SISTEMA DE DETECÇÃO E MÉTODO PARA CORREÇÃO DE RADIAÇÃO ELETROMAGNÉTICA POSICIONALMENTE DEPENDENTE DETECTADA A PARTIR DE OBJETOS EM UMA COLUNA DE FLUIDO

Description

DETECTION SYSTEM AND METHOD FOR CORRECTING POSITIONALLY DEPENDENT ELECTROMAGNETIC RADIATION DETECTED FROM OBJECTS IN A COLUMN OF FLUID Divided from BR112022017886-7 deposited on 03 / 15 / 2021 BACKGROUND

[001] Object discrimination devices and techniques distinguish between objects of different types, such as objects with different characteristics. These devices and techniques are particularly useful for analyzing and even classifying cells according to specified characteristics of interest. Some cell classification approaches rely on the light emitted by cells, or colored cells, to determine their type. In some implementations, cells moving in a fluid column are exposed to an excitation source to generate an output electromagnetic radiation for detection. Cells of a first type, or those with a particular characteristic, produce different output electromagnetic radiation in some characteristics, for example, wavelength and / or intensity, compared to other cells. Such differences serve as the basis for cell type discrimination and classification. SUMMARY

[002] Some embodiments refer to a discrimination system for distinguishing between different types of objects based on electromagnetic radiation emitted from objects arranged in a fluid column. A fluid column-forming structure creates a fluid column containing objects in different positions within the fluid column, and an excitation source generates electromagnetic radiation by excitation directed at the objects in the fluid column within a measurement region. The objects inside the fluid column Petition 870260049819, dated 05 / 25 / 2026, page 9 / 123 2 / 38 emit outgoing electromagnetic radiation in response to excitation electromagnetic radiation. An optical arrangement measures the outgoing electromagnetic radiation from the objects, and a detector generates an electrical signal responsive to the intensity of the outgoing electromagnetic radiation. An analyzer includes instructions stored in it i) to normalize the intensity of the outgoing electromagnetic radiation represented in the electrical signal based on the object's position in the fluid column, and ii) to discriminate a first type of object from other objects.

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

[004] According to other modalities, an object discrimination method begins with the creation of a fluid column containing objects in different positions within the fluid column. Electromagnetic radiation by excitation is directed towards objects in the fluid column in a measurement region. The objects in the measurement region emit outgoing electromagnetic radiation in response to the electromagnetic radiation by excitation, which is collected and used to generate an electrical signal responsive to the intensity of the outgoing electromagnetic radiation. The intensity of the outgoing electromagnetic radiation represented in the electrical signal is normalized based on the object's position in the fluid column, and a first type of object is discriminated from other objects. Petition 870260049819, dated 05 / 25 / 2026, page 10 / 123 3 / 38 BRIEF DESCRIPTION OF THE DRAWINGS

[005] FIG. 1 is a diagram of a discrimination system according to certain modalities.

[006] FIG. 2 shows a cross-section of an xy plane of the fluid column in the measurement region of the system in FIG. 1.

[007] FIG. 3 shows the light emanating from an object located near the center of the fluid column with substantially uniform light refraction at the fluid-air interface of the fluid column relative to the optical apparatus functioning as the collecting optical element.

[008] FIG. 4 shows the light emanating from an object located in an upper portion of the elliptical core of the fluid column exhibiting non-uniform light refraction at the fluid-air interface relative to the optical apparatus functioning as the collecting optical element.

[009] FIG. 5 illustrates the geometry used to develop an analytical formula for the angular dependence of light ray density in the plane on a position function x.

[0010] FIG. 6 provides a family of graphs showing the angular dependence of radiance for different object positions in a fluid column.

[0011] FIG. 7 provides a family of graphs of the relative intensity of light collected from the fluid column in relation to the object's position along the geometric x-axis for different numerical apertures of the collecting optical element.

[0012] FIG. 8 is a flowchart of an approach to identifying moving objects in a fluid column by correcting for the positional variation of the detected outgoing light according to some modalities.

[0013] FIG. 9 provides a schematic representation for a mode that includes position and intensity detection from a single detector. Petition 870260049819, dated 05 / 25 / 2026, page 11 / 123 4 / 38

[0014] FIG. 10 provides a schematic representation of a modality that includes a first detector to detect intensity and a second detector to determine the position of the object.

[0015] FIG. 11 illustrates the output of a simulation to determine the location of the object with a sandwich-type detector.

[0016] FIG. 12 illustrates the results of an experiment that improves intensity measurements by correcting detected values ​​based on the position of sperm nuclei.

[0017] FIG. 13 illustrates the results of an experiment that improves intensity measurements by correcting detected values ​​based on the position of live sperm cells.

[0018] FIG. 14 illustrates the results of an experiment that seeks to improve the performance of a misaligned instrument with a correction factor based on the position of sperm nuclei.

[0019] FIG. 15 provides a family of graphs of the angular dependence of radiance for different object positions and shows exclusion regions.

[0020] FIG. 16 shows the relative intensity of light collected from the fluid column in relation to the object's position along the geometric x-axis when no angle is excluded, when rays that have angles between -0.3 rad and +0.3 rad are excluded, and when rays that have angles between -0.4 rad and +0.4 rad are excluded.

[0021] FIG. 17 illustrates the results of an experiment that uses both a correction factor and an element to optically reduce the positional dependence of intensity measurements.

[0022] The figures are not necessarily to scale. Similar numbers used in the figures refer to similar components. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure identified with the same number. Petition 870260049819, dated 05 / 25 / 2026, page 12 / 123 5 / 38 DETAILED DESCRIPTION

[0023] The embodiments described in this document refer to devices, systems, and methods for discriminating different types of objects. The objects emit output light in response to an excitation light that is directed at the objects in a fluid column, such as a flow stream. As used in this document, the term emit refers to both reflected and fluoresced electromagnetic radiation, such as light. As used in this document, the term light refers to electromagnetic radiation at wavelengths in the visible spectrum, as well as electromagnetic radiation at wavelengths in the infrared and ultraviolet spectra. Such output electromagnetic radiation may include light reflected or fluoresced directly from an object, as well as light reflected or fluoresced by a coloration or dye associated with the object.In some implementations, cell types are distinguished based on the intensity of the outgoing electromagnetic radiation emanating from objects. The intensity can be determined as a peak intensity or even as a total intensity, such as the integrated area under an intensity signal. The specific modalities described in this document refer to the distinction between X-chromosome sperm cells and Y-chromosome sperm cells. The other modalities refer to the distinction of 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.

[0024] It will be observed that the approaches of this disclosure can be applied more generally to distinguish any objects of different types provided that the outgoing electromagnetic radiation emanating from one type of object generates a discernible difference in at least Petition 870260049819, dated 05 / 25 / 2026, page 13 / 123 6 / 38 less a characteristic compared to electromagnetic radiation emanating from another type of object. In some examples given, the fluid column is a flow stream that has a curved boundary or interface where refraction of electromagnetic radiation can occur. For example, the curved boundary of the fluid column may generally be circular in cross-section. A fluid column may be bounded by solid walls, as inside a cuvette or inside a microfluidic channel, or it may be jetted in air, as in an air-jet type flow cytometer. Objects may move along the fluid column through a central core formed by a coating fluid that at least partially surrounds the central core. In the case of sperm sorting applications, the central core may comprise a sample fluid core stream containing sperm cells.The core current can generally be conditioned in a ribbon shape or it can have a generally elliptical cross-section for the purpose of orienting aspherical sperm cells. The electromagnetic radiation emanating from objects encounters at least one optical refraction limit between the objects and other materials, such as at the interface between the fluid column and air.

