Patent Description:
<CIT> discloses a prior art discrimination system.

The invention is defined by a discrimination system according to claim <NUM> and the corresponding method according to claim <NUM>.

Some embodiments are directed to a discrimination system for discriminating between different types of objects based on electromagnetic radiation emanating from objects disposed within a fluid column. A fluid column forming 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. Objects within the fluid column emanate output electromagnetic radiation in response to the excitation electromagnetic radiation. An optical arrangement collects output electromagnetic radiation from the objects and a detector generates an electrical signal responsive to the intensity of the output electromagnetic radiation. An analyzer includes instructions stored thereon i) to normalize the intensity of the output electromagnetic radiation represented in the electrical signal based on the position of the object in the fluid column, and ii) to discriminate a first type of object from other objects.

In accordance with some embodiments of a detection system, an optical arrangement collects output electromagnetic radiation from objects in a fluid column and a detector generates an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical arrangement. An analyzer has instructions stored thereon i) to normalize the intensity of the output electromagnetic radiation represented in the electrical signal based on the position of the object in the fluid column, and ii) to discriminate a first type of object from other objects.

In accordance with other embodiments, a method of discriminating objects begins by creating a fluid column containing objects at differing positions within the fluid column. Excitation electromagnetic radiation is directed toward objects in the fluid column at a measurement region. Objects at the measurement region emanate output electromagnetic radiation in response to the excitation electromagnetic radiation, which is collected and used to generate an electrical signal responsive to the intensity of the output electromagnetic radiation. The intensity of the output electromagnetic radiation represented in the electrical signal are normalized based on the position of the object in the fluid column and a first type of object is discriminated from other objects.

Embodiments described herein relate to devices, systems and methods for discriminating between different types of objects. The objects emanate output light in response to an excitation light that is directed toward the objects in a fluid column, such as a flow stream. As used herein the term "emanate" refers to both reflected and fluoresced electromagnetic radiation, such as light. As used herein the term "light" refers to both electromagnetic radiation at wavelengths in the visible spectrum as well as electromagnetic radiation at wavelengths in the infrared and ultraviolet spectrums. Such output electromagnetic radiation may include light reflected or fluoresced directly from an object as well as light reflected or fluoresced by a stain or dye associated the object. In some implementations, cell types are distinguished based on the intensity of the output electromagnetic radiation emanating from the objects. Intensity can be determined as a peak intensity or even as a total intensity, such as the integrated area under an intensity signal. Specific embodiments described herein are directed to distinguishing between X-chromosome sperm cells and Y-chromosome sperm cells. Further embodiments are concerned with distinguishing viable X-chromosome bearing sperm cells from objects other than viable X-chromosome bearing sperm cells, including Y-chromosome bearing sperm cells and non-viable cells of both sexes.

It will be appreciated that the approaches of this disclosure can be applied more generally to distinguishing between any objects of different types so long as the output electromagnetic radiation emanating from one object type generates a discernable difference in at least one characteristic when compared to the electromagnetic radiation emanating from another object type. The invention is directed to the case wherein the objects are sperm cells. According to the invention, the fluid column is a flow stream that has a curved boundary or interface where refraction of electromagnetic radiation may occur. The curved boundary of the fluid column is generally circular in cross section. The fluid column can be bounded by solid walls, such as within a cuvette or within a microfluidic channel, or may be jetted into the air, such as in a jet-in-air flow cytometer. The objects move along the fluid column through a central core shaped by a sheath fluid that at least partially surrounds the central core. According to the invention, the central core may comprise a core stream of sample fluid containing sperm cells. The core stream has a generally elliptical cross section for the purpose of orienting aspherical sperm cells. Electromagnetic radiation emanating from the objects encounters at least one optical refraction boundary between the objects and other materials, such as at the interface between the fluid column and air.

Due at least in part to the different refractive properties of sheath fluid and air, the light collection efficiency external to the fluid column of light emanating from objects within the column depends upon the position of the objects for such systems. Light collection efficiency that varies with position is detrimental in applications where the light emanating from the objects must be precisely quantified and such precision is limited by random (not directly observable) position fluctuations of the objects. In the case of sex differentiating sperm specifically, such systems seek to differentiate very bright and closely related fluorescence intensities. Sperm cells and sperm nuclei are generally stained with Hoechst <NUM> to make such differentiations. Hoechst <NUM> is a bright, cell permeable dye that binds selectively with the A-T base pairs in the minor grove of double stranded nuclear DNA. The stoichiometric staining of sperm cells with Hoechst <NUM> differentiates X-chromosome and Y-chromosome as having slightly different amounts of nuclear DNA. For example, many domestic animals have about a four percent difference. When sperm cells are properly stained and oriented, this small difference can be distinguished by the fluorescence intensity of the Hoechst <NUM> associated with the nuclear DNA of the sperm cells when they are irradiated with an appropriate excitation source, such as a laser operating at or near a wavelength of <NUM>.

This four percent difference is difficult to detect for several reasons. First, sperm nuclear DNA resides within the sperm head, which is aspherical or has a paddle-like shape in most species. This asymmetry causes sperm to fluoresce differently out the flat side and more narrow side. Indeed, this fluctuation exceeds the four percent difference in DNA content, meaning sperm must be oriented in order to be differentiated based on nuclear chromosomal content. Orienting geometries tend to produce a core stream having a ribbon shape, or an elliptical cross section. This elliptical cross section provides sperm larger than normal latitude for placement in one axis.

The approaches disclosed herein enhance the precision of systems that may be limited by such fluctuations, such as jet-in-air flow cytometers. As described in more detail below, the positional variability of light intensity collected from objects in a fluid column can be addressed with an algorithm that corrects for the dependence of intensity on position.

The approaches outlined herein 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 emanating the light from the other side of the interface, wherein the interface causes a variation in the emanating light ray paths in a manner dependent on the object's position relative to the detector. Approaches herein correct for positional variation within the fluid column thus providing more accurate measurements for distinguishing types of objects.

