves sperm sorting accuracy at normal speeds but also increases the overall speed with respect to throughput with little loss of fidelity.

[0041] A method for identifying an object moving through a fluid column in the presence of positional variability is shown in the flowchart of FIG. 8. The process includes creating (510) a fluid column containing the object at different positions within the fluid column. The fluid column can be a coaxial flow of fluid generated by a jet-in air-flow cytometer. Such a fluid column can include a core flow having an elliptical cross-section with a major axis along which the object can be placed. The core flow can be coaxially contained within a sheath flow. In some embodiments, the fluid column can have an air-fluid interface, thereby causing refraction. In other embodiments, the fluid column can be formed within a cuvette or a microfluidic channel. In such cases, there can be a liquid-glass interface and perhaps a glass-air interface, and the emitted light can be refracted twice. Such doubly refracted light is expected to benefit greatly from the angle-dependence correction of certain embodiments.

[0042] The process continues by generating (520) excitation electromagnetic radiation and directing (530) the excitation electromagnetic radiation towards the object in the measurement region within the fluid column. The object within the fluid column emits output electromagnetic radiation in response to the excitation electromagnetic radiation in the measurement region. The output electromagnetic radiation is collected (540) from the objects in the fluid column, including objects having different positions within the fluid column in the measurement region, and the detector generates (550) an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical configuration.

[0043] Next, an analyzer or other suitable means normalizes (560) the intensity represented by the output signal based on the position of the object in the fluid column. The normalization can be performed by correction, whereby signals generated off the central axis, such as those directed towards and including the second and third positions of FIG. 2, are amplified by appropriate correction factors based on those positions. The magnitude of the appropriate correction factor can be seen in FIG. 7. Once normalized by correction, the method continues by discriminating (570) the first type of object from other objects. The discrimination can be performed by a flow cytometer analyzer and may include one or more additional operations. For example, a univariate histogram showing the distribution of fluorescence intensity can be generated. Using the corrected signal and further calculated values, a bivariate histogram can also be generated. Such corrected and calculated values can be compared to a gating region in the flow cytometer analyzer or to a look-up table to discriminate the first type of object from other types of objects.

[0044] An exemplary object, sperm, can be discriminated as either sperm having an X chromosome or sperm having a Y chromosome. Further, the sperm can be stained with a DNA-selective dye in addition to a quenching dye. The quenching dye typically penetrates membrane-compromised cells such as dead or dying sperm cells and greatly reduces the fluorescence generated by the DNA-selective dye associated with those compromised cells. Such quenched cells are effectively removed from the closely associated population being discriminated / sorted. In this way, the system can discriminate live or viable sperm cells from sperm cells that are dying or compromised. The system can also discriminate sperm having an X chromosome from all other cells, discriminate sperm having a Y chromosome from all other cells, or even simultaneously discriminate sperm having an X chromosome and sperm having a Y chromosome from all other sperm cells.

[0045] FIG. 9 shows a first embodiment of a discrimination system substantially similar to the discrimination system shown in FIGS. 1 and 2, in which output electromagnetic radiation 161 emitted from an object 172a disposed in a measurement region is collected by an optical collection configuration 190. The optical collection configuration 190 may include a collection lens that focuses the modified output electromagnetic radiation onto a detector 185. In the illustrated embodiment, the detector functions in the same manner as a position detector 186 that measures the characteristics of the modified output electromagnetic radiation and identifies the position of the object 172a within the core flow 151 of the fluid column 150.

[0046] A detector 185 suitable for identifying the characteristics of the modified output electromagnetic radiation 162 and identifying the position of the object 172a in the measurement region may include a split detector or a detector array such as a PMT, SiPM, pin photodiode, etc. These detectors may be disposed on the image plane or Fourier plane of the object to identify the position of the object. On the image plane, the detector directly measures the position of the object, while on the Fourier plane, the position information is extracted from the lateral intensity distribution (e.g., left-right asymmetry).