[0025] Due at least in part to the different refractive properties of the coating fluid and air, the light-gathering efficiency of external light emanating from objects inside the column depends on the position of the objects for such systems. Light-gathering efficiency that varies with position is detrimental in applications where the light emanating from objects must be precisely quantified and such precision is limited by random (not directly observable) positional oscillations of the objects. In the case of sex differentiation spermatozoa specifically, such systems seek to differentiate very bright and strictly fluorescence intensities. Petition 870260049819, dated 05 / 25 / 2026, page 14 / 123 7 / 38 related. Sperm cells and sperm nuclei are commonly stained with Hoechst 33342 to make such differentiations. Hoechst 33342 is a clear, cell-permeable dye that selectively binds to AT base pairs in the minor groove of double-stranded nuclear DNA. Stoichiometric staining of sperm cells with Hoechst 33342 differentiates X chromosome and Y chromosome as having slightly different amounts of nuclear DNA. For example, many domestic animals have a difference of about four percent. When sperm cells are properly stained and oriented, this small difference can be distinguished by the fluorescence intensity of Hoechst 33342 associated with the nuclear DNA of the sperm cells when irradiated with a suitable excitation source, such as a laser operating at or near a wavelength of 355 nm.

[0026] This four percent difference is difficult to detect for several reasons. First, sperm nuclear DNA resides in the sperm head, which is aspherical or spade-shaped in most species. This asymmetry causes the sperm to fluoresce differently on the flat side and the narrower side. In fact, this oscillation exceeds the four percent difference in DNA content, meaning that the sperm must be oriented in order to be differentiated based on nuclear chromosome content. Orientation geometries tend to produce a nuclear stream that has a ribbon shape or an elliptical cross-section. This elliptical cross-section provides sperm with larger than normal latitude for positioning on a geometric axis.

[0027] The approaches disclosed in this document improve the accuracy of systems that may be limited by such oscillations, such as air-jet flow cytometers. As described in more detail below, the positional variability of light intensity Petition 870260049819, dated 05 / 25 / 2026, page 15 / 123 The 8 / 38 density collected from objects in a fluid column can be resolved with an algorithm that corrects for the intensity dependence on position.

[0028] The approaches presented in this document are particularly applicable to flow cytometry. However, the approaches can be applied to any system where light is collected on one side of an interface from objects emitting light on the other side of the interface, where the interface causes a variation in the paths of emitted light rays in a manner dependent on the object's position relative to the detector. The approaches in this document correct for the positional variation in the fluid column, thus providing more accurate measurements to distinguish types of objects.

[0029] The jet-in-air flow cytometer system 100 schematically illustrated in FIG. 1 is a type of discrimination system that can be used to discuss the concepts of disclosure. The jet-in-air flow cytometer system 100 includes a fluid column-forming structure that creates a flow stream comprising a fluid column 150 that is jetted from the outlet nozzle 160 of the chamber 110 at a high velocity, for example, about 20 m / s. The fluid column 150 expelled from the outlet nozzle 160 may be nearly circular in cross-section and may have a diameter of about 10 μm to about 100 μm in some implementations. In some embodiments, the interior of the chamber 110 and / or the outlet nozzle 160 are configured with an internal geometry that hydrodynamically guides the spermatozoa within the fluid column. As non-limiting examples, nozzles such as those described in U.S. Patent 6,782,768 and 6,263.745, can be incorporated for the purpose of guiding the spermatozoon and generating the coaxial flow of a fluid column. The fluid column 150 is composed of a core stream. Petition 870260049819, dated 05 / 25 / 2026, page 16 / 123 9 / 38 151 in a cladding stream 152 wherein the arrows in FIG. 1 indicate the flow direction of the core and cladding streams 151, 152. The cladding stream 152 may have a generally circular cross-section, while the core stream has a generally elliptical cross-section, with a major and a minor geometric axis.

[0030] Inside chamber 110, a sample injection element 111 introduces core stream 151 containing objects 171, 172 which may be of multiple types. The core stream 151 is connected by a coating stream 152 comprising the coating fluid and is shaped by hydrodynamic forces in chamber 110. The coating stream 152 surrounds at least partially the core stream 151, and the coating stream 152 and the core stream 151 do not substantially mix. The inclined or angled walls 115 of chamber 110 exert forces that shape the core stream 151 and accelerate objects 171, 172 in the core stream 151. The movement of the jacket stream 152 constrains objects 171, 172 in the core stream 151 to move them toward the center of the fluid column 150 when the fluid column 150 is ejected from chamber 110.The fluid column 150 delivers objects 171, 172 to a measuring region 175 of the fluid column 150, for example, in a single row.

[0031] As objects traverse the measurement region 175 of the fluid column 150, light from an excitation source 180 provides excitation light to the objects 171, 172. The excitation source 180 may provide light in a broad wavelength band or in a narrow wavelength band. For example, the excitation source 180 may be a laser. Any laser suitable for producing a response from the object or a dye associated with the object may be employed. Pulsed lasers and wave lasers Petition 870260049819, dated 05 / 25 / 2026, page 17 / 123 10 / 38 of the continuous waveform are each well suited to produce appropriate responses. In some configurations, the electromagnetic radiation generated by the excitation source, such as excitation light, can be modified by an optical element 181. For example, the excitation light can be focused on the measurement region 175 by one or more lenses 181. The lenses can be used to focus the excitation electromagnetic radiation into a suitable beam shape focused on the measurement region. Objects 172a in the measurement region 175 emit light, for example, diffuse or fluorescent light, in response to the excitation source 180.

[0032] Objects of a first type 171 will emit outgoing electromagnetic radiation that differs in at least one characteristic from the outgoing electromagnetic radiation emitted by objects of the second type 172. For example, in some situations, objects of the first type 171 will emit light with a higher intensity than the light emitted by objects of the second type 172.

[0033] An optical collection arrangement 190 is positioned to collect the outgoing electromagnetic radiation 161 emanating from the object 172a within the measurement region 175 that crosses the optical refraction limit of the fluid column 150 at the fluid-air interface 153. In some embodiments, the optical arrangement 190 can be configured to modify the outgoing electromagnetic radiation 161 to provide modified outgoing electromagnetic radiation 162 that focuses the outgoing electromagnetic radiation emanating from the object 172a in the measurement region 175 onto a detector 185. In some embodiments, the optical collection arrangement 190 may include an element that reduces the positional dependence of the outgoing electromagnetic radiation 161. The detector 185 receives the modified outgoing electromagnetic radiation 162 and, in response, generates an electrical signal representative of characteristics of the modified outgoing electromagnetic radiation. As an example, the Petition 870260049819, dated 05 / 25 / 2026, page 18 / 123 11 / 38 detector 185 can be a forward fluorescence detector. Obviously, other detectors can be incorporated to detect features of interest, such as scattering, decay, phase shifts, or other features of interest. As non-limiting examples, the detector can be a photomultiplier tube (PMT), a silicon photomultiplier tube (SiPM), a photodiode array, or a sandwich-type detector. In some embodiments, detector 185 can represent more than one detector. In some embodiments, a second position detector can be used. In other embodiments, a side detector can be employed to detect side scatter or side fluorescence. Still other embodiments may incorporate a position detector and a side detector in addition to detector 185.

