System and method for correcting position-dependent electromagnetic radiation detected from an object in a fluid column - Patents.com

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 technology]

[0001] Object discrimination devices and techniques distinguish between different types of objects, such as objects with different properties. These devices and techniques are particularly useful for analyzing and even sorting cells according to a specified property of interest. Some cell sorting techniques rely on light emitted from cells or stained cells to identify the type of cell. In some embodiments, cells moving through a column of fluid are exposed to an excitation source to generate output electromagnetic radiation for detection. Cells of a first type or with a particular property generate output electromagnetic radiation that differs in some property, e.g., wavelength and / or intensity, compared to other cells. Such differences serve as a basis for discriminating and sorting cell types. Summary of the Invention [Means for solving the problem]

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

[0003] According to some embodiments of the detection system, the optical arrangement collects output electromagnetic radiation from objects in the fluid column, the detector generates an electrical signal responsive to an intensity of the output electromagnetic radiation collected by the optical arrangement, and the 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 objects in the fluid column, and ii) discriminate the first type of objects from other objects.

[0004] According to another embodiment, a method of discriminating between objects begins by creating a fluid column including objects at different locations within the fluid column. An excitation electromagnetic radiation is directed at the objects in the fluid column in a measurement region. The objects in the measurement region emit output electromagnetic radiation in response to the excitation electromagnetic radiation, which is collected and used to generate an electrical signal responsive to an intensity of the output electromagnetic radiation. The intensity of the output electromagnetic radiation, represented by the electrical signal, is normalized based on the location of the objects in the fluid column, and a first type of object is discriminated from other objects. The present specification also provides, for example, the following items: (Item 1) 1. A discrimination system comprising: a fluid column forming structure for creating a fluid column containing objects at different positions therein; an excitation source that generates excitation electromagnetic radiation directed at objects in the fluid column at a measurement region, the objects in the fluid column emitting output electromagnetic radiation in response to the excitation electromagnetic radiation; an optical arrangement for collecting output electromagnetic radiation from the object; a detector that produces an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical arrangement; an analyzer storing instructions: i) 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) for discriminating a first type of object from other objects; A discrimination system comprising: (Item 2) 2. The discrimination system of claim 1, wherein the detector includes a first detector, and the system further comprises a second detector for detecting the position of an object in the fluid column. (Item 3) 3. The discrimination system of claim 2, wherein the second detector includes a position detector. (Item 4) 4. The discrimination system of claim 3, wherein the position detector is a detector selected from the group of a camera, a CCD, a PSD, a SiPM split detector, and a photodiode array. (Item 5) 2. The discrimination system of claim 1, wherein the detector includes a first detector that i) generates an electrical signal responsive 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. (Item 6) 6. The discrimination system of claim 5, wherein the first detector is not located in an image plane of the optical configuration. (Item 7) 6. The discrimination system of claim 5, wherein the first detector comprises a detector selected from the group consisting of a PMT split detector, a SiPM split detector, a photodiode array, an array of PMTs, and an array of SiPMs. (Item 8) 2. The discrimination system of claim 1, wherein the fluid column includes objects in a core flow of generally elliptical cross-section contained within a sheath fluid of generally circular cross-section, the objects in the fluid column being disposed at different positions on a major axis of the generally elliptical cross-section of the core flow. (Item 9) 9. The discrimination system of claim 8, wherein the instructions stored in the analyzer apply a correction to the intensity of the output electromagnetic radiation represented by the electrical signal, normalizing the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the object in the fluid column. (Item 10) 10. The discrimination system of claim 9, wherein a correction factor for each position on the major axis of the core flow is determined, and the correction factor is applied to each event based on the position on the major axis of the core flow. (Item 11) 2. The discrimination system of claim 1, wherein the first type of object includes an object selected from the group consisting of live sperm having an X chromosome and live sperm having a Y chromosome. (Item 12) 2. The discrimination system of item 1, wherein the analyzer includes instructions for discriminating between live sperm having an X chromosome and live sperm having a Y chromosome. (Item 13) 2. The discrimination system of claim 1, wherein the object comprises sperm disposed within an elliptical column of sample fluid, the sample fluid being formed coaxially with an outer layer of sheath fluid, the sheath fluid having a generally cylindrical shape. (Item 14) 2. The discrimination system of claim 1, further comprising an element that modifies the output electromagnetic radiation collected by the optical arrangement to increase uniformity of the output electromagnetic radiation of objects at different positions. (Item 15) 1. A detection system comprising: an optical arrangement for collecting output electromagnetic radiation from the object in the fluid column; a detector that produces an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical arrangement; an analyzer storing instructions: i) for normalizing the intensity of the output electromagnetic radiation represented by the electrical signal based on a position of the object in the fluid column; and ii) for discriminating a first type of object from other objects; A detection system comprising: (Item 16) 16. The detection system of claim 15, wherein the detector comprises 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 an object in the fluid column based on a signal from the second detector. (Item 17) Item 17. The detection system of item 16, wherein the second detector includes a position detector. (Item 18) Item 18. The detection system of item 17, wherein the position detector is a detector selected from the group consisting of a camera, a CCD, a PSD, a SiPM split detector, and a photodiode array. (Item 19) Item 16. The detection system of item 15, wherein the detector comprises a first detector that i) generates an electrical signal responsive 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. (Item 20) 20. The detection system of claim 19, wherein the first detector comprises a detector selected from the group consisting of a PMT split detector, a SiPM split detector, a photodiode array, an array of PMTs, and an array of SiPMs. (Item 21) 20. The detection system of claim 19, wherein the first detector is positioned outside an image plane of the optical configuration. (Item 22) 16. The detection system of claim 15, wherein the fluid column includes objects in a core flow of generally elliptical cross-section contained within a sheath fluid of generally circular cross-section, the objects in the fluid column being positioned at different positions on a major axis of the generally elliptical cross-section of the core flow, and the instructions stored in the analyzer normalize the intensity of the output electromagnetic radiation represented by the electrical signal based on the position of the object in the fluid column, applying a correction to the intensity of the output electromagnetic radiation represented by the electrical signal. (Item 23) 23. The detection system of claim 22, wherein a correction factor for each position on the major axis of the core flow is determined, and the correction factor is applied to each event based on the position on the major axis of the core flow. (Item 24) 16. The detection system of claim 15, wherein the first type of object comprises an object selected from the group consisting of live sperm carrying an X chromosome and live sperm carrying a Y chromosome. (Item 25) Creating a fluid column containing objects at different positions therein; Producing excitation electromagnetic radiation; directing the excitation electromagnetic radiation at an object in the fluid column at a measurement region, the object in the fluid column emitting output electromagnetic radiation in response to the excitation electromagnetic radiation; collecting output electromagnetic radiation from the object; generating an electrical signal responsive to an 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; Distinguishing a first type of object from other objects; The method includes: (Item 26) 26. The method of claim 25, further comprising detecting the position of an object in the fluid column. (Item 27) 26. The method of claim 25, wherein the fluid column includes objects in a core flow of generally elliptical cross-section contained within a sheath fluid of generally circular cross-section, the objects in the fluid column being disposed at different positions on a major axis of the generally elliptical cross-section of the core flow. (Item 28) 28. The method of claim 27, further comprising applying a correction to the intensity of the output electromagnetic radiation represented by the electrical signal. (Item 29) 29. The method of claim 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) 26. The method of claim 25, wherein the first type of objects comprises objects selected from the group consisting of live sperm carrying an X chromosome and live sperm carrying a Y chromosome. (Item 31) 26. The method of claim 25, wherein the analyzer includes instructions for discriminating between live sperm carrying an X chromosome and live sperm carrying a Y chromosome. (Item 32) 26. The method of claim 25, further comprising modifying the output electromagnetic radiation to increase uniformity of the output electromagnetic radiation collected from the object at different positions. [Brief description of the drawings]