[0047] In flow cytometry applications, detectors that are very sensitive (up to single photon counting) and fast (objects move at about 20 m / s through 10 μm) are often required. Detectors with the required speed and sensitivity are typically detectors that provide internal gain. In a photomultiplier tube (PMT) or silicon photomultiplier (SiPM), also known as a pixelated avalanche photodiode, a single photon creates a cascade of up to about 10 6 electrons. Both detector types are also commercially available as detector arrays. Since SiPMs are fabricated by standard techniques on a silicon wafer, they may be more suitable for use in a detector array suitable for object position identification. Some detectors, such as SiPMs, may be particularly suitable for placement on the Fourier plane to distribute light over a larger area of the detector.

[0048] Figure 10 shows an alternative embodiment in which a beam splitter 191 or other suitable optical system redirects a portion of the power of the modified output electromagnetic radiation 162. Most of the modified output electromagnetic radiation 162 is directed towards and focused onto a detector 185. In this embodiment, the detector 185 includes a first detector 176 that detects symmetric properties. The first detector 176 can be any detector conventionally suitable for quantifying specific symmetric properties. In typical flow cytometry applications, photodiodes, photomultiplier tubes (PMTs), and silicon photomultiplier tubes may be particularly well-suited for detecting scattered or fluorescent emission electromagnetic intensity.

[0049] The beam splitter 191 can include a dielectric mirror 197, but those skilled in the art will understand that other suitable optical components such as cube beam splitters, prism beam splitters, etc. can be used to redirect a portion of the power of the modified output electromagnetic radiation 162. Regardless of the manner in which the output power is split, a first beam portion 164 is directed towards the detector along a first path, and a second beam portion 165 is directed towards a second detector 173 in the form of a position detector 177 along a different path. The position detector can be a position-sensitive device ("PSD") such as a camera, an isotropic sensor, or a charge-coupled device (CCD), a split detector, a PMT, a detector array of SiPMs, pin photodiodes, etc.

[0050] Referring to FIG. 11, a simulation was performed to show the feasibility of the segmentation detector for identifying position information in a flow cytometry system. The simulation employed a segmented SiPM detector including 3 mm SiPM detectors mounted side by side. The edge where the detector contacts was calibrated as the central x coordinate position, and the beam axis of the interrogation laser and the symmetric center of the fluid column were simulated. A 1.5 mm spot size was swept across the segmented detector from x positions from about -12 mm to 12 mm, and the relative intensity was measured and recorded by each detector. The first graph 601 shows the relative intensity recorded in the beam spot from one of the detectors for x positions in the range from about -12 mm to about 12 mm, where the x position corresponds to the plane of the SiPM detector. Graph 602 shows the corresponding relative intensity detected by other detectors for the beam spot in the range of x positions from about -12 mm to about 12 mm. As can be seen, the position difference between the two detectors leads to different measured intensities based on the x position of the 1.5 mm spot. These differences are correlated with the position and can be transformed through processing means to approximate the position information. Noise was included in the simulation, but the noise was independent of the intensity. At the maximum intensity, the noise corresponds to a coefficient of variation of 0.8%. The simulation demonstrated that in a segmented detector configuration, the x position can be identified based on the relative intensity detected by each SiPM in the segmented detector configuration. Those skilled in the art will understand that the embodiments of the present invention are not limited to this configuration, and other detector configurations suitable for identifying the position of particles in a fluid column are also contemplated for use herein. As a mere example, other detectors may be employed in a segmented detector configuration. Those skilled in the art will understand that the detector should have low noise because the combined signal must have a sufficiently low coefficient of variation.

[0051] Figure 12 shows the results of an experiment incorporating position correction of sperm nuclei in a fluid column leading to a significant improvement in the discrimination of sperm nuclei with X and Y chromosomes. Sperm nuclei stained with Hoechst 33342 were processed through a Cytonome Genesis III sperm sorting instrument. The instrument is equipped with a SiPM segmented detector. The sample and sheath pressure were adjusted to establish an event rate of 35,000 events per second. The nuclei were examined with a Coherent Genesis CW-355 laser at an average power of 150 mW. Plot 610 shows a bivariate histogram of the sum of fluorescence intensities from each detector in the segmented detector plotted against the position delta of the nuclei in the fluid column. As described above, the range of the position delta represents the major axis of the elliptical core flow in which the nuclei can enter the measurement region. The population of sperm nuclei 612 with the X chromosome is seen as a crescent. As expected, the measured intensity is maximum near position delta 0 and curves downward and decreases as the nuclei move away from the central position. The population of nuclei 614 with the Y chromosome appears as a second crescent directly below the X population. Here too, the highest intensity is seen near position delta 0 and the relative intensity decreases significantly as the nuclei move away from the central position.