[0034] In some situations, the amplitude of the electrical signal may be different for different types of objects. 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 discriminate between objects of the first type 171 and objects of the second type 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. As an example, a lateral detector may be employed at 90 degrees relative to detector 185 to detect lateral diffusion or lateral fluorescence. In the case of sperm classification, lateral fluorescence allows the analyzer 187 to properly differentiate an oriented sperm from a non-oriented sperm.

[0035] The analyzer 187 may include a processor 188 that has executable instructions stored within it. In addition to these known instructions 198 for the purpose of collecting, comparing, and manipulating detector signal information, the processor may include instructions Petition 870260049819, dated 05 / 25 / 2026, page 19 / 123 12 / 38 Instructions 192 for normalizing the intensity value of the output electromagnetic radiation represented in the detector's electrical signal based on the object's position 172a in the fluid column 150 in the measurement region 175. The intensity value can be normalized in several ways. For example, hand-drawn lines or curves can be entered by a user in a graphical user interface based on the initial sampling of data including fluorescence intensities and positional information.

[0036] Processor 188 may also include instructions 182 for discriminating objects. FIG. 2 shows a cross-section of an xy plane of the fluid column 150 in the measuring region 175 shown in FIG. 1. In the xy cross-section of the measuring region 175, the core stream 151 is elliptical in shape, and the fluid of the core stream 151 comprises at least one object 172a suspended in a buffer solution, which may also be referred to as a sample. The jacket stream 152 substantially surrounds the core stream 151. In a particular example used for this discussion in this disclosure, objects 171, 172 are sperm cells and system 100 is implemented to discriminate X-chromosome sperm from Y-chromosome sperm.

[0037] A focused laser beam generated by excitation source 180 irradiates the sperm cell 172a within the measuring region 175. Cells 171, 172 are colored with a fluorescent dye, and the electromagnetic radiation from excitation causes cell 172a within the measuring region 175 to emit fluorescent output electromagnetic radiation. The purpose of the generally elliptical core current 151 is to orient a sperm cell 172a such that the flat sides of the sperm cell face left and right, as shown in FIG. 2. In this orientation, the flat sides of the sperm cell 172a face the left and right. Petition 870260049819, dated 05 / 25 / 2026, page 20 / 123 13 / 38 being 180 and for the optical collection arrangement 190, respectively. When each cell 171,172 is presented in a similar orientation in the measurement region 175, the random variability based on orientation can be significantly reduced. However, the elliptical cross-section that aids in this orientation also provides significant latitude relative to the cell's position within the fluid column 150.

[0038] To obtain the desired orientation, the elliptical nucleus stream 151 has a major geometric axis that is parallel to the geometric x-axis shown in FIG. 2. The sperm cell 172a can assume various positions along the geometric x-axis within the nucleus stream 151. FIG. 2 shows three possible representative positions for the sperm cell 172a in the elliptical nucleus 151, although it is possible to observe that spermatozoa can be located anywhere between the positions shown. In the orientation shown in FIG. 2, the first possible position for the sperm cell 172a in the nucleus stream 151 consists approximately in the center of the elliptical nucleus 151 (on the optical geometric axis 199 of the optical collecting arrangement 190), a second possible position consists at the top of the nucleus stream 151 (above the optical geometric axis 199), and a third possible position consists at the bottom of the nucleus stream 151 (below the optical geometric axis 199).A position-dependent refraction of the outgoing light rays emanating from the sperm cell 172a occurs at the fluid-air interface 153 at different positions within the nucleus stream 151. As used in this document, relative position terms such as top, bottom, superior and inferior are to be understood as descriptive of the relationships between the features shown in the figures and are not limiting to the claims, especially the position of the spermatozoon in a nucleus stream 151. Petition 870260049819, dated 05 / 25 / 2026, page 21 / 123 14 / 38

[0039] When the sperm cell 172a is located in the first position and the fluid column 150 has a circular cross-section as shown in FIG. 2, the in-plane light rays emanating from the sperm cell 172a are normally incident approximately at the fluid-air interface 153. The rays emanating from points on the sperm cell 172a distant from its center, or the rays emanating out of the plane of the figure, are not exactly normally incident at the interface 153; these rays are not considered in this simplified discussion, but a person skilled in the art can observe how the discussion could be generalized to include them. Then, up to the point where any refraction of light occurs at the fluid-air interface 153, it occurs in a more uniform manner with respect to the detector 185.

[0040] The diagram in FIG. 3 shows the uniform light refraction of the outgoing electromagnetic radiation 298 emanating from a sperm cell 172a as the electromagnetic radiation crosses the interface 153 when the sperm cell 172a is in position 1a inside the elliptical nucleus 151 shown in FIG. 2. Correspondingly, the density in the plane of the light rays 298 exiting the fluid column 150 in FIG. 3 is uniform with respect to the angle of the rays. The uniform angular density of the light rays corresponds to the uniform radiance as a function of the angle of the rays.

[0041] In contrast, when a sperm cell 172a is off the optical geometric axis 199 and is closer to the top or bottom of the elliptical nucleus 151, for example, in the 2a and 3a positions of the elliptical nucleus 151 shown in FIG. 2, at least some of the outgoing rays emanating from the sperm cell 172a encounter the fluid-air interface 153 at an oblique angle. These outgoing rays are reflected in a non-uniform manner at the fluid-air interface 153 in contrast to the normal incidence situation described above. The rays Petition 870260049819, dated 05 / 25 / 2026, page 22 / 123 15 / 38 more oblique are the most severely reflected. The refraction of light rays causes the radiance distribution of fluorescent light exiting the fluid column 150 and crossing the fluid-air interface 153 to become non-uniform and vary with the position of the cell 172a along the geometric x-axis. That is, this refraction alters the radiance distribution of outgoing electromagnetic radiation emanating from the sperm cell 172a out of the fluid column 150.

[0042] For example, when cell 172a is located outside the optical geometric axis 199, for example, in the 2a or 3a positions shown in FIG. 2, the density of light rays and therefore the radiance on the air side of the interface 153 is greater at positive or negative ray angles, respectively, relative to the optical geometric axis 199 compared to the radiance on the air side of the interface 153 at angles parallel to the optical geometric axis 199 or at negative or positive ray angles, respectively. Positive and negative refer to the sign of the ray angle γ in FIG. 5. FIG. 4 is a diagram illustrating light rays 299 emanating from a cell 172a and exiting the fluid column 150 and traversing the fluid-air interface 153 when cell 172a is located in the 2a position of the elliptical core 151.In this situation, the density of light rays, or radiance, at positive ray angles is greater than the density of light rays parallel to the optical geometric axis 199 or at negative ray angles. For an optical system with a predetermined numerical aperture (NA), the amount of light collected by the system from cells of the same type (e.g., the 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 results in inaccuracies in determining the cell type.

[0043] With reference to FIG. 5, an analytical formula for the density of light rays as a function of the angle of the rays γ and position Petition 870260049819, dated 05 / 25 / 2026, page 23 / 123 16 / 38 Sperm density x is determined using Snell's law, where y is the angle of a light ray, relative to the optical geometric axis, emanating from the object after retraction at the fluid-air interface. This analysis considers only rays within the two-dimensional cross-section, or tangential to it, of the flow stream.