The "jet-in-air" flow cytometer system <NUM> illustrated schematically in <FIG> is one type of discrimination system that can be used to discuss the concepts of the disclosure. The "jet-in-air" flow cytometer system <NUM> includes a fluid column forming structure that creates a flow stream comprising a fluid column <NUM> that jets out of the exit nozzle <NUM> of the chamber <NUM> at high velocity, e.g., about <NUM>/s. The fluid column <NUM> expelled from the exit nozzle <NUM> can be roughly circular in cross-section and may have a diameter of about <NUM> to about <NUM> in some implementations. In some embodiments, the interior of the chamber <NUM> and/or the exit nozzle <NUM> are configured with an internal geometry that hydrodynamically orients sperm within the fluid column. As a non-limiting examples the nozzles like those described in <CIT> and <CIT> may be incorporated for the purpose of orienting sperm and generating the coaxial flow of a fluid column. The fluid column <NUM> is composed of a core stream <NUM> within a sheath stream <NUM> where the arrows in <FIG> indicate the direction of flow of the core and sheath streams <NUM>, <NUM>. The sheath stream <NUM> may have a generally circular cross section, while the core stream has generally elliptical cross section, with a major and a minor axis.

Within the chamber <NUM>, a sample injection element <NUM> introduces the core stream <NUM> containing objects <NUM>, <NUM> which may be of multiple types. The core stream <NUM> is bounded by a sheath stream <NUM> comprising sheath fluid and shaped by hydrodynamic forces in the chamber <NUM>. The sheath stream <NUM> at least partially surrounds the core stream <NUM>, and the sheath stream <NUM> and the core stream <NUM> do not substantially mix. The sloping or angled walls <NUM> of the chamber <NUM> impart forces that shape the core stream <NUM> and accelerate objects <NUM>, <NUM> within the core stream <NUM>. The movement of the sheath stream <NUM> constrains the objects <NUM>, <NUM> in the core stream <NUM> to move toward the center of the fluid column <NUM> when the fluid column <NUM> is ejected from the chamber <NUM>. The fluid column <NUM> delivers the objects <NUM>, <NUM> to a measurement region <NUM> of the fluid column <NUM>, e.g., in single file.

As the objects pass through the measurement region <NUM> of the fluid column <NUM>, light from an excitation source <NUM> provides excitation light to the objects <NUM>, <NUM>. The excitation source <NUM> can provide light in a broad wavelength band or in a narrow wavelength band. For example, the excitation source <NUM> 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 continuous wave lasers are each well suited to produce appropriate responses. In some configurations, electromagnetic radiation generated by the excitation source, such as excitation light, may be modified by an optical element <NUM>. For example, the excitation light may be focused on the measurement region <NUM> by a one or more lenses <NUM>. Lenses may be used to focus the excitation electromagnetic radiation into a suitable beam shape focused on the measurement region. Objects 172a in the measurement region <NUM> emanate light, e.g., scattered or fluorescent light, in response to the excitation source <NUM>.

Objects of a first type <NUM> will emanate output electromagnetic radiation that differs in at least one characteristic as compared to output electromagnetic radiation that emanates from objects of the second type <NUM>. For example, in some scenarios, objects of the first type <NUM> will emanate light having a higher intensity than the light that emanates from objects of the second type <NUM>.

An optical collection arrangement <NUM> is positioned to collect the output electromagnetic radiation <NUM> emanating from the object 172a within the measurement region <NUM> that crosses the optical refraction boundary of the fluid column <NUM> at the fluid-air interface <NUM>. In some embodiments, the optical arrangement <NUM> may be configured to modify the output electromagnetic radiation <NUM> to provide modified output electromagnetic radiation <NUM> that focuses output electromagnetic radiation emanating from the object 172a in the measurement region <NUM> onto a detector <NUM>. In some embodiments, the optical collection arrangement <NUM> may include an element that reduces the positional dependence of the output electromagnetic radiation <NUM>. The detector <NUM> receives the modified output electromagnetic radiation <NUM> and, in response, generates an electrical signal representative of characteristics of the modified output electromagnetic radiation. As but an example, the detector <NUM> may be a forward fluorescence detector. Of course, other detectors may be incorporated to detect characteristics of interest, such as scatter, decay, phase shifts or other characteristics of interest.

According to the invention the detector is a SiPM split detector.

In some scenarios, the amplitude of the electrical signal may be different for different object types. The electrical signal is used by an analyzer <NUM> to distinguish between different types of objects <NUM>, <NUM>. For example, the analyzer <NUM> may be configured to compare the amplitude of the electrical signal to a threshold to discriminate between objects of the first type <NUM> and objects of the second type <NUM>. The analyzer <NUM> may include one or more analog circuits and/or digital processors for manipulating one or more signals from one or more detectors. As but one example, a side detector may be employed <NUM> degrees relative to the detector <NUM> to detect side scatter or side fluorescence. In the case of sperm sorting, side fluorescence allows the analyzer <NUM> to differentiate properly oriented sperm from unoriented sperm.

The analyzer <NUM> may include a processor <NUM> having executable instructions stored thereon. In addition to those instructions <NUM> known for the purpose of collecting, comparing and manipulating information from detector signals, the processor may include instructions <NUM> for normalizing the intensity value of the output electromagnetic radiation in the represented in the electrical signal from the detector based on the position of the object 172a in the fluid column <NUM> at the measurement region <NUM>. The intensity value may be normalized in any number of ways. As but one example, hand drawn lines or curves may be input by a user into a graphical user interphase based on an initial sampling of data including fluorescence intensities and positional information.