[0005] [Figure 1]FIG. 1 is a diagram of a discrimination system in accordance with certain embodiments. [Diagram 2] 2 shows an xy-plane cross-section of a fluid column in the measurement region of the system of FIG. 1. [Diagram 3] 1 illustrates light emitted from an object located near the center of a fluid column having substantially uniform refraction of light at the fluid-air interface of the fluid column to an optical device functioning as collection optics. [Figure 4] 1 shows light emitted from an object placed on top of the elliptical core of a fluid column exhibiting non-uniform refraction of light at the fluid-air interface to an optical device acting as a collection optic. [Diagram 5] 1 shows the geometry used to develop an analytical expression for the angular dependence of the in-plane ray density as a function of position x. [Figure 6] A family of graphs is provided that shows the angular dependence of the emission light at different object positions within the fluid column. [Figure 7] 1 provides a family of graphs of relative intensity of light collected from a fluid column versus object position along the x-axis for different numerical apertures of the collection optics. [Figure 8] 1 is a flow diagram of a technique for identifying objects moving through a fluid column by correcting detected output light for position variations, according to some embodiments. [Figure 9] An overview of one embodiment is provided that involves detection of position and intensity from a single detector. [Figure 10] An overview of one embodiment is provided that includes a first detector that detects intensity and a second detector that identifies object location. [Figure 11] 13 shows the output of a simulation of object localization using a split detector. [Figure 12] 1 shows the results of one experiment that improves intensity measurements by correcting detected values ​​based on the position of the sperm nucleus. [Figure 13] The results of one experiment are presented to improve intensity measurements by correcting the detected values ​​based on the location of live sperm cells. [Figure 14]We present the results of one experiment that attempts to improve the performance of a misaligned instrument using a correction factor based on nucleus position. [Figure 15] A family of graphs of the angular dependence of the emission at different positions on the object is provided, showing the exclusion region. [Figure 16] Figure 14 shows the relative intensity of light collected from the fluid column with respect to object position along the x-axis when no angles are excluded, when rays with angles between -0.3 rad and +0.3 rad are excluded, and when rays with angles between -0.4 rad and +0.4 rad are excluded. [Figure 17] The results of one experiment utilizing both a correction factor and an element for optically reducing the position dependence of the intensity measurements are presented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The figures are not necessarily to scale. Like numbers used in the figures refer to like 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 labeled with the same number.

[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 and fluoresced electromagnetic radiation, such as light. As used herein, the term "light" refers to both electromagnetic radiation in wavelengths in the visible spectrum and in wavelengths in the infrared and ultraviolet spectrum. Such output electromagnetic radiation may include light reflected or fluoresced directly from the object and light reflected or fluoresced by a dye or pigment associated with the object. In some implementations, cell types are differentiated based on the intensity of the output electromagnetic radiation emitted from the object. The intensity may 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 differentiation of X-chromosome and Y-chromosome sperm cells. Further embodiments relate to the differentiation of live X-chromosome-bearing sperm cells from objects other than live X-chromosome-bearing sperm cells, including sperm cells bearing a Y chromosome and non-viable cells of both sexes.