[0044] We want to solve for the density of light rays with respect to angle γ, which can be used to determine the density of rays at the entrance pupil of an optical collection system for each sperm position x. This can be written as follows: ç(r)· (1)

[0045] For the purposes of this document, it can be assumed that the sperm cell emits light uniformly in all directions, then the density of emitted light rays in relation to the angle Θ is: = Vn.(2) that is, uniformly distributed from 0 = — ^ to 0 = p By geometric analysis: „ -1 / COS ®-x\ $=tcm ;(3) tx = - 0 — 0; e(4) = ^-0- / ?,(5) where the angles χ,0, φ,α,ρ, and the distance x are shown in FIG. 5. Since the flux current has a refractive index ή, Snell's law produces another relationship between the angles: sin(fi) = nsin <x.(6) Petition 870260049819, dated 05 / 25 / 2026, p. 24 / 123 17 / 38

[0046] The density of light rays external to the interface ι^ζβ) is related to the density of light rays internal to the interface la(a) following the formula, with τΐατ) representing the average, in both polarizations, of the transmission through the interface: = í«o)n?o|^l(7'

[0047] Transmission is related to Fresnel reflection coefficients for sep polarization, and with the following formulas: Γ(κ) = 1 -(8) (9) Mk) = ---7---!( Icdjk+ hcü^ / íI o MK)=—— (W ICÜ5 / í4- HCOJKl

[0048] Using Eq. (7) with the above relations and the following additional relations: / 0(@) =ísw|^|,(12) / „W = íoC0)|^|,e(13) W = Í.W |^[(14) gives an expression for the ray density with respect to γ: i,fr> = W |^| r(y)(15)

[0049] Now, the NA of the optical collection arrangement is given by the sine of the maximum angle of the rays y0, so that it is possible to solve for this angle in terms of NA: Petition 870260049819, dated 05 / 25 / 2026, page 25 / 123 18 / 38 y0= sen1(NA) (16)

[0050] Finally, the relative collected light intensity, as a function of sperm position x, is given by integrating the Eq. (15) from -y0 to y0e normalizing with that integral value ax = o: r Xo Relative Intensity =rr~y°JyCÍF—. (17)

[0051] Using the formula for ray density distribution from Eq. (15), the angular dependence of ray density (radiance) for different sperm positions can be plotted as in FIG. 6. In FIG. 6, each of the lines represents the ray density as a function of angle γ for a given sperm position x, where angle γ is in radians. The plots correspond to a series of positions that are in a symmetrical range around x = a, (corresponding to graph 404 in FIG. 6), which is where the ray density (radiance) is uniform as a function of angle. When x is positive (e.g., 2a position in FIG. 2, corresponding to graph 402), the relative radiance is greater for positive ray angles γ and smaller for negative ray angles γ, and the opposite is true when x is negative (for example, 3aposition in FIG. 2, corresponding to graph 403).

[0052] If the numerical aperture of the optical collecting element (optical collecting arrangement 190 in FIGS. 1 and 2) is large, for example, close to one, the variation in the collected optical intensity with respect to position for the light emitted from an object inside the elliptical core is relatively small. This is because essentially all the light emitted from the object is directed to the right. Petition 870260049819, dated 05 / 25 / 2026, page 26 / 123 19 / 38 would be collected by the collecting optical element, regardless of the exact ray direction, and the total amount of light emitted is invariant with respect to the object's position (given uniform excitation). In contrast, a small numerical aperture results in a relatively large variation in collected intensity with respect to the object's position, due to the fact that changes in the object's position affect the radiance distribution, and a small numerical aperture means that only a portion of this variable radiance distribution is collected. Practical systems may have NAs that are significantly smaller than one, for example, less than 0.5 or less than 0.3. The family of graphs provided in FIG. 7 illustrates the relative intensity of light collected from an object, as a function of object position x, through collecting optical elements with different NAs. FIG. 6 illustrates the range of angles γ captured by the different numerical apertures of FIG. 7.

[0053] In the family of graphs in FIG. 7, graph 412 illustrates the relative intensity with respect to position along the geometric x-axis for the optical collection element (e.g., the optical collection arrangement 190 shown in FIGS. 1 and 2) that has a numerical aperture (NA) of 0.2; graph 414 shows the relative intensity with respect to position along the geometric x-axis for the optical collection element that has an NA of 0.4; graph 416 shows the relative intensity with respect to position along the geometric x-axis for the optical collection element that has an NA of 0.6; graph 418 shows the relative intensity with respect to position along the geometric x-axis for the optical collection element that has an NA of 0.8; and graph 419 shows the relative intensity with respect to position along the geometric x-axis for the optical collection element that has an NA of 0.9. This is evident from FIGS.6 and 7 that the optical collection element that has smaller NAs produces a greater variation in intensity. Petition 870260049819, dated 05 / 25 / 2026, page 27 / 123 20 / 38 density of light collected in relation to the object's position compared to the collecting optical element with larger NAs. Additionally, the collecting optical element with larger NAs collects light rays that have a wider range of refraction angles than the collecting optical element with smaller NAs and therefore has a higher overall collection efficiency.

[0054] Regarding sperm discrimination or classification application in particular, it can be understood that the largest elliptical geometric axis of the core stream 151 (FIGS. 1 and 2) can be about 50 μm in length, providing a sperm of about 25 μm in latitude to move in any direction. Referring again to FIG. 7, it can be observed that an NA of 0.2 captures only about 90% of a relative object intensity when the object is about 17 μm off-center. Similarly, an NA of 0.4 captures only 92% of the relative intensity for objects that are about 17 μm off-center, and an NA of 0.6 captures slightly more than 94% of the relative intensity at the same position. It can be further observed that the NA of the optical collection element for a sperm classifier can be between about 0.3 and 0.6. Although FIG.7. Illustrating the benefit of increasingly larger numerical apertures, such numerical apertures are progressively more expensive and have a shallower depth of field, meaning that the larger aperture must be positioned closer to the nozzle. However, there is a limit to the proximity at which the collecting optical element can be positioned in sperm sorting applications. In typical sperm sorting instruments, apertures can be between approximately 0.5 and 0.6. The modalities described in this document correct the positional dependence on measured intensity, allowing a smaller numerical aperture collecting optical element to exhibit similar performance. Petition 870260049819, dated 05 / 25 / 2026, page 28 / 123 21 / 38 thousand to a larger aperture collecting optical element.

[0055] Spermatozoa located in nuclear stream 151 in positions near the 2nd and 3rd positions of FIG. 2, therefore, emit a significantly lower overall intensity of electromagnetic radiation that is ultimately detected for analysis and discrimination. In fact, nuclear stream 151 may have a larger elliptical geometric axis that is about 50 μm long at high event rates (on the magnitude of 60,000 events per second and greater). Some spermatozoa will be off-center by 20 μm or even as far as about 25 μm on either side of the 1st position. In the context of extremely clear and closely related fluorescence signals, this variation can obscure the almost 4% difference in colored nuclear DNA that differentiates X-chromosome-bearing spermatozoa from Y-chromosome-bearing spermatozoa.