The processor <NUM> may also include instructions <NUM> for discriminating objects. <FIG> shows an x-y plane cross section of the fluid column <NUM> in the measurement region <NUM> depicted in <FIG>. In the x-y cross section of the measurement region <NUM>, the core stream <NUM> is elliptical in shape, and the fluid of the core stream <NUM> comprises at least one object 172a suspended in a buffer solution, which may also be referred to as sample. The sheath stream <NUM> substantially surrounds the core stream <NUM>. In a particular example used for this discussion in this disclosure, the objects <NUM>, <NUM> are sperm cells and the system <NUM> is implemented to discriminate X-chromosome sperm from Y-chromosome sperm.

A focused laser beam generated by the excitation source <NUM> irradiates the sperm cell 172a within the measurement region <NUM>. The cells <NUM>, <NUM> are stained with a fluorescent dye, and the excitation electromagnetic radiation causes the cell 172a within the measurement region <NUM> to emanate fluorescent output electromagnetic radiation. The purpose of the generally elliptical core stream <NUM> is to orient a sperm cell 172a such that the flat sides of the sperm cell are facing to the left and the right as shown in <FIG>. In this orientation, the flat sides of the sperm cell 172a face the laser <NUM> and the optical collection arrangement <NUM>, respectively. When each cell <NUM>,<NUM> is presented in a similar orientation at the measurement region <NUM>, random variability based on orientation can be greatly reduced. However, the elliptical cross section which aids in this orientation also provides significant latitude with respect to the position of the cell within the fluid column <NUM>.

To obtain the desired orientation, the elliptical core stream <NUM>, presents a major axis that parallels the x-axis depicted in <FIG>. The sperm cell 172a can take any number of positions along the x-axis within the core stream <NUM>. <FIG> shows three representative possible positions for the sperm cell 172a in the elliptical core <NUM>, although it may be appreciated sperm may be located anywhere in between the depicted positions. In the orientation shown in <FIG>, the first possible position for the sperm cell 172a in the core stream <NUM> is approximately at the center of the elliptical core <NUM> (on the optical axis <NUM> of the optical collection arrangement <NUM>), a second possible position is at the top of the core stream <NUM> (above the optical axis <NUM>), and a third possible position is at the bottom of the core stream <NUM> (below the optical axis <NUM>). A position-dependent refraction of the output light rays emanating from the sperm cell 172a occurs at the fluid-air interface <NUM> at the different positions within the core stream <NUM>. As used herein terms of relative position such as "top," "bottom," "upper," and "lower" should be understood as descriptive regarding the relationships between depicted features in the figures and not limiting on the claims, especially the position of sperm in a core stream <NUM>.

When the sperm cell 172a is located at the first position and the fluid column <NUM> has a circular cross section as shown in <FIG>, the in-plane rays of light emanating from the sperm cell 172a are approximately normally incident on the fluid-air interface <NUM>. Rays that emanate from points of the sperm cell 172a away from its center, or rays that emanate out of the plane of the figure, are not exactly normally incident on the interface <NUM>; these rays are not considered in this simplified discussion, but one of ordinary skill in the art can see how the discussion could be generalized to include them. Thus, to the extent any refraction of light occurs at the fluid-air interface <NUM> it occurs in a more uniform manner with respect to the detector <NUM>.

The diagram of <FIG> shows uniform light refraction of the output electromagnetic radiation <NUM> emanating from a sperm cell 172a as the electromagnetic radiation crosses the interface <NUM> when the sperm cell 172a is at the <NUM>st position within the elliptical core <NUM> shown in <FIG>. Correspondingly, the in-plane density of the light rays <NUM> exiting the fluid column <NUM> in <FIG> is uniform with respect to ray angle. Uniform angular density of light rays corresponds to uniform radiance as a function of ray angle.

In contrast, when a sperm cell 172a is off the optical axis <NUM> and is nearer to the top or bottom of the elliptical core <NUM>, e.g., at the <NUM>nd and <NUM>rd positions of the elliptical core <NUM> shown in <FIG>, at least some of the output rays emanating from the sperm cell 172a encounter the fluid-air interface <NUM> at an oblique angle. These output rays are refracted in a non-uniform fashion at the fluid-air interface <NUM> in contrast to the normal incidence scenario described above. The most oblique rays are the most severely refracted. Refraction of the light rays causes the radiance distribution of the fluorescent light exiting the fluid column <NUM> across the fluid-air interface <NUM> to become non-uniform and to vary with position of the cell 172a along the x axis. That is, this refraction changes the radiance distribution of output electromagnetic radiation emanating from sperm cell 172a outside of the fluid column <NUM>.

For example, when the cell 172a is located off the optical axis <NUM>, e.g., at the <NUM>nd or <NUM>rd positions shown in <FIG>, the density of light rays and thus the radiance on the air side of the interface <NUM> is higher at positive or negative ray angles, respectively, with respect to the optical axis <NUM> when compared to the radiance on the air side of the interface <NUM> at angles parallel to the optical axis <NUM> or at negative or positive ray angles, respectively. Positive and negative refer to the sign of the ray angle γ in <FIG>. <FIG> is a diagram illustrating light rays <NUM> emanating from a cell 172a and exiting the fluid column <NUM> through the fluid-air interface <NUM> when the cell 172a is located at the <NUM>nd position of the elliptical core <NUM>. In this scenario, the density of light rays, or radiance, at positive ray angles is greater than the density of the light rays parallel to optical axis <NUM> 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) may vary depending on whether the cell is in the first position or the second position. The positional dependence of the system collection efficiency leads to inaccuracies in determining cell type.

With reference to <FIG>, an analytical formula for the light ray density as a function of ray angle γ and sperm position x is determined using Snell's law, where γ is the angle of a light ray, with respect to the optical axis, emanating from the object after refraction at the fluid-air interface. This analysis considers only rays within, or tangential to, the two-dimensional cross-section of the flow stream.

We wish to solve for the density of the light rays with respect to the angle γ, which we can use to determine the density of rays at the entrance pupil of an optical collection system for each sperm position x. This can be written: <MAT>.