[0008] It will be appreciated that the techniques of the present disclosure may be more generally applied to distinguish between any objects of different types, so long as the output electromagnetic radiation emitted from one object type produces a distinguishable difference in at least one characteristic when compared to the electromagnetic radiation emitted from another object type. In some examples provided, the fluid column is a flow stream having a curved boundary or interface where refraction of electromagnetic radiation may occur. For example, the curved boundary of the fluid column may generally be of circular cross section. The fluid column may be bounded by a solid wall, such as in a cuvette or a microfluidic channel, or may be jetted into air, such as in a jet-in-air flow cytometer. The object may travel along the fluid column through a central core shaped by a sheath fluid at least partially surrounding the central core. In sperm sorting applications, the central core may include a core flow of sample fluid containing sperm cells. The core flow may be generally ribbon-shaped or may have a generally elliptical cross section to orient aspherical sperm cells. Electromagnetic radiation emitted from an object encounters at least one optically refractive boundary between the object and another material, such as the interface between a fluid column and air.

[0009] At least in part due to the differing refractive indices of the sheath fluid and air, the collection efficiency outside the fluid column of light emitted from an object within the column depends on the object's position in such a system. Light collection efficiency that varies with position is detrimental in applications where the light emitted from an object must be precisely quantified and such precision is limited by random (not directly observable) positional variations of the object. In the case of sex-differentiated sperm, in particular, such systems attempt to distinguish between very bright and closely related fluorescent intensities. Sperm cells and sperm nuclei are typically stained with Hoechst 33342 to make such a distinction. Hoechst 33342 is a bright, cell-permeant 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 distinguishes between X and Y chromosomes as having slightly different amounts of nuclear DNA. For example, many livestock have a difference of about 4%. If the sperm cells are appropriately 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 illuminated with an appropriate excitation source, such as a laser operating at or near a wavelength of 355 nm.

[0010] This 4% difference is difficult to detect for several reasons. First, sperm nucleus DNA resides within sperm heads that in most species are aspherical or have a paddle-like shape. This asymmetry causes sperm to fluoresce differently from the flatter and narrower sides. In practice, this variation exceeds the 4% difference in DNA content, meaning that sperm must be oriented in order to differentiate based on nuclear chromosome content. Orientation geometries tend to produce core streams with ribbon-shaped or elliptical cross sections. This elliptical cross section provides sperm with more freedom than they would normally have if positioned uniaxially.

[0011] The techniques disclosed herein enhance the precision of systems that may be limited by such variations, such as jet-in-air flow cytometers. As described in more detail below, positional variability in the light intensity collected from objects in the fluid column can be addressed using algorithms that correct for the dependence of intensity on position.

[0012] The techniques outlined herein are particularly applicable to flow cytometry. However, the techniques are applicable to any system in which light is collected from an object that emits light from one side of an interface, and the interface varies the path of the emitted light beam depending on the object's position relative to the detector. The techniques herein compensate for positional variations within the fluid column, and therefore provide more accurate measurements for distinguishing object types.

[0013] A "jet-in-air" flow cytometer system 100, shown diagrammatically in FIG. 1, is one type of discrimination system that can be utilized to explore the concepts of the present disclosure. The "jet-in-air" flow cytometer system 100 includes a fluid column forming a structure that produces a flow stream including a fluid column 150 that ejects from an outlet nozzle 160 of a chamber 110 at high velocity, e.g., about 20 m / s. The fluid column 150 ejected from the outlet nozzle 160 can be roughly circular in cross section, and in some implementations can have a diameter of about 10 μm to about 100 μm. In some embodiments, the chamber 110 and / or the outlet nozzle 160 are configured with an internal geometry that hydrodynamically orients the sperm within the fluid column. As a 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 the sperm and generate a coaxial flow of the fluid column. 1 indicate the flow directions of the core flow 151 and the sheath flow 152. The sheath flow 152 may have a generally circular cross-section, while the core flow has a generally elliptical cross-section, with a major axis and a minor axis.

[0014] In the chamber 110, the sample injection element 111 introduces a core flow 151 containing objects 171, 172, which may be of multiple types. The core flow 151 is bounded by a sheath flow 152 containing a sheath fluid, and is shaped by fluid forces in the chamber 110. The sheath flow 152 at least partially surrounds the core flow 151, and the sheath flow 152 and the core flow 151 do not substantially mix. The inclined or slanted walls 115 of the chamber 110 provide a force that shapes the core flow 151 and accelerates the objects 171, 172 within the core flow 151. The movement of the sheath flow 152 confines the objects 171, 172 to the core flow 151, and they move towards the center of the fluid column 150 as the fluid column 150 is ejected from the chamber 110. The fluid column 150 carries the objects 171, 172 to a measurement region 175 of the fluid column 150, for example in a single file.

[0015] As the objects pass through the measurement area 175 of the fluid column 150, light from an excitation source 180 provides excitation light to the objects 171, 172. The excitation source 180 can provide light in a broad or narrow wavelength band. For example, the excitation source 180 can be a laser. Any laser suitable for generating a response from the object or a dye associated with the object may be employed. Pulsed and continuous wave lasers are each well suited for generating a suitable response. In some configurations, electromagnetic radiation generated by a historical source, such as an excitation light, can be modified by an optical element 181. For example, the excitation light can be focused by one or more lenses 181 into the measurement area 175. The lenses can be used to focus the excitation electromagnetic radiation into a suitable beam shape that focuses into the measurement area. The objects 172a in the measurement area 175 emit light, such as scattered light or fluorescent light, in response to the excitation source 180.

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

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

[0018] In some circumstances, the amplitude of the electrical signal may be different for different object types. The electrical signal is used by the 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 side detector may be employed at 90 degrees to the detector 185 to detect side scatter or side fluorescence. In the case of sperm sorting, the side fluorescence allows the analyzer 187 to distinguish properly oriented sperm from non-oriented sperm.

[0019] The analyzer 187 may include a processor 188 having executable instructions stored thereon. In addition to known instructions 198 for collecting, comparing, and manipulating information from the detector signals, the processor may include instructions 192 for normalizing intensity values ​​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 values ​​may be normalized in any manner. As merely one example, a hand-drawn line or curve may be entered by a user into a graphical user interface based on an initial sampling of data including the fluorescence intensity and position information.