[0056] Furthermore, increasing the number of events at a given sperm concentration in a buffer sample requires increasing the sample volume per unit time in the fluid column traversing the measurement region. Increasing the number of events detected per second thus also increases the elliptical cross-section of the nucleus stream within the fluid column, including the length of the longest geometric axis. As a natural consequence, and as those skilled in the art know, generally increasing the sorting speed by increasing the sample flow rate decreases the sensitivity of the sperm sorting equipment. Therefore, the modalities described in this document not only improve sperm sorting accuracy at common speeds but also provide sperm sorting at an overall increased speed in terms of productivity without suffering fidelity losses. Petition 870260049819, dated 05 / 25 / 2026, p. 29 / 123 22 / 38

[0057] An approach to identifying moving objects in a fluid column in the presence of positional variation is illustrated in the flowchart of FIG. 8. The process involves creating a fluid column containing objects in different positions within the fluid column. The fluid column can be a coaxial fluid stream created by a jet-in-air type flow cytometer. Such a fluid column may comprise a core stream with an elliptical cross-section that has a major geometric axis along which objects can be positioned. The core stream may be coaxially contained within a jacket stream. In some embodiments, the fluid column may have an air-fluid interface where refraction occurs. In other embodiments, the fluid column may be formed in a cuvette or a microfluidic channel. In such cases, there may be a liquid-glass interface and possibly a glass-air interface, and the emitted light may be reflected twice.It is expected that such twice-reflected light will significantly benefit from the angle-dependency correction of certain modalities.

[0058] The process continues by generating 520 electromagnetic radiation by excitation and directing 530 electromagnetic radiation by excitation to objects in the fluid column in a measurement region. Objects inside the fluid column emit outgoing electromagnetic radiation in response to the electromagnetic radiation by excitation in the measurement region. The outgoing electromagnetic radiation is collected 540 from objects in the fluid column, including objects that have a different position inside the fluid column in a measurement region, and a detector generates 550 an electrical signal responsive to the intensity of the outgoing electromagnetic radiation collected by the optical array.

[0059] Next, an analyzer or other suitable means normalizes the intensity represented by the output signal based on 560 Petition 870260049819, dated 05 / 25 / 2026, p. 30 / 123 23 / 38 position of the object in the fluid column. Normalization can be performed by means of a correction, whereby signals generated outside the central geometric axis, such as towards and including the second and third positions of FIG. 2, are amplified by an appropriate correction factor based on their position. The magnitude of appropriate correction factors can be seen in FIG. 7. After being normalized by correction, the method continues discriminating a first type of object from other objects. Discrimination can occur in a flow cytometer analyzer and may include one or more additional manipulations. For example, univariate histograms can be generated, illustrating a distribution of fluorescence intensities. Bivariate histograms can also be generated with the corrected signal and with additional calculated values.These corrected and calculated values ​​can be compared to gating regions in a flow cytometer analyzer or compared to lookup tables to discriminate a first type of object from other types of objects.

[0060] As illustrative examples, spermatozoa can be discriminated as X-bearing spermatozoa or Y-bearing spermatozoa. Furthermore, spermatozoa can be stained with a DNA-selective dye in addition to a secondary extinction dye. An extinction dye typically permeates sperm cells with compromised membranes, such as dead or dying sperm cells, and significantly reduces the fluorescence produced by the DNA-selective dye associated with those compromised cells. Such suppressed cells are effectively removed from closely related populations undergoing discrimination / classification. In this way, a system can discriminate live or viable sperm cells from dying or compromised sperm cells. The system tam Petition 870260049819, dated 05 / 25 / 2026, page 31 / 123 24 / 38 can also discriminate viable sperm carrying the X chromosome from all other cells, sperm carrying the Y chromosome from all other cells, or even simultaneously distinguish viable sperm carrying both the X chromosome and the Y chromosome from all other sperm cells.

[0061] FIG. 9 illustrates a first embodiment of the discrimination system substantially similar to the discrimination system shown in FIGS. 1 and 2, in which the outgoing electromagnetic radiation 161 emanating from an object 172a located in the measurement region is collected by an optical collection arrangement 190. The optical collection arrangement 190 may include a collection lens that focuses modified outgoing electromagnetic radiation onto a detector 185. In the embodiment shown, the detector functions to measure a characteristic of the modified outgoing electromagnetic radiation, as well as a position detector 186 to determine the position of the object 172a within the core stream 151 of the fluid column 150.

[0062] The detector 185 suitable for determining a characteristic of the modified output electromagnetic radiation 162 and for determining the object location 172a in the measurement region may comprise sandwich-type detectors or an array of PMT, SiPM, pin photodiode or similar detectors. These detectors may be located in the object image plane or in the Fourier plane to determine the object position. In the image plane, the detectors directly measure the object position, while in the Fourier plane the position information will be extracted from the lateral intensity distribution (e.g., left-right asymmetry).

[0063] Flow cytometry applications often require very sensitive (reduced to single photon count) and fast (objects move at ~20 m / s per 10 μm) detectors. Detectors with Petition 870260049819, dated 05 / 25 / 2026, page 32 / 123 25 / 38 The speed and sensitivity required are typically those detectors that provide internal gain. In a photomultiplier tube (PMT) or a silicon photomultiplier (SiPM), also known as a pixelated avalanche photodiode, a single photon creates a cascade of up to about 10⁶ electrons. Both detector types are commercially available as detector arrays. SiPMs may be more suitable for use in detector arrays suitable for determining object position since they are manufactured by standard techniques on silicon wafers. Some detectors, such as SiPMs, may be particularly suitable for placement in a Fourier plane in order to distribute light over a larger area of ​​the detector.

[0064] FIG. 10 illustrates an alternative embodiment in which a beam splitter 191, or other suitable optical element, redirects a fraction of the energy of the modified output electromagnetic radiation 162. Most of the modified output electromagnetic radiation 162 is directed to and focused on the detector 185. In this embodiment, the detector 185 comprises a first detector 176 for detecting a feature of interest. The first detector 176 can be any detector conventionally suitable for quantifying the particular feature of interest. In photodiodes of particular flow cytometer applications, photomultiplier tubes (PMTs) and silicon photomultipliers may be particularly suitable for detecting diffuse or fluorescent electromagnetic intensity.

[0065] The beam splitter 191 may comprise a dielectric mirror 197, however, those skilled in the art will observe that other suitable optical components, such as cubic beam splitters, prismatic beam splitters and the like, may be used to redirect a portion of the energy of the modified output electromagnetic radiation 162. Regardless of the manner in which Petition 870260049819, dated 05 / 25 / 2026, page 33 / 123 26 / 38 the output energy is split, a first beam fraction 164 is directed along a first path to the detector and a second beam fraction 165 is directed 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 a charged-coupled device (CCD), sandwich-type detectors, an array of PMT detectors, SiPM, pin photodiodes or similar.