For our purposes we can assume that the sperm cell emanates light uniformly in all directions, so the density of emanated light rays with respect to the angle θ is: <MAT> that is, uniformly distributed from <MAT> to <MAT>. By geometrical analysis: <MAT> <MAT> <MAT> wherein the angles γ, θ, ϕ, α, β, and the distance x are shown in <FIG>. As the flow stream has index of refraction n, Snell's law yields another relation between the angles: <MAT>.

The density of light rays external to the interface Iβ(β) is related to the density of light rays internal to the interface Iα(α) by the following formula, with T(α) representing the average, across both polarizations, of the transmission through the interface: <MAT>.

The transmission is related to the Fresnel reflection coefficients for s- and p-polarization, Rs(α) and Rp(α), with the following formulas: <MAT> <MAT> <MAT> <MAT>.

Using Eq. (<NUM>) with the above and the following additional relations: <MAT> <MAT> <MAT> we have an expression for the density of rays with respect to γ: <MAT>.

Now, the optical collection arrangement's NA is given by the sine of the maximum ray angle γ<NUM>, so we can solve for this angle in terms of NA: <MAT>.

Finally, the relative collected light intensity, as a function of sperm position x, is given by integrating Eq. (<NUM>) from -γ<NUM> to γ<NUM> and normalizing by that integral value at x = <NUM>: <MAT>.

Using the formula for ray density distribution of Eq. (<NUM>), the angular dependence of ray density (radiance) for different sperm positions can be plotted as in <FIG>. In <FIG>, each of the lines represents the density of rays as a function of angle γ for a given sperm position x, where the angle γ is in radians. The plots correspond to a series of positions that lie in a range symmetric about x = <NUM>, (corresponding to graph <NUM> in <FIG>), which is where the ray density (radiance) is uniform as a function of angle. When x is positive (e.g., <NUM>nd position in <FIG>, corresponding to graph <NUM>), relative radiance is higher for positive ray angles γ and lower for negative ray angles γ, and the opposite is true when x is negative (e.g., <NUM>rd position in <FIG>, corresponding to graph <NUM>).

If the numerical aperture of the collection optics (optical collection arrangement <NUM> in <FIG> and <FIG>) is large, e.g., approaching one, the variation in collected optical intensity with respect to position for light emanating from an object within the elliptical core is relatively small. This is because essentially all light emanating from the object and directed to the right would be collected by the collection optics, regardless of the exact ray direction, and the total amount of emanating light is invariant to object position (given uniform excitation). In contrast, a small numerical aperture results in a relatively large collected intensity variation with respect to object position, because changes in object position affect the radiance distribution, and a small numerical aperture implies only a portion of this changing radiance distribution is collected. Practical systems may have NAs that are significantly less than one, e.g., less than <NUM>, or less than <NUM>. The family of graphs provided in <FIG> illustrates the relative intensity of light collected from an object, as a function of object position x, through collection optics with different NAs. <FIG> illustrates the range of angles γ captured by the different numerical apertures of <FIG>.

In the family of graphs of <FIG>, graph <NUM> illustrates the relative intensity with respect to position along the x axis for collection optics (e.g., optical collection arrangement <NUM> shown in <FIG> and <FIG>) having a numerical aperture (NA) of <NUM>; graph <NUM> shows the relative intensity with respect to position along the x axis for collection optics having an NA of <NUM>; graph <NUM> shows the relative intensity with respect to position along the x axis for collection optics having an NA of <NUM>; graph <NUM> shows the relative intensity with respect to position along the x axis for collection optics having an NA of <NUM>; and graph <NUM> shows the relative intensity with respect to position along the x axis for collection optics having an NA of <NUM>. It is clear from <FIG> and <FIG> that collection optics having smaller NAs produce a larger variation in collected light intensity with respect to object position when compared to collection optics having larger NAs. Additionally, collection optics with larger NAs collect light rays having a wider range of refraction angles than collection optics having smaller NAs, and therefore have a higher overall collection efficiency.

With respect to sperm discrimination or sorting application in particular, it can be understood that the elliptical major axis of the core stream <NUM> (<FIG> and <FIG>) may be about <NUM> in length, providing sperm about <NUM> in latitude to move in either direction. Referring back to <FIG>, it can be seen a NA of <NUM> only captures about <NUM>% of an objects relative intensity when the object is about <NUM> off center. Similarly, a NA of <NUM> captures only <NUM>% of the relative intensity for objects that are about <NUM> off center and a NA of <NUM> captures a little more than <NUM>% of the relative intensity at the same position. It may be further appreciated that the NA of collection optics for a sperm sorter may be between about <NUM> and <NUM>. While <FIG> illustrates the benefit of increasingly large numerical apertures, such numerical apertures are increasingly expensive and have a shallower field of depth, meaning the larger aperture must be placed closer to the nozzle. There is, however, a limit on how close the collection optics can be placed in sperm sorting applications. In typical sperm sorting instruments, the apertures may be between about <NUM> and <NUM>. Embodiments described herein correct for the positional dependency on measured intensity allowing lower numerical aperture collection optics to perform like higher numerical aperture collection optics.

Sperm located in the core stream <NUM> at positions approaching the <NUM>nd and <NUM>rd positions of <FIG>, therefore, emanate a significantly lower overall intensity of electromagnetic radiation that is ultimately detected for analysis and discrimination. Indeed, the core stream <NUM> may have an elliptical major axis that is about <NUM> in length at high event rates (in the magnitude of <NUM>,<NUM> events per second and greater). Some sperm will be off center by <NUM> or even up to about <NUM> either side of the <NUM>st position. In the context of extremely bright and closely related fluorescence signals, this variation can overshadow the roughly <NUM>% difference in stained nuclear DNA differentiating X-chromosome bearing sperm from Y-chromosome bearing sperm.