[0020] The processor 188 may also include instructions 182 for discriminating between objects. Figure 2 shows an xy-plane cross-section of the fluid column 150 at the measurement region 175 shown in Figure 1. In the xy-section at the measurement region 175, the core stream 151 is elliptical in shape, and the fluid of the core stream 151 includes at least one object 172a suspended in a buffer solution, which may also be referred to as a sample. The sheath stream 152 substantially surrounds the core stream 151. In the particular example used for this discussion in 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] A focused laser beam generated by an excitation source 180 illuminates sperm cells 172a in the measurement region 175. The cells 171, 172 are stained with a fluorescent dye, and the excitation electromagnetic radiation causes the cells 172a in the measurement region 175 to emit fluorescent output electromagnetic radiation. The purpose of the generally elliptical core flow 151 is to orient the sperm cells 172a so that their flat sides 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 arrangement 190, respectively. If each cell 171, 172 is present in the measurement region 175 with a similar orientation, random variability based on orientation can be greatly reduced. However, the elliptical cross-section intended for this orientation also provides a large degree of freedom with respect to the position of the cells within the fluid column 150.

[0022] To obtain the desired orientation, the elliptical core stream 151 presents a major axis parallel to the x-axis shown in FIG. 2. The sperm cell 172a can assume any number of positions along the x-axis within the core stream 151. FIG. 2 shows three representative positions of the sperm cell 172a possible in the elliptical core 151, but it can be understood that the sperm may be positioned anywhere between the positions shown. In the orientation shown in FIG. 2, a first possible position for the sperm cell 172a in the core stream 151 is approximately in the center of the elliptical core 151 (on the optical axis 199 of the optical collection arrangement 190), a second possible position is above the core stream 151 (above the optical axis 199), and a third possible position is below the core stream 151 (below the optical axis 199). Position-dependent refraction of the output light beam emitted from the sperm cell 172a occurs at different positions at the fluid-air interface 153 within the core stream 151. As used herein, terms of relative position such as "above," "below," "upper," and "lower" should be understood as describing the relationship between the features shown, and do not limit the scope of the claims, particularly the location of the sperm in the core stream 151.

[0023] When sperm cell 172a is positioned in a first position and fluid column 150 has a circular cross section as shown in Figure 2, the in-plane light rays emitted from sperm cell 172a are generally perpendicular to fluid-air interface 153. Light rays emitted from points of sperm cell 172a away from the center or from outside the plane of the figure do not strike interface 153 strictly perpendicularly, and these rays are not considered in this simplified discussion, although one skilled in the art will recognize that the discussion herein can be generalized to include them. Therefore, to the extent that any refraction of light occurs at fluid-air interface 153, the refraction occurs more uniformly with respect to detector 185.

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

[0025] In contrast, when the sperm cell 172a is offset from the optical axis 199 and closer to the top or bottom of the elliptical core 151, such as in 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 encounter the fluid-air interface 153 at an oblique angle. These output light rays are non-uniformly refracted at the fluid-air interface 153, in contrast to the normal incidence situation described above. The most oblique light rays are refracted the most. The refraction of the light rays causes the radiance distribution of the fluorescent light exiting the fluid column 150 across the fluid-air interface 153 to be non-uniform and to vary with the position of the cell 172a along the x-axis. That is, this refraction changes the radiance distribution of the output electromagnetic radiation emitted from the sperm cell 172a to the outside of the fluid column 150.

[0026] For example, if the cell 172a is positioned off the optical axis 199, e.g., in the second or third position shown in FIG. 2, the density of rays, and therefore the distribution of radiance on the air side of the interface 153, is higher at positive or negative ray angles, respectively, with respect to the optical axis 199, when compared to the radiance on the air side of the interface 153 at angles parallel to the optical 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 illustrates rays 299 emanating from the cell 172a and exiting the fluid column 150 through the fluid-air interface 153, when the cell 172a is positioned in the second position of the elliptical core 151. In this situation, the density of rays or radiance at positive ray angles is higher than the density of rays parallel to the optical axis 199 or at negative ray angles. For an optical system with a given numerical aperture (NA), the amount of light collected by the system from a cell of the same type (e.g., collection efficiency) may vary depending on whether the cell is in a first location or a second location. The position dependence of the system collection efficiency introduces imprecision in identifying cell types.

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

[0028] We wish to solve for the ray density versus angle γ, which can be used to determine the 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 sperm cells emit light uniformly in all directions, so that the density of light rays generated with respect to the angle θ is given by: I θ (θ)=1 / π (2) That is,

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[0030] Density of rays outside the boundary surface I β (β) is the density of light inside the boundary surface I α(α), where T(α) represents the average across both polarities of the transmission through the interface.

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

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[0032] Using equation (7) together with the above and the following additional relations:

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[0033] Here, the NA of the optical collection configuration is given by the sine of the maximum ray angle γ 0 and therefore one can solve this angle in terms of the NA. γ0=sin -1 (NA) (16)

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

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[0035] Using the formula for the ray density distribution in Eq. (15), the angular dependence of ray density (radiance) at different sperm positions can be plotted as in FIG. 6. In FIG. 6, each line represents the ray density as a function of angle γ at a given sperm position x, where angle γ is in radians. The plot corresponds to a set of positions in the range symmetrically centered about x=0, where the ray density (radiance) is uniform as a function of 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 ray angles γ and lower at negative ray angles γ, and the opposite is true when x is negative (e.g., the third position in FIG. 2, corresponding to graph 403).