[0066] Returning to FIG. 11, a simulation was performed illustrating the feasibility of a sandwich-type detector for determining positional information in a flow cytometry system. The simulation employed a split SiPM detector comprising 3 mm SiPM detectors mounted side-by-side. The edge where the detectors meet was calibrated as a central x-coordinate position, simulating the geometric beam axis of an interrogating laser as well as the symmetrical center of a fluid column. An area size of 1.5 mm was encompassed by the sandwich-type detectors from an x-position between approximately -12 mm and 12 mm, and the relative intensity measured by each detector was recorded. A first graph 601 illustrates the relative intensity recorded for the beam area of ​​one of the detectors from x-positions in the range of approximately -12 mm to approximately 12 mm, where the x-position corresponds to a plane of the SiPM detector.Graph 602 illustrates the corresponding relative intensity detected by the other detector for the beam area in a range of x positions from approximately -12 mm to approximately 12 mm. As can be seen, the positional difference in the two detectors results in different measured intensities based on the x position of the 1.5 mm area. These differences correlate to position and can be translated by means of processing into approximate positional information. Noise was included in the simulation, but it was independent of in. Petition 870260049819, dated 05 / 25 / 2026, page 34 / 123 27 / 38 intensity. At maximum intensity, the noise corresponds to a 0.8% coefficient of variation. The simulation demonstrated that the position x can be determined in a sandwich-type detector array based on the relative intensity detected by each SiPM in a sandwich-type detector array. Those skilled in the art can verify that the embodiments of the present invention are not limited to this configuration and that other detector configurations suitable for determining the position of a particle in a fluid column are also contemplated for use in this document. As an example, other detectors can be employed in a sandwich-type detector array. Those skilled in the art will observe that the detectors must have low noise, since the combined signals must have a sufficiently low coefficient of variation.

[0067] FIG. 12 illustrates the result of an experiment incorporating positional correction for sperm nuclei in a fluid column, resulting in significant improvements in the differentiation of sperm nuclei bearing X and Y chromosomes. Sperm nuclei stained with Hoechst 33342 were processed using a Genesis III sperm sorting instrument manufactured by Cytonome. The instrument was equipped with a SiPM sandwich-type detector. Sample and coating pressure was adjusted to establish an event rate of 35,000 events per second. Nuclei were interrogated with a Genesis CW-355 Coherent laser at an average power of 150 mW. Plot 610 shows a bivariate histogram illustrating a sum of fluorescence intensities from each detector in the sandwich-type detector plotted against the positional delta of nuclei in the fluid column.As previously described, the positional delta band represents the major geometric axis of the elliptical core current along which the nodes... Petition 870260049819, dated 05 / 25 / 2026, page 35 / 123 28 / 38 nuclei can enter the measurement region. A population of 612 X-chromosome-bearing nuclei is seen in a crescent shape. As expected, the measured intensities are highest when close to a delta position of 0, with reductions decaying as the nuclei move to the opposite side of the central position. A population of 614 Y-chromosome-bearing nuclei is seen with a second crescent just below the X population, and again, the highest intensities are seen close to a delta position of 0, with significant losses in relative intensity as the nuclei move to the opposite side of the central position.

[0068] Plot 620 presents a univariate histogram of the summed fluorescence intensities that correspond to the intensities shown in plot 610. Although the distinct population of X-bearing nuclei 612 and the population of Y-bearing nuclei 614 can be observed, a comparison of plot 610 with plot 620 makes it evident that off-center X-bearing sperm nuclei progressively overlap with well-centered Y-bearing sperm nuclei. In fact, the peak-to-trough ratio is calculated at 76.8%.

[0069] According to embodiments of the invention, a correction factor 616 is illustrated as a curved line in plot 610. The correction factor 616 illustrates the degree of correction required for the detected fluorescence intensity to remove the variation introduced by the random positions of events. A corresponding correction was applied to the fluorescence sum values ​​shown in plot 630 to produce a corrected population of X-chromosome-bearing nuclei 632 and a corrected population of Y-chromosome-bearing nuclei 634. The corrected population of X-chromosome-bearing nuclei 632 forms a generally rectangular shape and Petition 870260049819, dated 05 / 25 / 2026, page 36 / 123 Plot 29 / 38 no longer demonstrates oscillation based on the position of the nuclei in the fluid column. A more distinct gap can be seen in plot 630 between the corrected population of X-bearing nuclei 632 and a corrected population of Y-bearing nuclei 634. Plot 640 illustrates the corresponding univariate histogram, which has a peak-to-trough ratio of 94% between the corrected population of X-bearing nuclei 632 and the corrected population of Y-bearing nuclei 634. The clear contrast between plot 620 and plot 640 is visually evident. Furthermore, the difference is quantifiable at 17.2 percentage points higher.

[0070] FIG. 13 illustrates the results of an example incorporating correction according to the modalities described in this document. Live spermatozoa stained with Hoechst 33342 were processed using a Genesis III sperm sorting instrument manufactured by Cytonome. Sample and coating pressures were adjusted to achieve an event rate of 43,000 events per second, and the spermatozoa were analyzed with a Coherent Genesis CW-355 laser operated at an average power of 100 mW. Plot 710 illustrates a bivariate histogram of the summed fluorescence intensity and the relative positions of live spermatozoa in the nuclear stream. Again, the population of X-carrying spermatozoa 712 can be seen as a first population above a population of Y-carrying spermatozoa 714. A correction factor 716 to normalize the summed intensity values ​​is also shown in plot 710.Plot 720 illustrates the univariate histogram of uncorrected summed intensities and demonstrates a peak-to-trough ratio of 75.3% between the population of spermatozoa carrying chromosome X 712 and the population of spermatozoa. Petition 870260049819, dated 05 / 25 / 2026, page 37 / 123 30 / 38 carriers of Y chromosome 714.

[0071] Plot 730 provides a type of bivariate histogram common in sperm sorting applications. In this case, a corrected forehead fluorescence intensity is plotted against a side fluorescence. A forehead versus side fluorescence histogram is useful for sorting live sperm because side fluorescence provides information about the orientation of each cell. In contrast, sperm nuclei are sonicated and removed from the aspherical sperm head. Thus, orientation is not a problem during sperm nuclei sorting. For this reason, nuclei are easier to sort and are more frequently used to calibrate sperm sorting flow cytometers. Plot 730 shows a corrected population of X-bearing sperm 732 and a corrected population of Y-bearing sperm 734

[0072] In a manner very similar to the previous example, plot 740 still correlates the geometric Y-axis to the corrected forward fluorescence of graph 730. In the univariate plot of graph 740, the corrected population of X-bearing spermatozoa 732 and the corrected population of Y-bearing spermatozoa 734 can be observed as more distinct peaks that have a machine-calculated peak-to-valley ratio of 81.0%. Again, the corrected histogram shows a significant improvement over plot 720, demonstrating the value of the positional correction of live spermatozoa.

[0073] In another aspect, the modalities described in this document can provide systems and methods that substantially facilitate an alignment process in a flow cytometer. In the case of spermatozoa, for example, the measurement region, detect Petition 870260049819, dated 05 / 25 / 2026, page 38 / 123 31 / 38 and even the structure that forms the coating flow must be properly and precisely aligned to generate and collect signals clear enough to differentiate very clearly and closely related X and Y chromosome-bearing sperm populations. Even in a precise and proper alignment, the oriented sperm in a fluid column can assume various positions along the major geometric axis of the nuclear stream. As described above in relation to FIGS. 3-7, this means that even when the flow cytometer components are in perfect alignment, there is an angular dependence of the detected output electromagnetic radiation. This angular dependence introduces noise-like variations due to the fact that cells may be randomly positioned in the nuclear stream.