Furthermore, increasing the number of events at a given sperm concentration within a sample of buffer requires increasing the volume of sample per unit time in the fluid column passing through the measurement region. Increasing the number of events detected per second in this manner also increases the elliptical cross section of the core stream within the fluid column, including the length of the major axis. As a natural consequence, and as those of skill in the art are aware, generally increasing the sorting speed by increasing the flow rate of sample decreases the sensitivity of sperm sorting equipment. Therefore, embodiments described herein not only improve sperm sorting precision at customary speeds, but may also provide for sperm sorting at increased overall speed in terms of throughput without suffering losses in fidelity.

An approach for identifying objects traveling in a fluid column in the presence of positional variation is illustrated in the flow diagram of <FIG>. The process includes creating <NUM> a fluid column containing objects at differing positions within the fluid column. The fluid column may be a coaxial stream of fluid created by a jet-in-air flow cytometer. Such a fluid column may comprise a core stream with an elliptical cross section having a major axis along which objects may be positioned. The core stream may be coaxially contained within a sheath stream. In some embodiments the fluid column may have an air-fluid interface whereby refraction occurs. In other embodiments the fluid column may be formed within a cuvette or a microfluidic channel. In such cases there may be a liquid-glass interface and possibly a glass-air interface and emanating light may be refracted twice. Such twice refracted light is expected to benefit greatly from the angular dependency correction of certain embodiments.

The process continues by generating <NUM> excitation electromagnetic radiation and directing <NUM> the excitation electromagnetic radiation toward objects in the fluid column at a measurement region. Objects within the fluid column emanate output electromagnetic radiation in response to the excitation electromagnetic radiation at the measurement region. The output electromagnetic radiation is collected <NUM> from the objects in the fluid column, including objects having different position within the fluid column at a measurement region and a detector generates <NUM> an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical arrangement.

Next, an analyzer or other suitable means normalizes <NUM> the intensity represented by the output signal based on the position of the object in the fluid column. The normalization may be performed by means of a correction, whereby signals generated off the central axis, such as toward and including the second and third positions of <FIG>, are amplified by an appropriate correction factor based on their position. The magnitude of appropriate correction factors can be seen in <FIG>. Once normalized by correction the method continues by discriminating <NUM> a first type of object from other objects. The discrimination may take place in a flow cytometer analyzer and may include one or more additional manipulations. For example, univariate histograms may be generated illustrating a distribution of fluorescence intensities. Bivariate histograms may also be generated with the corrected signal and with further calculated values. Such corrected and calculated values may be compared against gating regions in a flow cytometer analyzer or compared against look-up-tables to discriminate a first type of object from other types of objects.

As exemplary objects sperm may be discriminated as either X-chromosome bearing or Y-chromosome bearing sperm. Further, sperm may be stained with a DNA selective dye in addition to a secondary quenching dye. A quenching dye typically permeates membrane compromised sperm cells, such as dead or dying sperm cells, and greatly reduces the fluorescence produced by the DNA selective dye associated with those compromised cells. Such quenched cells are effectively removed from the closely related populations undergoing discrimination/sorting. In this way a system can discriminate live or viable sperm cells from dying or compromised sperm cells. The system may also discriminate viable X-chromosome bearing sperm from all remaining cells, Y-chromosome bearing sperm from all remaining cells, or even simultaneously viable X-chromosome bearing sperm and Y-chromosome bearing sperm from all other sperm cells.

<FIG> illustrates a first embodiment of the discrimination system substantially similar to the discrimination system depicted in <FIG> and <FIG> in which output electromagnetic radiation <NUM> emanating from an object 172a located in the measurement region is collected by an optical collection arrangement <NUM>. The optical collection arrangement <NUM> may include a collection lens that focuses a modified output electromagnetic radiation onto a detector <NUM>. In the depicted embodiment, the detector functions to both measure a characteristic of the modified output electromagnetic radiation as well as a position detector <NUM> for determining the position of the object 172a within the core stream <NUM> of the fluid column <NUM>.

The detector <NUM> suitable for determining both a characteristic of the modified output electromagnetic radiation <NUM> and for determining the location of the object 172a in the measurement region comprises a split SiPM detector.

This detector may be located in the image plane of the object or in the Fourier plane to determine the position of the object. In the image plane the detectors directly measure the position of the object, whereas in the Fourier plane the position information will be extracted from the lateral intensity distribution (e.g. the left-right asymmetry).

Flow cytometry applications often require very sensitive (down to single photon counting) and fast (objects are moving with ~<NUM>/s through <NUM>) detectors. Detectors with the requisite speed and sensitivity are typically those detectors that provide an internal gain. In photomultiplier tube (PMT) or a silicon photomultiplier (SiPM), also known as pixelated avalanche photodiode, a single photon creates a cascade of up to about <NUM><NUM> electrons. Both detector types are commercially available as detector arrays. SiPM may be better suited for use in detector arrays suitable for determining the position of the object because they are fabricated by standard techniques on silicon wafer. Some detector, such as SiPMs may be particularly well suited to be placed in a Fourier plane in order to distribute light over a larger area of the detector.

<FIG> illustrates an alternative embodiment not belonging to the invention in which a beam splitter <NUM>, or other suitable optics, redirect a fraction of the power of the modified output electromagnetic radiation <NUM>. The majority of the modified output electromagnetic radiation <NUM> is directed to and focused on the detector <NUM>. In this embodiment the detector <NUM> comprises a first detector <NUM> for detecting a characteristic of interest. The first detector <NUM> may be any detector conventionally suited to quantify the particular characteristic of interest. In typical flow cytometer applications photodiodes, photomultiplier tubes (PMTs) and silicon photomultipliers may be particularly well suited to detect scattered or fluoresced electromagnetic intensity.