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

[0037] In the family of graphs in FIG. 7, graph 412 shows the relative intensity with respect to position along the x-axis for a collection optic with 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 position along the x-axis for a collection optic with a NA of 0.4, graph 416 shows the relative intensity with respect to position along the x-axis for a collection optic with a NA of 0.6, graph 418 shows the relative intensity with respect to position along the x-axis for a collection optic with a NA of 0.8, and graph 419 shows the relative intensity with respect to position along the x-axis for a collection optic with a NA of 0.9. From FIGS. 6 and 7, it is clear that a collection optic with a smaller NA has a larger variation in collected light intensity with respect to object position compared to a collection optic with a larger NA. Furthermore, a collection optic with a larger NA collects light rays with a wider range of refractive angles than a collection optic with a smaller NA, and therefore has a higher overall collection efficiency.

[0038] With particular reference to sperm discrimination and sorting applications, it can be seen that the elliptical major axis of the core stream 151 (FIGS. 1 and 2) can be about 50 μm long, providing the sperm with about 25 μm of freedom to move in either direction. Referring again to FIG. 7, it can be seen that an NA of 0.2 captures only about 90% of the object relative intensity when the object is offset from center by about 17 μm. Similarly, an NA of 0.4 captures only 92% of the relative intensity of an object offset from center by about 17 μm, and an NA of 0.6 captures just above 94% of the relative intensity at the same location. It can further be seen that the NA of the sperm sorter collection optics can be about 0.3 to about 0.6. FIG. 7 shows the benefits of increasingly larger numerical apertures, but such numerical apertures are increasingly expensive, have a shallower depth of field, and mean that larger apertures must be placed closer to the nozzle. However, in sperm sorting applications, there is a limit to how close the collection optics can be placed. In a typical sperm sorting instrument, the aperture may be about 0.5 to 0.6. The embodiments described herein correct for position dependence on the measured intensity, allowing smaller numerical aperture collection optics to perform like higher numerical aperture collection optics.

[0039] Thus, sperm positioned in the core stream 151 at positions approaching the second and third positions of FIG. 2 emit electromagnetic radiation of much lower overall intensity that is ultimately detected for analysis and discrimination. In fact, the core stream 151 may have an elliptical major axis that is about 50 μm long with a high event rate (on the order of 60,000 events per second or greater). Some sperm may be displaced from the center by 20 μm or even about 25 μm to either side of the first position. In the context of extremely bright and closely related fluorescent signals, this variation may overshadow the roughly 4% difference in stained nuclear DNA that distinguishes X-bearing sperm from Y-bearing sperm.

[0040] Furthermore, to increase the number of events at a given sperm concentration in a sample of buffer, the sample volume per unit time in the fluid column passing through the measurement region must be increased. Increasing the number of detected events per second in this way also increases the elliptical cross-sectional area of ​​the core flow in the fluid column, including the length of the major axis. As a corollary, and as those skilled in the art will recognize, increasing the sorting speed by increasing the sample flow rate generally decreases the sensitivity of the sperm sorting device. Thus, the embodiments described herein may not only improve sperm sorting accuracy at normal speeds, but also provide sperm sorting at increased overall speeds in terms of throughput without significant loss of fidelity.

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

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

[0043] The analyzer or other suitable means then normalizes the intensity represented by the output signal based on the position of the object in the fluid column (560). The normalization may be performed by a correction whereby signals generated off the central 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 the appropriate correction factor can be seen in FIG. 7. Once normalized by the correction, the method continues by discriminating the first type of object from other objects (570). The discrimination may be performed in a flow cytometer analyzer and may include one or more additional operations. For example, a univariate histogram may be generated showing the distribution of the fluorescence intensity. A bivariate histogram may also be generated using the corrected signal and further calculated values. Such corrected and calculated values ​​may be compared to gating regions in the flow cytometer analyzer or compared to a look-up table to discriminate the first type of object from other types of objects.

[0044] Exemplary objects, sperm, may be differentiated as either sperm bearing an X chromosome or sperm bearing a Y chromosome. Furthermore, the sperm may be stained with a DNA-selective dye in addition to the secondary quenching dye. The quenching dye typically permeates membrane-weakened sperm cells, such as dead or dying sperm cells, greatly reducing the fluorescence generated by the DNA-selective dye associated with those weakened cells. Such quenching cells are effectively removed from the closely associated population undergoing differentiation / sorting. In this manner, the system can differentiate live or viable sperm cells from dying or weakened sperm cells. The system can also differentiate live sperm bearing an X chromosome from all remaining cells, and sperm bearing a Y chromosome from all remaining cells, or even simultaneously differentiate live sperm bearing an X chromosome and sperm bearing a Y chromosome from all other sperm cells.

[0045] 9 illustrates a first embodiment of a discrimination system substantially similar to that 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 arrangement 190. The optical collection arrangement 190 may include a collection lens that focuses the modified output electromagnetic radiation onto a detector 185. In the illustrated embodiment, the detector functions similarly to a position detector 186 that measures a characteristic of the modified output electromagnetic radiation and locates the position of the object 172a within the core flow 151 of the fluid column 150.

[0046] Detectors 185 suitable for determining the properties of the modified output electromagnetic radiation 162 and determining the position of the object 172a in the measurement region may include split detectors or detector arrays such as PMTs, SiPMs, pin photodiodes, etc. These detectors may be positioned in the image plane or the Fourier plane of the object to determine the object's position. In the image plane, the detectors directly measure the object's position, while in the Fourier plane, position information is extracted from the lateral intensity distribution (e.g., left-right asymmetry).

[0047] Flow cytometry applications often require detectors that are very sensitive (up to single photon counts) and fast (objects move at about 20 m / s through 10 μm). Detectors with the required speed and sensitivity are typically detectors that offer internal gain. In photomultiplier tubes (PMTs) or silicon photomultiplier tubes (SiPMs), also known as pixelated avalanche photodiodes, a single photon can count up to about 10 6 Both detector types are commercially available as detector arrays. SiPMs are fabricated by standard techniques on silicon wafers and may therefore be better suited for use in detector arrays suitable for object localization. Some detectors, such as SiPMs, may be particularly well suited to being placed at the Fourier plane to distribute the light over a larger area of ​​the detector.