[0074] In commercial sperm sorting applications, technicians typically perform numerous stroke adjustments followed by numerous fine adjustments to multiple components across multiple geometric axes to align the instrument. Due to the instrument's sensitivity to each adjustment, the closely related nature of the detected signals, and the number of adjustments, such alignments can be time-consuming for technicians operating sperm sorting instruments. When changing samples, machine alignments for commercial sperm sorting can take several minutes, even up to five minutes. After unclogging the nozzle or otherwise removing, replacing, or adjusting other components requiring calibration, a technician may take 5 minutes, 15 minutes, and in rare cases, up to 30 minutes to get an instrument into proper alignment for commercial sperm sex sorting.

[0075] FIG. 14 illustrates the results of an example in which the alignment process is greatly reduced to discriminate nuclei Petition 870260049819, dated 05 / 25 / 2026, p. 39 / 123 32 / 38 sperm nuclei. Sperm nuclei stained with Hoechst 33342 were processed using a Genesis III sperm classifier manufactured by Cytonome. The instrument was equipped with a SiPM sandwich-type detector. Forward fluorescence detection was aligned for less than one minute, resulting in irregular alignment. Sperm nuclei were processed at an event rate of 33,000 nuclei per second and analyzed with a Coherent Genesis CW-355 laser operated at an average power of 150 mW. Plot 810 illustrates the bivariate histogram showing summed forward fluorescence plotted against the position detected by each event by SiPM. Unsatisfactory alignment is evident in each of the populations of X-bearing nuclei 812 and Y-bearing nuclei 814.In unsatisfactory alignment, the crescent shapes are asymmetrical and the fluorescence intensity values ​​drop drastically in the positive x direction compared to the negative x direction. The population of nuclei bearing Y chromosome 814 demonstrates the same inclination.

[0076] The correction factor 816 is illustrated as a line between the two populations. This correction factor 816 illustrates the degree of correction that will be applied to summed fluorescence values ​​at each location x. Indicated differently, the correction factor 816 represents a curved line that will be normalized by correction to a flat line. Each summed fluorescence value at a corresponding position x along the line receives the same magnitude of increase or decrease as the correction factor 816.

[0077] The distortion caused by irregular alignment is most pronounced in the fluorescence intensity histogram of plot 820, where increased overlap results in a peak-to-valley ratio of 72.3% among the population of chrom-bearing nuclei. Petition 870260049819, dated 05 / 25 / 2026, page 40 / 123 33 / 38 X chromosome 812 and the population of nuclei carrying Y chromosome 814.

[0078] In plot 830, the summed value of forward fluorescence is plotted on a bivariate histogram against the detected position of each event. It can be observed, again, that by normalizing the fluorescence intensity values ​​with a correction factor of 816 based on the cell position, two clear cell populations emerge. A corrected population of X-chromosome-bearing nuclei 832 and a corrected population of Y-chromosome-bearing nuclei 834 are more clearly and distinctly grouped in plot 830. Above all, the orthogonal relationship of these populations translates into the univariate fluorescence intensity histogram observed in plot 840, where two distinct univariate peaks have a calculated peak-to-value ratio of 94.4%.

[0079] In addition to the use of correction, some embodiments disclosed in this document include elements that reduce the variation in collected light intensity relative to the object's position in a flux stream. Some embodiments described in this document can provide modified output light that has less than about 3%, or less than about 2%, or even less than about 1% of measured intensity variation for a deviation in the object's position that is less than 60% of a flux stream radius distant from a flux stream center along a geometric axis perpendicular to the optical geometric axis. Many applications are sensitive to intensity measurement errors, which can arise from a variety of sources.Due to the difficulty of reducing intensity oscillations by precisely controlling the position of objects in the flow stream, it is useful instead to reduce the variation in collected light intensity relative to the object's position through careful design of the optical collection array. For co-applications... Petition 870260049819, dated 05 / 25 / 2026, page 41 / 123 34 / 38 In X / Y sperm classification, it frequently occurs that two or more cell populations must be separated based on the difference in fluorescence intensity measured between the populations. If random positional oscillations result in oscillations in collected light intensity that are greater in magnitude than the nominal difference in fluorescence intensity of the two populations, it is not possible to distinguish them with high yield and high purity simultaneously. The difference in fluorescence intensity between X and Y sperm cells is typically a small percentage (e.g., ~4% for bovine sperm). Current sperm sorting systems can theoretically achieve high throughput by increasing the nuclear stream flow rate, but this can have the effect of increasing the nuclear stream width.Consequently, there is a very large uncertainty regarding the position of spermatozoa within the nucleus of the flow stream. This positional uncertainty and the resulting oscillations in the collected fluorescence intensity limit the maximum productivity of current sperm sorting systems to levels that do not mask the small difference in fluorescence intensity between X and Y spermatozoa.

[0080] An approach to intensity-position correction can be understood with reference to FIGS. 6 and 7. The keys in FIG. 6 highlight integration regions that correspond to the fluorescence collection optical element with a given NA. Plots of the variation in collected intensity relative to object position for the NAs in FIG. 6 are provided in FIG. 7. In FIG. 7, for a given NA, integration in the fluorescence collection region is performed in such a way that the intensity of the collected light can be plotted as a function of each sperm position. It is evident from FIG. 7 that increasing the NA of the collection optical element helps to Petition 870260049819, dated 05 / 25 / 2026, page 42 / 123 35 / 38 reduce the influence of the object's position on the fluorescence intensity collected by the optical collection element.

[0081] In some embodiments, the optical collecting element (e.g., optical collecting arrangement 190 in FIGS. 1 and 2) can be modified with elements that reduce the variation in collected light intensity relative to the object position, as described above. Such 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 optical collecting element operates by masking certain rays in angle space, i.e., the optical collecting element selectively collects, attenuates, and / or blocks rays from different angles γ in order to achieve a desired intensity versus position profile. In practice, an angle-space masking function can be applied to a pupil (e.g., entrance pupil, exit pupil, or aperture stop) of an optical system, where the position of a ray intersection with the pupil plane corresponds to the angle γ.In some modes, the optical collection arrangement achieves a desired profile, for example, a flatter intensity-versus-position profile, by preferentially collecting wider-angle light rays (directed to the opposite side of the optical geometric axis) instead of excluding certain narrower-angle light rays.

[0082] FIGS. 15 and 16 illustrate how excluding low-angle reflected rays at a given NA causes the intensity-versus-position curve to flatten. Excluding low-angle rays excludes the rays that produce the most variation in the intensity-versus-position profile, while the angular variation of radiance at high positive angles tends to cancel the corresponding variation at high negative angles. FIG. 15 shows plots of the relative radiance versus angle of the rays, γ, for different object positions along Petition 870260049819, dated 05 / 25 / 2026, page 43 / 123 36 / 38 of the geometric x-axis where the angle γ is in radians. In FIG. 15, each graph corresponds to a position of the object, x, in the core of a flux stream, as indicated in FIG. 5. The keys in FIG. 15 show the portion of the light rays that will be excluded by the collecting optical element for each position x, when rays that have an angle magnitude of less than 0.3 rad are excluded (lower key in FIG. 15) and when rays that have an angle magnitude of less than 0.4 rad are excluded (upper key in FIG. 15).

[0083] FIG. 16 shows the relative collected light intensity versus object position along the geometric x-axis when no angle is excluded (graph 900), when rays with angles between -0.3 rad and +0.3 rad are excluded (graph 903), and when rays with angles between -0.4 rad and +0.4 rad are excluded (graph 904). Graph 16 shows that when rays with smaller angles are excluded, the graph of relative intensity versus position exhibits less variation in intensity with respect to position.