[0052] Plot 620 presents a univariate histogram of the combined fluorescence intensity corresponding to the intensity charted in Plot 610. Distinct populations of nuclei 612 with the X chromosome and nuclei 614 with the Y chromosome can be seen, but comparison of Plot 610 with Plot 620 reveals that sperm nuclei with the X chromosome displaced from the center increasingly overlap with sperm nuclei with the Y chromosome placed in the center. In fact, the peak-to-valley ratio is calculated to be 76.8%.

[0053] According to an embodiment of the present invention, the correction factor 616 is shown as a curve in plot 610. The correction factor 616 indicates the degree of correction required for the detected fluorescence intensity in order to remove the variations introduced by the random positions of the events. The corresponding correction was applied to the fluorescence sum value shown in plot 630 to generate a corrected population 632 of nuclei with X chromosomes and a corrected population 634 of nuclei with Y chromosomes. The corrected population 632 of nuclei with X chromosomes generally forms a rectangular shape and no longer shows variations based on the positions of the nuclei in the fluid column. In plot 630, a clear gap can be seen between the corrected population 632 of nuclei with X chromosomes and the corrected population 634 of nuclei with Y chromosomes. Plot 640 shows the corresponding univariate histogram, which has a peak-to-valley ratio of 94% between the corrected population 632 of nuclei with X chromosomes and the corrected population 634 of nuclei with Y chromosomes. The significant contrast between plot 620 and plot 640 is visually apparent. Furthermore, the difference can be quantified at a higher 17.2 percentage point.

[0054] Figure 13 shows the results of an example incorporating the correction according to the embodiment described herein. Raw sperm stained with Hoechst 33342 were processed through a Cytonome Genesis III sperm sorting instrument. The sample and sheath pressure were adjusted to reach an event rate of 43,000 events per second, and the sperm were examined using a Coherent Genesis CW-355 laser operating at an average power of 100 mW. Plot 710 shows a bivariate histogram of the combined fluorescence intensity and relative position of raw sperm in the core flow. Again, the population 712 of sperm with X chromosomes can be seen as the first population above the population 714 of sperm with Y chromosomes. A correction factor 716 for normalizing the combined intensity values is also shown in plot 710. Plot 720 shows a univariate histogram of the uncorrected combined intensity, which shows a peak-to-valley ratio of 75.3% between the population 712 of sperm with X chromosomes and the population 714 of sperm with Y chromosomes.

[0055] Plot 730 provides a type of bivariate histogram that is common in sperm sorting applications. In this case, the corrected forward fluorescence intensity is plotted against the side fluorescence. Since the side fluorescence provides information about the orientation of each cell, the forward fluorescence versus side fluorescence histogram is useful for sorting live sperm. In contrast, the sperm nucleus is sonicated and removed from the aspherical sperm head. Thus, when sorting sperm nuclei, orientation is not an issue. For this reason, nuclei are easily sorted and are often used for calibration of sperm sorting flow cytometers. Plot 730 shows a corrected population 732 of sperm with X chromosomes and a corrected population 734 of sperm with Y chromosomes.

[0056] Similar to the previous example, Plot 740 still correlates on the Y-axis with the corrected forward fluorescence of Graph 730. In the univariate plot of Graph 740, the corrected population 732 of sperm with X chromosomes and the corrected population 734 of sperm with Y chromosomes can be seen as clearer peaks with a machine-calculated peak-to-valley ratio of 81.0%. And here too, the corrected histogram shows a significant improvement over Plot 720, which shows the values of the position correction of live sperm.