[0048] 10 illustrates 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. The majority of the modified output electromagnetic radiation 162 is directed to and focused on a detector 185. In this embodiment, the detector 185 includes a first detector 176 that detects the property of interest. The first detector 176 can be any detector conventionally suited for quantifying the particular property of interest. In a typical flow cytometer application, photodiodes, photomultiplier tubes (PMTs), and silicon photomultiplier tubes may be particularly well suited for detecting scattered or fluorescent emission electromagnetic intensity.

[0049] Beam splitter 191 may include a dielectric mirror 197, although one skilled in the art will appreciate that other suitable optical components, such as a cube beam splitter, a prism beam splitter, or the like, may 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 along a first path to a detector, and a second beam portion 165 is directed along a different path to a second detector 173 in the form of a position detector 177. The position detector can be a camera, an isotropic sensor or a position sensitive device ("PSD") such as a charge coupled device (CCD), a split detector, a PMT, a SiPM, a detector array of pin photodiodes, or the like.

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

[0051] FIG. 12 shows the results of one experiment incorporating position correction of sperm nuclei in the fluid column leading to a significant improvement in the discrimination of sperm nuclei bearing X and Y chromosomes. Hoechst 33342 stained sperm nuclei were processed through a Genesis III sperm sorting instrument from Cytonome. The instrument is equipped with a SiPM split detector. Sample and sheath pressures were adjusted to establish an event rate of 35,000 events per second. Nuclei were interrogated using a Coherent Genesis CW-355 laser at an average power of 150 mW. Plot 610 shows a bivariate histogram showing the sum of the fluorescence concentration from each detector in the split detector plotted against the position delta of the nuclei in the fluid column. As mentioned previously, the range of position delta represents the major axis of the elliptical core flow that nuclei may enter the measurement region. The population of sperm nuclei 612 bearing X chromosomes is seen in a crescent shape. As expected, the measured intensity is greatest near position delta 0 and curves downwards and decreases as the nuclei move away from the central position. The cluster of Y-chromosome-bearing nuclei 614 is visible as a second crescent just below the X cluster, and again, the highest intensity is seen near position delta 0, with a large drop in relative intensity as the nuclei move away from the central position.

[0052] Plot 620 presents a univariate histogram of the combined fluorescence intensity corresponding to the intensities charted in plot 610. A clear population of X-chromosome bearing nuclei 612 and Y-chromosome bearing nuclei 614 can be seen, however, comparison of plot 610 with plot 620 reveals that the off-center, X-chromosome bearing sperm nuclei increasingly overlap with the centrally located, Y-chromosome bearing sperm nuclei. 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 to remove the variation introduced by the random position of the events. The corresponding correction was applied to the fluorescence summation values ​​shown in plot 630 to generate a corrected population of nuclei bearing an X chromosome 632 and a corrected population of nuclei bearing a Y chromosome 634. The corrected population of nuclei bearing an X chromosome 632 forms a generally rectangular shape and no longer shows variation based on the position of the nuclei in the fluid column. In plot 630, a clear gap can be seen between the corrected population of nuclei bearing an X chromosome 632 and the corrected population of nuclei bearing a Y chromosome 634. Plot 640 shows the corresponding univariate histogram, with a peak-to-valley ratio of 94% between the corrected population of nuclei bearing an X chromosome 632 and the corrected population of nuclei bearing a Y chromosome 634. The striking contrast between plot 620 and plot 640 is visually apparent. Moreover, the difference is quantifiable at a higher 17.2 percentage points.

[0054] FIG. 13 shows an example result incorporating correction according to embodiments described herein. Live sperm stained with Hoechst 33342 were processed through a Genesis III sperm sorting instrument from Cytonome. Sample and sheath pressures 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 live sperm in the core stream. Again, the population of sperm bearing an X chromosome 712 can be seen as the first population above the population of sperm bearing a Y chromosome 714. A correction factor 716 for normalizing the combined intensity values ​​is also shown on plot 710. Plot 720 shows a univariate histogram of uncorrected combined intensity, showing a peak-to-valley ratio of 75.3% between the population of sperm bearing an X chromosome 712 and the population of sperm bearing a Y chromosome 714.

[0055] Plot 730 provides a type of bivariate histogram common in sperm sorting applications. In this case, corrected forward fluorescence intensity is plotted against side fluorescence. Forward fluorescence vs. side fluorescence histograms are useful for sorting live sperm because the side fluorescence provides information about the orientation of each cell. In contrast, sperm nuclei are sonicated and removed from the aspherical sperm heads. Thus, orientation does not matter when sorting sperm nuclei. Because of this, the nuclei are easy to sort and are often used to calibrate sperm sorting flow cytometers. Plot 730 shows a corrected population 732 of sperm bearing an X chromosome and a corrected population 734 of sperm bearing a Y chromosome.

[0056] Much like 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 of X-chromosome-bearing sperm 732 and the corrected population of Y-chromosome-bearing sperm 734 can be seen as more distinct peaks with a machine-calculated peak-to-valley ratio of 81.0%. And again, the corrected histogram represents a significant improvement over plot 720, which shows the location-corrected values ​​for live sperm.