[0084] FIG. 17 illustrates the results of an experiment incorporating a software-based positional correction and a hardware-based element in the light collection path that reduces the variation in collected light intensity relative to the object's position, as described. Sperm nuclei stained with Hoechst 33342 were processed using a Genesis III sperm sorter manufactured by Cytonome. The sperm sorter was equipped with a SiPM sandwich-type detector that has a wire positioned in the light collection path to exclude low-angle electromagnetic radiation produced from the sperm nuclei. Wires and other suitable elements for blocking low-angle electromagnetic radiation are described in US Patent Application No. 16 / 133,531.

[0085] Sample and coating pressures were adjusted Petition 870260049819, dated 05 / 25 / 2026, page 44 / 123 37 / 38 to achieve an event rate of 60,000 events per second, and the nuclei were analyzed with a Coherent Genesis CW-355 laser operated at an average power of 90 mW. In plot 1010, it can be observed that the wire somewhat mitigates the effect of intensity dependence on the position of the nuclei in the fluid column. However, there is still a significant decrease in relative intensity as the nuclei move increasingly in the positive direction along the geometric x-axis. A population of X-chromosome-bearing nuclei 1012 and a population of Y-chromosome-bearing nuclei 1014 are observed sinking significantly in the positive direction on the geometric x-axis. The corresponding peak-to-trough ratio calculated from the fluorescence intensity histogram of plot 1020 is 81.5%. Again, X-chromosome-bearing nuclei located towards one end of the fluid column are not sufficiently detected.As a result, the summed fluorescence intensity of the nuclei at this end has intensity values ​​similar to the Y-chromosome-bearing nuclei in the Y-chromosome-bearing nuclei population 1014. This skewness is evident in the univariate histogram of plot 1020 in the form of a downward-shifting bump and an exaggerated peak in the Y-chromosome-bearing nuclei population 1014.

[0086] A correction factor of 1016 is illustrated in plot 1010. For each position, a correction value is added to the corresponding correction factor of detected fluorescence intensity. Plot 1030 illustrates a bivariate histogram that has a corrected population of X-chromosome-bearing nuclei 1032 and a corrected population of Y-chromosome-bearing nuclei 1034, which are more distinct rectangular populations. Plot 1040 provides the corresponding univariate histogram of summed intensity values. Petition 870260049819, dated 05 / 25 / 2026, page 45 / 123 38 / 38 corrected independent of the location of each event. The corrected population of X-chromosome-bearing nuclei 1032 and the corrected population of Y-chromosome-bearing nuclei 1034 are more distinct, having nearly equal peak heights and a peak-to-trough ratio of 92.6%.

[0087] The preceding description of various modalities was presented for illustrative and descriptive purposes only, not for limitation. The modalities revealed are not intended to be exhaustive or to limit the possible implementations of the modalities revealed. Many modifications and variations are possible in light of the above teachings.

Claims

1. Detection system, characterized in that it comprises: a collection optic that collects outgoing electromagnetic radiation from X-bearing and Y-bearing spermatozoa within a fluid column; a detector that generates an electrical signal responsive to the intensity of the outgoing electromagnetic radiation collected by the collection optic; and an analyzer that has instructions stored therein i) to normalize the intensity of the outgoing electromagnetic radiation represented in the electrical signal based on the position of the spermatozoa in the fluid column, and ii) to discriminate between X-bearing and Y-bearing spermatozoa.

2. Detection system, according to claim 1, characterized in that the detector comprises a first detector and in that the system further comprises a second detector that detects the position of spermatozoa in the fluid column, wherein optionally, the second detector comprises a position detector, wherein optionally, the position detector is selected from the group of: a camera, a CCD, PSD, SiPM sandwich-type detector and photodiode array.

3. Detection system, according to claim 1, characterized in that the detector comprises a first detector that: i) generates an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the collection optics and ii) detects the position of spermatozoa in the measurement region, wherein, optionally, the first detector is not located in a plane of the image of the collection optics, and / or, optionally, the first detector comprises a detector selected from the group consisting of: PMT sandwich type detector, SiPM sandwich type detector and photodiode array, a PMT array and a SiPM array.

4. Detection system, according to claim 1, characterized in that the instructions stored in the analyzer to normalize the intensity of the output electromagnetic radiation represented in the electrical signal based on the position of the spermatozoa in the measurement region applies a correction to the intensity of the output electromagnetic radiation represented in the electrical signal and in that, optionally, the correction factor is determined for each position on a major geometric axis of a nucleus current in the measurement region, and in that the correction factor is applied to the electrical signal representing each spermatozoon based on its detected position on the major geometric axis of the nucleus current.

5. Detection system, according to claim 1, characterized in that the analyzer includes instructions for discriminating viable spermatozoa carrying the X chromosome or viable spermatozoa carrying the Y chromosome from other cells.

6. Discrimination system, according to claim 1, characterized in that the spermatozoa are located in an elliptical column of sample fluid, where the sample fluid is formed coaxially with an outer layer of coating fluid, and the coating fluid comprises a generally cylindrical shape.

7. Detection system, according to claim 1, characterized in that it further comprises an element that modifies the output electromagnetic radiation to increase the uniformity of the output electromagnetic radiation collected by the collection optics. Petition 870260049819, dated 05 / 25 / 2026, page 48 / 123 3 / 4 of sperm cells in different positions.

8. Method, characterized in that it comprises: flowing cells through a measurement region at different positions relative to an optical axis of a collection optic; generating electromagnetic radiation by excitation; directing the electromagnetic radiation by excitation to the cells in the measurement region, wherein the cells emit outgoing electromagnetic radiation in response to the electromagnetic radiation by excitation; collecting outgoing electromagnetic radiation from X-bearing spermatozoa and Y-bearing spermatozoa within a measurement region; generating an electrical signal responsive to the intensity of the collected outgoing electromagnetic radiation and responsive to the position of the cell passing through the measurement region relative to the optical axis of the collection optic;To normalize the intensity of the output electromagnetic radiation represented in the electrical signal by adjusting the detected intensity of the output electromagnetic radiation based on the position of the spermatozoa in the measurement region relative to the optical axis of the collection optics, and to discriminate between spermatozoa carrying the X chromosome and spermatozoa carrying the Y chromosome.

9. Method according to claim 8, characterized in that it further comprises detecting the position of spermatozoa in the measurement region, wherein, optionally, the analyzer includes instructions for discriminating viable spermatozoa bearing the X chromosome or viable spermatozoa bearing the Y chromosome from other cells. Petition 870260049819, dated 05 / 25 / 2026, p. 49 / 123 4 / 4 10. A method according to claim 8, characterized in that the spermatozoa in the measurement region are located in a generally elliptical cross-section of a core stream contained within a generally circular cross-section of a coating fluid, and in that the spermatozoa in the fluid column are located at different positions on a major geometric axis of the generally elliptical cross-section of the core stream, the method further comprising, optionally, applying a correction to the intensity of the output electromagnetic radiation represented in the electrical signal, the method further comprising, optionally, determining a correction factor for each position on the major geometric axis of the core stream and applying the correction factor to each event based on the position on the major geometric axis of the core stream.

11. Method, according to claim 8, characterized in that it further comprises an element that modifies the outgoing electromagnetic radiation to increase the uniformity of the outgoing electromagnetic radiation collected by the collection optics from spermatozoa in different positions.

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