[0057] In another aspect, the embodiments described herein may provide a system and method that substantially facilitate the alignment process in a flow cytometer. For example, in the case of sperm, in order to generate and collect a signal that is clear enough to distinguish between sperm populations with very bright and closely related X and Y chromosomes, the measurement region, the detector, and even the structure that forms the sheath flow must be aligned appropriately and precisely. Even with precise and proper alignment, the oriented sperm in the fluid column can take any number of positions along the long axis of the core flow. As described above with respect to FIGS. 3-7, this means that there is an angular dependence on the detected output electromagnetic radiation even when the components of the flow cytometer are aligned. Since the cells can be randomly located within the core flow, this angular dependence introduces fluctuations such as noise.

[0058] In commercial sperm sorting applications, technicians typically make several coarse adjustments on multiple axes for multiple components to align the equipment, followed by several fine adjustments. Due to the susceptibility of the equipment to the effects of each adjustment, the highly correlated nature of the detected signals, and the number of possible adjustments, such alignment can be a time-consuming task for the technician operating the sperm sorting equipment. When switching samples, aligning the machine for commercially sorting sperm can take several minutes, up to five minutes at most. After removing nozzle blockages or otherwise removing and replacing or adjusting other components that require calibration, it can take five minutes, 15 minutes, or in rare cases, up to 30 minutes of the technician's time to align the equipment to a position suitable for commercial sperm sex sorting.

[0059] Figure 14 shows the results of an example where the alignment process for discriminating sperm nuclei was significantly reduced. Sperm nuclei stained with Hoechst 33342 were processed through a Cytonome Genesis III sperm sorter. The device was equipped with SiPM segmentation detection. Coarse alignment was performed by aligning the forward fluorescence detection in less than one minute. Sperm nuclei were run at an event rate of 33,000 nuclei per second, and the sperm nuclei were examined using a Coherent Genesis CW-355 laser operating at an average power of 150 mW. Plot 810 shows a bivariate histogram showing the combined forward fluorescence plotted against the position detected by the SiPM for each event. Misalignment is evident in each of the population 812 of nuclei with X chromosomes and the population 814 of nuclei with Y chromosomes. In the case of poor alignment, the crescent meter is asymmetric, and the fluorescence intensity values drop dramatically in the positive x-direction compared to the negative x-direction. The population 814 of nuclei with Y chromosomes shows the same skew.

[0060] Correction factor 816 is shown as a line between the two populations. This correction factor 816 indicates the degree of correction performed on the combined fluorescence value for the nuclear x location. In other words, correction factor 816 represents a curve that is normalized by correction to a flat line. Each combined fluorescence value at the corresponding x position along the line undergoes the same increase or decrease in magnitude as correction factor 816.

[0061] The distortion caused by rough alignment is more prominent in the histogram of the fluorescence intensity of plot 820. In plot 820, due to the increase in duplication, the peak-to-valley ratio between the population 812 of nuclei with X chromosomes and the population 814 of nuclei with Y chromosomes becomes 72.3%.

[0062] In plot 830, the corrected forward fluorescence sum values are plotted on a bivariate histogram against the detected positions of each event. Here too, by normalizing the fluorescence intensity values using the correction factor 816 based on the cell positions, it can be seen that two distinct populations of cells emerge. The corrected population 832 of nuclei with X chromosomes and the corrected population 834 of nuclei with Y chromosomes are clearer and are clearly grouped in plot 830. Importantly, the orthogonality of these populations leads to the univariate fluorescence intensity histogram seen in plot 840, where two distinct univariate peaks have a calculated peak-to-valley ratio of 94.4%.