[0057] In another aspect, the embodiments described herein may provide systems and methods that substantially facilitate the alignment process in a flow cytometer. For example, in the case of sperm, the measurement area, detectors, and even the structures forming the sheath flow must be appropriately and precisely aligned to generate and collect a signal clear enough to distinguish between sperm populations with very bright and tightly associated X and Y chromosomes. Even with precise and proper alignment, oriented sperm in a fluid column can take any number of positions along the long axis of the core flow. As discussed above with respect to Figures 3-7, this means that even when aligned with the components of a flow cytometer, there is an angular dependency on the detected output electromagnetic radiation. This angular dependency introduces noise-like variability, since cells can be randomly located within the core flow.

[0058] In commercial sperm sorting applications, a technician typically makes several coarse adjustments in multiple axes on multiple components followed by several fine adjustments to align the instrument. Due to the sensitivity of the instrument to each adjustment, the very closely related nature of the signals detected, and the number of possible adjustments, such alignment can be a time-consuming task for the technician operating the sperm sorting instrument. When switching samples, aligning the machine for commercial sperm sorting can take several minutes, even up to 5 minutes. After unclogging or otherwise clearing nozzles and replacing or adjusting other components that require calibration, it can take 5, 15, or even in rare cases 30 minutes of technician time to properly align the instrument for commercial sperm sex sorting.

[0059] FIG. 14 shows the results of one example where the alignment process for discriminating sperm nuclei was greatly reduced. Hoechst33342 stained sperm nuclei were processed through a Genesis III sperm sorter from Cytonome. The instrument was equipped with SiPM split detection. Coarse alignment was performed with forward fluorescence detection aligned in less than one minute. The sperm nuclei were run at an event rate of 33,000 nuclei per second and interrogated 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 each event by the SiPM. Misalignment is evident in each of the populations of nuclei bearing an X chromosome 812 and nuclei bearing a Y chromosome 814. With poor alignment, the crescents are asymmetric and the fluorescence intensity values ​​drop dramatically in the positive x direction compared to the negative x direction. The population of nuclei bearing a Y chromosome 814 shows the same skew.

[0060] A correction factor 816 is shown as a line between the two populations. This correction factor 816 indicates the degree of correction performed on the summed fluorescence values ​​at the nuclear x-locations. In other words, the correction factor 816 represents a curve that is normalized by correction to a flat line. Each summed fluorescence value at a corresponding x-location along the line is increased or decreased by an amount equal to the correction factor 816.

[0061] The distortion caused by the rough alignment is more pronounced in the fluorescence intensity histogram of plot 820, where the increased overlap results in a peak-to-valley ratio of 72.3% between the population of X-chromosome-bearing nuclei 812 and the population of Y-chromosome-bearing nuclei 814.

[0062] In plot 830, the corrected forward fluorescence sum values ​​are plotted in a bivariate histogram versus the detected position of each event. Again, by normalizing the fluorescence intensity values ​​using the correction factor 816 based on the cell's position, it can be seen that two distinct populations of cells emerge. The corrected population of nuclei bearing an X chromosome 832 and the corrected population of nuclei bearing a Y chromosome 834 are more distinct and are clearly grouped together in plot 830. Importantly, the orthogonal relationship of these populations leads to a 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 using compensation, some embodiments described herein include elements that reduce collected light intensity variations with respect to object position in the flow stream. Some embodiments described herein can provide modified output light with measured intensity variations of less than about 3%, less than about 2%, or even less than about 1% for object position deviations that are less than 60% of the flow stream's radius 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 sources. Due to the difficulty in reducing intensity variations by precisely controlling the position of objects in the flow stream, it is useful instead to reduce the collected light intensity variations with respect to object position by carefully designing the optical collection configuration. In applications such as X / Y sperm sorting, it is often necessary to separate two or more cell populations based on the difference in measured fluorescence intensity between the populations. If random positional variations lead to a variation in collected light intensity that is greater than the nominal difference in the fluorescence intensity of the two populations, it is not possible to distinguish the two populations with both high yield and high purity. The fluorescence intensity difference between X and Y sperm cells is typically only a few percent (e.g., about 4% for bovine sperm). Current sperm sorter systems could 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. Thus, there is a large uncertainty of sperm position within the core of the flow stream. This position uncertainty and the resulting variability in collected fluorescence intensity limits the maximum throughput of current sperm sorter systems to a level that does not mask the small fluorescence intensity difference between X and Y sperm.

[0064] One approach to intensity-position correction can be understood with reference to Figures 6 and 7. The brackets in Figure 6 highlight the integration region corresponding to the fluorescence collection optics with a given NA. A graph of collected intensity variation versus object position for the NA of Figure 6 is provided in Figure 7. In Figure 7, for a given NA, integration over the fluorescence collection area is performed so that the intensity of collected light can be plotted as a function of each sperm position. From Figure 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 optics (e.g., optical collection configuration 190 in Figs. 1 and 2) may be modified with elements that reduce collected light intensity variation with respect to object position as described above. Some embodiments are described in more detail in U.S. Patent Application Serial No. 16 / 133,531, which is incorporated herein by reference. According to some such embodiments, the collection optics operates by masking certain configurations in "angle space", i.e., the collection optics selectively collects, attenuates, and / or blocks light rays from different angles γ to achieve a desired intensity vs. position profile. In practice, the "angle space" masking function can be applied to the pupil of the optics (e.g., entrance pupil, exit pupil, or aperture stop), and the location where the light rays intersect the pupil plane corresponds to the angle γ. In some embodiments, the collection optics achieves a desired, e.g., flatter, intensity vs. position profile by prioritizing the collection of high-angle (pointing away from the optical axis) light rays over the exclusion of certain low-angle rays.

[0066] 15 and 16 show how excluding low angle refracted rays at a given NA flattens the intensity vs. position curve. excluding low angle rays excludes rays that produce the largest variation in the intensity vs. position profile, while the angular variation in radiance at large positive angles tends to cancel the corresponding variation at large negative angles. FIG. 15 shows a plot of relative radiance vs. ray angle γ at different positions of the object along the x-axis, where 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 that are excluded by the collection optics at each position x when rays with angles less than 0.3 rad in magnitude are excluded (lower bracket in FIG. 15) and when rays with angles less than 0.4 rad in magnitude are excluded (upper bracket in FIG. 15).