The beam splitter <NUM> may comprise a dielectric mirror <NUM>, however those of skill in the art will appreciate other suitable optical components such as cube beam splitters, prism beam splitters and the like may be used for redirecting a portion of the modified output electromagnetic radiation <NUM> power. Regardless of the manner in which the output power is split, a first beam fraction <NUM> is directed along a first path to the detector and a second beam fraction <NUM> is directed along a different path to a second detector <NUM> in the form of a position detector <NUM>. The position detector can be a camera, a position sensitive device ("PSD") such as an isotropic sensor or a charged coupled device (CCD), split detectors, a detector array of PMTs, SiPM, pin photodiodes or the like.

Turning to <FIG>, a simulation was performed illustrating the viability of a split detector for determining positional information in a flow cytometry system. The simulation employed a split SiPM detector comprising <NUM> SiPM detectors mounted side by side. The edge at which the detectors met was calibrated as a central x coordinate position, simulating the beam axis of an interrogation laser as well as the symmetric center of a fluid column. A <NUM> spot size was swept across the split detectors from an x position between about -<NUM> to <NUM> and the relative intensity was measured by each detector was recorded. A first graph <NUM> illustrates the relative intensity recorded for the beam spot from one of the detectors from x positions ranging from about -<NUM> to about <NUM>, where the x position corresponds to a plane of the SiPM detector. A graph <NUM> illustrates the corresponding relative intensity detected by the other detector for the beam spot in a range of x positions from about -<NUM> to about <NUM>. As can be seen, the positional difference in the two detectors results in differing measured intensities based on the x position of the <NUM> spot. These differences correlate to position and can be translated through processing means to approximate positional information. Noise was included in the simulation, but it was independent of intensity. At max intensity the noise corresponds to <NUM>% coefficient of variation. The simulation demonstrated that x position can be determined in a split detector arrangement based on the relative intensity detected by each SiPM in a split detector arrangement. Those of skill in the art can appreciate, embodiments of the present invention are not limited to this configuration and that other detector configurations suitable for determining the position of a particle within a fluid column are also contemplated for use herein. As but one example, other detectors may be employed in a split detector arrangement. Those of skill in the art will appreciate that detectors should have low noise, as the combined signals must have a sufficiently low coefficient of variation.

<FIG> illustrates the result of an experiment incorporating positional correction for sperm nuclei in a fluid column resulting in significant improvements in differentiating X and Y-chromosome bearing sperm nuclei. Sperm nuclei stained with Hoechst <NUM> were processed through a Genesis III sperm sorting instrument manufactured by Cytonome. The instrument was outfitted with a SiPM split detector. Sample and sheath pressure were adjusted to establish an event rate of <NUM>,<NUM> events per second. Nuclei were interrogated with a Coherent Genesis CW-<NUM> laser at an average power of 150mW. Plot <NUM> depicts a bivariate histogram illustrating a sum of fluorescence intensives from each detector in the split detector plotted against the positional delta of nuclei in the fluid column. As previously described, the range of the positional delta represents the major axis of the elliptical core stream along which nuclei may enter the measurement region. A population of X-chromosome bearing nuclei <NUM> is seen in a crescent shape. As expected, measured intensities are greatest near a position delta of <NUM> with reductions curving downward as nuclei move away from the central position. A population of Y-chromosome bearing nuclei <NUM> is seen as a second crescent just below the X population and, again, the highest intensities are seen near a position delta of <NUM> with significant losses in relative intensity as the nuclei move away from the central position.

Plot <NUM> presents a univariate histogram of the summed fluorescence intensities that corresponds to the intensities charted in plot <NUM>. While the distinct population of X-chromosome bearing nuclei <NUM> and population of Y-chromosome bearing nuclei <NUM> can be seen, a comparison of plot <NUM> with plot <NUM> makes apparent that off center X-chromosome bearing sperm nuclei increasingly overlap with the well centered Y-chromosome bearing sperm nuclei. Indeed, the peak to valley ratio is calculated at <NUM>%.

In accordance with embodiments of the invention, a correction factor <NUM> is illustrated as a curved line in plot <NUM>. The correction factor <NUM> illustrates the degree of correction required to 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 depicted in plot <NUM> to produce a corrected population of X-chromosome bearing nuclei <NUM> and a corrected population of Y-chromosome bearing nuclei <NUM>. The corrected population of X-chromosome bearing nuclei <NUM> form a generally rectangular shape and no longer demonstrates fluctuation based on the position of the nuclei in the fluid column. A more distinct gap can be seen in plot <NUM> between the corrected population of X-chromosome bearing nuclei <NUM> and a corrected population of Y-chromosome bearing nuclei <NUM>. Plot <NUM> illustrates the corresponding univariate histogram, which has a <NUM>% peak to valley ratio between the corrected population of X-chromosome bearing nuclei <NUM> and the corrected population of Y-chromosome bearing nuclei <NUM>. The stark contrast between plot <NUM> and plot <NUM> is visually apparent. Furthermore, the difference is a quantifiable with at <NUM> percentage points higher.

<FIG> illustrates the results of an example incorporating correction in accordance with embodiments described herein. Live sperm stained with Hoechst <NUM> were processed through a Genesis III sperm sorting instrument manufactured by Cytonome. Sample and sheath pressures were adjusted to reach an event rate of <NUM>,<NUM> events per second and the sperm was interrogated with a Coherent Genesis CW-<NUM> laser operated at an average power of 100mW. Plot <NUM> illustrates a bivariate histogram of the summed fluorescence intensity and the relative positions of live sperm in the core stream. Again, the population of X-chromosome bearing sperm <NUM> can be seen as a first population above a population of Y-chromosome bearing sperm <NUM>. A correction factor <NUM> for normalizing the summed intensity values is also depicted in plot <NUM>. Plot <NUM> illustrates the univariate histogram of uncorrected summed intensities and demonstrates a peak to valley ratio of <NUM>% between the population of X-chromosome bearing sperm <NUM> and the population of Y-chromosome bearing sperm <NUM>.