[0063] In addition to the use of corrections, some embodiments described herein include elements that reduce the collected light intensity variations related to the object position in the flow stream. Some embodiments described herein can provide modified output light having a measured intensity variation of less than about 3%, less than about 2%, or even less than about 1% for object position shifts that are less than 60% of the radius of the flow stream away from the center of the flow stream along an axis perpendicular to the optical axis. Many applications are susceptible to intensity measurement errors, which can arise from a variety of causes. Due to the difficulty in reducing intensity variations by precisely controlling the position of the object within the flow stream, it is useful instead to carefully design the optical collection configuration to reduce the variations in the collected light intensity related to the object position. In applications such as X / Y sperm sorting, it is often desirable to separate two or more cell populations based on the difference in measured fluorescence intensity between the populations. If random position variations lead to a variation in the collected light intensity that is greater than the nominal difference in fluorescence intensity between the two populations, it is not possible to distinguish between the two populations with both high yield and high purity. The fluorescence intensity difference between X sperm and Y sperm is typically only a few percent (e.g., about 4% in bovine sperm). Current sperm sorting systems can theoretically achieve high throughput by increasing the flow rate of the core stream, but this has the effect of increasing the width of the core stream. Therefore, there is a large uncertainty in the sperm position within the core of the flow stream. This position uncertainty and the resulting variation in the collected fluorescence intensity limit the maximum throughput of current sperm sorting systems to a level that does not obscure the small fluorescence intensity difference between X sperm and Y sperm.

[0064] One approach to intensity-position correction can be understood with reference to FIGS. 6 and 7. The brackets in FIG. 6 highlight the integration region corresponding to the fluorescence collection optics using a given NA. A graph of the collected intensity variation versus object position for the NA of FIG. 6 is provided in FIG. 7. In FIG. 7, for a given NA, the integration over the fluorescence collection region is performed such that the intensity of the collected light can be plotted as a function of each sperm position. From FIG. 7, it is clear that increasing the NA of the collection optics helps to reduce the effect of object position on the fluorescence intensity collected through the collection optics.

[0065] In some embodiments, the collection optical system (e.g., the optical collection configuration 190 in FIGS. 1 and 2) can be modified using elements that reduce the variation in the collected light intensity with respect to the object position as described above. Some embodiments are described in more detail in U.S. Patent Application No. 16 / 133,531, which is incorporated herein by reference. According to some such embodiments, the collection optical system operates by masking a particular configuration in the "angular space", i.e., the collection optical system selectively collects, attenuates, and / or blocks rays from different angles γ in order to achieve a desired intensity versus position profile. In practice, the "angular space" masking function can be applied to the pupil of the optical system (e.g., the entrance pupil, the exit pupil, or the aperture stop), and the position where the pupil plane intersects the ray corresponds to the angle γ. In some embodiments, the collection optical configuration achieves a desired, e.g., flatter intensity versus position profile by prioritizing the collection of high-angle (directed away from the optical axis) rays over the exclusion of particular low-angle rays.

[0066] FIGS. 15 and 16 show how the exclusion of low-angle refracted rays at a given NA flattens the intensity versus position curve. Excluding low-angle rays excludes the rays that produce the largest variations in the intensity versus position profile, while the angular variations in the radiance at large positive angles tend to cancel out the corresponding variations at large negative angles. FIG. 15 shows a plot of relative radiance versus ray angle γ at different positions of the object along the x-axis, where the angle γ is in radians. In FIG. 15, each graph corresponds to an object position x within the core of the flow stream as shown in FIG. 5. The brackets in FIG. 15 indicate the portion of the rays excluded by the collection optical system at each position x when rays having an angle magnitude less than 0.3 rad are excluded (lower bracket in FIG. 15) and when rays having an angle magnitude less than 0.4 rad are excluded (upper bracket in FIG. 15).

[0067] FIG. 16 shows the relationship between the relative collected light intensity and the position of the object along the x-axis when the angle is not excluded (graph 900), when light rays having an angle of -0.3 rad to +0.3 rad are excluded (graph 903), and when light rays having an angle of -0.4 rad to +0.4 rad are excluded (graph 904). Graph 16 shows that when lower angle light rays are excluded, the relative intensity vs. position graph shows lower intensity fluctuations with respect to position.

[0068] FIG. 17 shows the results of an experiment incorporating software-based position correction and hardware-based elements in the collection optical path to reduce the collected light intensity variation with respect to the object position as described. Nuclei of sperm stained with Hoechst 33342 were processed through a Cytonome Genesis III sperm sorter. The sperm sorter was coupled with a SiPM segmented detector with wires arranged in the collection optical path to exclude low collection angle electromagnetic radiation generated from the nuclei of sperm. Wires and other elements suitable for blocking low collection angle electromagnetic radiation are described in U.S. Patent Application No. 16 / 133,531.