[0067] 16 shows the relationship between relative collected light intensity and object position along the x-axis when no angles are 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 lower angle rays are excluded, the relative intensity versus position graph shows lower intensity variation with position.

[0068] FIG. 17 shows the results of one experiment incorporating software-based position correction as well as hardware-based elements in the collection light path that reduce the collected light intensity variation with object position as described. Hoechst33342 stained sperm nuclei were processed through a Genesis III sperm sorter from Cytonome. The sperm sorter was matched to a SiPM split detector with wires placed in the collection path to filter out the low collection angle electromagnetic radiation generated from the sperm nuclei. Wires and other elements suitable for blocking low collection angle electromagnetic radiation are described in U.S. Patent Application Serial No. 16 / 133,531.

[0069] The sample and sheath pressures were adjusted to reach an event rate of 60,000 events per second, and the nuclei were interrogated using a Coherent Genesis CW-355 laser operating at an average power of 90 mW. In plot 1010, it can be seen that the wires reduce the effect of the intensity dependence on the nucleus position within the fluid column. However, there is still a large drop in relative intensity as the nuclei move further along the x-axis in the positive direction. The population of nuclei bearing an X chromosome 1012 and the population of nuclei bearing a Y chromosome 1014 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 in plot 1020 is 81.5%. Again, the nuclei bearing an X chromosome located towards one end of the fluid column are poorly detected. As a result, the combined fluorescence intensity of the nuclei at this end has a similar intensity value as the centrally located Y chromosome bearing nuclei within the population of nuclei bearing a Y chromosome 1014. This skew is evident in the univariate histogram of plot 1020 in the form of a downwardly shifting shoulder and an exaggerated peak in the population of nuclei 1014 with Y chromosomes.

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

[0071] The above description of various embodiments has been presented for purposes of illustration and description and not limitation. The disclosed embodiments are not intended to be exhaustive or to limit the possible implementations of the disclosed embodiments. Many modifications and variations are possible in light of the above teachings.

Claims

1. 1. A discrimination system comprising: collection optics for collecting output electromagnetic radiation from the X chromosome-bearing sperm and the Y chromosome-bearing sperm within the measurement region; a detector that produces an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the collection optics; an analyzer storing i) instructions for normalizing the intensity of the output electromagnetic radiation represented by the electrical signal based on a position of a cell in the measurement region, and ii) instructions for discriminating between sperm bearing an X chromosome and sperm bearing a Y chromosome; Equipped with A discrimination system, wherein the sperm cells in the measurement region are located at different positions on the major axis of a generally elliptical cross-section of the core stream.

2. 2. The discrimination system of claim 1, wherein the detector includes a first detector, the system further comprising a second detector for detecting the location of the sperm cells in the measurement region.

3. The discrimination system of claim 2 , wherein the second detector comprises a position detector.

4. The discrimination system of claim 3 , wherein the position detector is a detector selected from the group of a camera, a CCD, a PSD, a SiPM split detector, and a photodiode array.

5. 2. The discrimination system of claim 1, wherein the detector comprises a first detector: i) generating an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the collection optics; and ii) detecting the position of a sperm cell in the measurement region.

6. The discrimination system of claim 5 , wherein the first detector is not located at an image plane of the collection optics.

7. The discrimination system of claim 5 , wherein the first detector comprises a detector selected from the group consisting of a split PMT detector, a split SiPM detector, a photodiode array, an array of PMTs, and an array of SiPMs.

8. 2. The discrimination system of claim 1, wherein 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 sperm cell in the measurement region apply a correction to the intensity of the output electromagnetic radiation represented by the electrical signal.

9. A discrimination system as described in claim 8, wherein an amount of correction is determined for each position on the long axis of the core flow in the measurement area, and the correction is applied to the electrical signal representing each sperm cell based on the detected position on the long axis of the core flow.

10. 2. The discrimination system of claim 1, wherein the analyzer includes instructions for discriminating between live sperm carrying an X chromosome and other cells or live sperm carrying a Y chromosome.

11. 2. The discrimination system of claim 1, wherein the sperm cells are disposed within an elliptical column of sample fluid, the sample fluid being formed coaxially with an outer layer of sheath fluid, the sheath fluid having a generally cylindrical shape.

12. The discrimination system of claim 1 , further comprising an element that modifies the output electromagnetic radiation to increase uniformity of the output electromagnetic radiation collected by the collection optics for cells at different locations.

13. A method for collecting output electromagnetic radiation from X-chromosome-bearing sperm and Y-chromosome-bearing sperm within a measurement region, the sperm cells within said measurement region being positioned at different locations on a major axis of a generally elliptical cross-section of a core stream; generating an electrical signal responsive to an intensity of the collected output electromagnetic radiation; normalizing the intensity of the output electromagnetic radiation represented by the electrical signal based on a position of the sperm cell within the measurement region; Distinguishing between sperm carrying an X chromosome and sperm carrying a Y chromosome, A method comprising:

14. The method of claim 13, further comprising detecting the position of a sperm cell in the measurement area.

15. The method of claim 13 , further comprising applying a correction to the intensity of the output electromagnetic radiation represented by the electrical signal.

16. 16. The method of claim 15, 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.

17. 14. The method of claim 13, wherein the analyzer includes instructions for discriminating between live sperm carrying an X chromosome and live sperm carrying a Y chromosome.

18. 14. The method of claim 13, further comprising an element for modifying the output electromagnetic radiation to increase uniformity of the output electromagnetic radiation collected from cells at different locations.

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