Plot <NUM> provides a type of bivariate histogram common in sperm sorting applications. In this case, a corrected forward fluorescence intensity is plotted against a side fluorescence. A forward fluorescence vs side fluorescence histogram is useful for sorting live sperm because the side fluorescence provides information on the orientation of each cell. In contrast, sperm nuclei are sonicated and removed from the aspherical sperm head. As such, orientation is not an issue when sorting sperm nuclei. For this reason, nuclei are easier to sort and are often used to calibrate sperm sorting flow cytometers. Plot <NUM> depicts a corrected population of X-chromosome bearing sperm <NUM> and a corrected population of Y-chromosome bearing sperm <NUM>.

Much like the previous example, plot <NUM> still correlates in the Y axis to the corrected forward fluorescence of graph <NUM>. In the univariate plot of graph <NUM>, the corrected population of X-chromosome bearing sperm <NUM> and the corrected population of Y-chromosome bearing sperm <NUM> can be seen as more distinct peaks having a machine calculated peak to valley ratio of <NUM>%. And again, the corrected histogram presents a significant improvement over plot <NUM> demonstrating the value of positional correction for live sperm.

In another aspect, embodiments described herein may provide systems and methods that substantially ease an alignment process in a flow cytometer. In the case of sperm for example, the measurement region, detectors, and even the structure forming the sheath flow must be properly and precisely aligned in order to generate and collect sufficiently clear signals for differentiating the very bright and closely related X and Y-chromosome bearing sperm populations. Even in a precise and proper alignment, oriented sperm in a fluid column can assume any number positions along the major axis of core stream. As described above with respect to <FIG>, this means that even when the components of the flow cytometer are in perfect alignment, there is an angular dependency to the detected output electromagnetic radiation. This angular dependency introduces noise like variations because the cells may be randomly positioned within the core stream.

In commercial sperm sorting applications, technicians typically undertake a number of course adjustments followed by a number of fine adjustments for multiple components in multiple axis in order to align the instrument. Due to the sensitivity of the instrument to each adjustment, the very closely related nature of the detected signals, and the number of possible adjustments, such alignments can be time consuming tasks for technicians operating sperm sorting instruments. When switching between samples machine alignments for commercially sorting sperm can take a few minutes, even up to five minutes. After declogging a nozzle or otherwise removing, replacing or adjusting other components that require calibration, it may take a technician <NUM> minutes, <NUM> minutes, and in rare cases as long as <NUM> minutes in order put an instrument in suitable alignment for commercially sex sorting sperm.

<FIG> illustrates the results of an example in which the alignment process is greatly reduced for discriminating sperm nuclei. Sperm nuclei stained with Hoechst <NUM> were processed through a Genesis III sperm sorter manufactured by Cytonome. The instrument was fit with an SiPM split detector. The forward fluorescence detection was aligned for less than one minute resulting in a rough alignment. Sperm nuclei were run at an event rate of <NUM>,<NUM> nuclei per second and interrogated with a Coherent Genesis CW-<NUM> laser operated at an average power of 150mW. Plot <NUM> illustrates the bivariate histogram showing the summed forward fluorescence plotted against the position detected by each event by the SiPM. The poor alignment is evident in each of the population of X-chromosome bearing nuclei <NUM> and the population of Y-chromosome bearing nuclei <NUM>. In poor alignment the crescent shapes are asymmetric and the fluorescence intensity values drop dramatically in the positive x direction as compared to the negative x direction. The population of Y-chromosome bearing nuclei <NUM> demonstrate the same skew.

The correction factor <NUM> is illustrated as a line between the two populations. This correction factor <NUM> illustrates the degree of correction that will be performed to summed fluorescence values at each x location. Stated differently, the correction factor <NUM>, represents a curved line that will be normalized by correction to a flat line. Each summed fluorescence value at a corresponding x position along line receives the same magnitude of increase or decrease as the correction factor <NUM>.

The distortion caused by rough alignment is more pronounced in the histogram of fluorescence intensities of plot <NUM>, where increased overlap results in a peak to valley ratio of <NUM>% between the population of X-chromosome bearing nuclei <NUM> and the population of Y-chromosome bearing nuclei <NUM>.

In plot <NUM>, the corrected forward fluorescence summed value is plotted in a bivariate histogram against the detected position of each event. It can be seen, again, that by normalizing the fluorescence intensity values with a correction factor <NUM> based on the position of the cells, two clean populations of cells emerge. A corrected population of X-chromosome bearing nuclei <NUM> and a corrected population of Y-chromosome bearing nuclei <NUM> are more clearly and distinctly grouped in plot <NUM>. Importantly, the orthogonal relationship of these populations translates in the univariate fluorescence intensity histogram seen in plot <NUM>, where two distinct univariate peaks have a calculated peak to value ratio of <NUM>%.

In addition to the use of correction, some embodiments disclosed herein include elements that reduce the variation in collected light intensity with respect to object position in a flow stream. Some embodiments described herein can provide modified output light that has less than about a <NUM>%, or less than about a <NUM>%, or even less than about a <NUM>% measured intensity variation for a deviation in position of the object that is less than <NUM>% of a radius of the flow stream away from a center of the flow stream along an axis perpendicular to the optical axis. Many applications are sensitive to intensity measurement errors, which may arise from a variety of sources. Due to the difficulty in reducing intensity fluctuations by precisely controlling the position of objects within the flow stream, it is useful to instead reduce the variation in collected light intensity with respect to object position by careful design of the optical collection arrangement. For applications such as X/Y sperm sorting, it is often the case that two or more cell populations are to be separated based on the difference in measured fluorescence intensity between the populations. If the random position fluctuations lead to fluctuations 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 simultaneously high yield and high purity. The fluorescence intensity difference between X and Y sperm cells is typically only a few percent (e.g., ~<NUM> % for bovine sperm). Current sperm sorter systems can in theory 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. Consequently, there would be a large uncertainty of the sperm position within the core of the flow stream. This position uncertainty and the resultant fluctuations in collected fluorescence intensity limit the maximum throughput of current sperm sorter systems to levels which do not obscure the small fluorescence intensity difference between X and Y sperm.