[0069] The sample and sheath pressure were adjusted to reach an event rate of 60,000 events per second, and the nuclei were examined using a Coherent Genesis CW-355 laser operating at an average power of 90 mW. In plot 1010, it can be seen that the wire reduces the influence of intensity dependence on the nuclear position within the fluid column. However, as the nuclei move further in the positive direction along the x-axis, there is still a significant decrease in the relative intensity. The population 1012 of nuclei with X chromosomes and the population 1014 of nuclei with Y chromosomes are seen to droop significantly in the positive direction of the x-axis. The corresponding peak-to-valley ratio calculated from the fluorescence intensity histogram of plot 1020 is 81.5%. Here too, nuclei with X chromosomes placed towards one end of the fluid column are not detected sufficiently. As a result, the total fluorescence intensity of the nuclei at this end has an intensity value similar to that of the nuclei with Y chromosomes placed in the center within the population 1014 of nuclei with Y chromosomes. This skew is evident in the univariate histogram of plot 1020 in the form of a downward-shifted shoulder and an exaggerated peak of the population 1014 of nuclei with Y chromosomes.

[0070] The correction factor 1016 is shown in graph 1010. For each position, the correction value is added to the correction factor-corresponding detected fluorescence intensity. Plot 1030 shows a bivariate histogram having a corrected population 1032 of nuclei with X chromosomes and a corrected population 1034 of nuclei with Y chromosomes, which are more distinct rectangular populations. Plot 1040 provides the corresponding univariate histogram of the corrected total intensity values independent of the location of each event. The corrected population 1032 of nuclei with X chromosomes and the corrected population 1034 of nuclei with Y chromosomes are more distinct and have approximately equal peak heights and a peak-to-valley ratio of 92.6%.

[0071] The above description of the various embodiments is presented for purposes of illustration and explanation, not limitation. The disclosed embodiments are not exhaustive, i.e., there is no intention to limit the possible embodiments to those disclosed. Many changes and modifications are possible in light of the above teachings.

Claims

**Claim 1**: A discrimination system, comprising: a collection optical system that collects output electromagnetic radiation from sperm having an X chromosome and sperm having a Y chromosome within a measurement region; a split detector that generates an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the collection optical system, the electrical signal being responsive to the intensity of the output electromagnetic radiation collected by the collection optical system; an analyzer that stores instructions for determining the position of sperm cells in the measurement region and instructions for discriminating between sperm having an X chromosome and sperm having a Y chromosome; The discrimination system comprising the above. **Claim 2**: The discrimination system according to claim 1, wherein the split detector is not disposed on the image plane of the collection optical system. **Claim 3**: The discrimination system according to claim 1, wherein the split detector includes a detector selected from the group consisting of a PMT split detector and a SiPM split detector. **Claim 4**: The discrimination system according to claim 1, wherein the analyzer further stores instructions for normalizing the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the sperm cells in the measurement region, and applies a correction to the intensity of the output electromagnetic radiation represented by the electrical signal. **Claim 5**: The discrimination system according to claim 4, wherein the amount of correction for each position on the major axis of the core flow in the measurement region is determined, and the correction is applied to the electrical signal representing each sperm cell based on the detected position on the major axis of the core flow. **Claim 6**: The discrimination system according to claim 1, wherein the sperm cells are disposed within an elliptical column of sample fluid, the sample fluid is formed coaxially with an outer layer of sheath fluid, and the sheath fluid generally has a cylindrical shape. **Claim 7**: The discrimination system according to claim 1, further comprising an element that modifies the output electromagnetic radiation to increase the uniformity of the output electromagnetic radiation collected by the collection optical system for sperm cells at different positions. **Claim 8**: The discrimination system according to claim 1, wherein the split detector is disposed on the Fourier plane. **Claim 9**: The discrimination system according to claim 1, further comprising a mask for attenuating low and angular refracted rays of the output electromagnetic radiation. **Claim 10**: The discrimination system according to claim 1, wherein the mask includes wires.

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