One approach for intensity-position correction may be understood with reference to <FIG> and <FIG>. The brackets in <FIG> highlight regions of integration that correspond to fluorescence collection optics with a given NA. Graphs of the collected intensity variation with respect to object position for the NAs of <FIG> are provided in <FIG>. In <FIG>, for a given NA, integration over the fluorescence collection region is performed such that the intensity of collected light can be plotted as a function of each sperm position. It is evident from <FIG> that increasing the NA of the collection optics helps to decrease the influence of object position on the fluorescence intensity gathered via the collection optics.

In some embodiments collection optics (e.g., the optical collection arrangement <NUM> in <FIG> and <FIG>) may be modified with elements that reduce collected light intensity variation with respect to object position as described above. Such embodiments are described in more detail in <CIT>.

According to some such embodiments, the collection optics operate by masking certain rays in "angle space", that is, the collection optics selectively collect, attenuate, and/or block rays from different angles γ in order to achieve a desired intensity vs. position profile. In practice, an "angle space" masking function can be applied at 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 embodiments, the collection optical arrangement achieves a desired, e.g., flatter, intensity vs. position profile by preferentially collecting higher angle (pointing away from the optical axis) light rays to the exclusion of certain lower angle light rays.

<FIG> and <FIG> illustrate how excluding low-angle refracted rays, at a given NA, causes the intensity-vs-position curve to flatten out. Excluding the low angle rays excludes the rays that produce the most variation in the intensity vs. position profile, whereas the angular variation of radiance at high positive angles tends to cancel the corresponding variation at high negative angles. <FIG> shows plots of the relative radiance vs. ray angle, γ, for different positions of the object along the x axis where the angle γ is in radians. In <FIG>, each graph corresponds to an object position, x, within the core of a flow stream, as indicated in <FIG>. The brackets in <FIG> show the portion of the light rays that will be excluded by the collection optics for each position x, when rays having angle magnitude less than <NUM> rad are excluded (bottom bracket in <FIG>) and when rays having angle magnitude less than <NUM> rad are excluded (top bracket in <FIG>).

<FIG> shows the relative collected light intensity vs. position of the object along the x axis when no angles are excluded (graph <NUM>), when rays having angles between -<NUM> rad and +<NUM> rad are excluded (graph <NUM>) and when rays having angles between -<NUM> rad and +<NUM> rad are excluded (graph <NUM>). Graph <NUM> shows that when lower angle rays are excluded, the relative intensity vs. position graph exhibits less intensity variation with respect to position.

<FIG> illustrates the results of an experiment incorporating both a software based positional correction and a hardware based element in the collection light path that reduces collected light intensity variation with respect to object position as described. Sperm nuclei stained with Hoechst <NUM> were processed through a Genesis III sperm sorter manufactured by Cytonome. The sperm sorter was fit with an SiPM split detector having a wire placed in the light collection path in order to exclude low collection angle electromagnetic radiation produced from the sperm nuclei. Suitable wires and other elements for blocking low collection angle electromagnetic radiation are described in <CIT>.

Sample and sheath pressures were adjusted to reach an event rate of <NUM>,<NUM> events per second and the nuclei was interrogated with a Coherent Genesis CW-<NUM> laser operated at an average power of 90mW. In plot <NUM> it can be seen the wire mitigates some effect of the intensity dependence on nuclei position within the fluid column. There is still however, a significant decrease in relative intensity as nuclei move further in the positive direction along the x axis. A population of X-chromosome bearing nuclei <NUM> and a population of Y-chromosome bearing nuclei <NUM> are seen sagging significantly in the positive direction in the x axis. The corresponding peak to valley ratio calculated from the fluorescence intensity histogram of plot <NUM> is <NUM>%. Again, X-chromosome bearing nuclei that are located toward one end of the fluid column are not sufficiently detected. As a result, the summed fluorescence intensity of the nuclei at this end have similar intensity values as centered Y-chromosome bearing nuclei within the population of Y-chromosome bearing nuclei <NUM>. This skew is evident in the univariate histogram of plot <NUM> in the form of a shoulder shifting downward and an exaggerated peak of the population of Y-chromosome bearing nuclei <NUM>.

A correction factor <NUM> is illustrated on graph <NUM>. For each position, a correction value is added to the detected fluorescence intensity corresponding correction factor. Plot <NUM> illustrates a bivariate histogram having a corrected population of X-chromosome bearing nuclei <NUM> and a corrected population of Y-chromosome bearing nuclei <NUM>, which are more distinct rectangular populations. Plot <NUM> provides the corresponding univariate histogram of corrected summed intensity values independent of the location of each event. The corrected population of X-chromosome bearing nuclei <NUM> and the corrected population of Y-chromosome bearing nuclei <NUM> are more distinct having roughly equal peaks heights and a peak to valley ratio of <NUM>%.

The foregoing description of various embodiments has been presented for the purposes of illustration and description.

Claim 1:
A discrimination system, comprising:
a fluid column (<NUM>) forming structure that creates a fluid column containing sperm cells within a
generally elliptical cross section of core stream (<NUM>) contained within a generally circular cross section of sheath fluid, wherein the sperm cells are at differing positions within the fluid column;
an excitation source (<NUM>) that generates excitation electromagnetic radiation directed toward sperm cells in the fluid column (<NUM>) at a measurement region, where the sperm cells within the fluid column emanate output electromagnetic radiation in response to the excitation electromagnetic radiation;
an optical arrangement (<NUM>) that collects output electromagnetic radiation from the sperm cells;
a SiPM split detector (<NUM>) that i) generates an electrical signal responsive to the intensity of the output electromagnetic radiation collected by the optical arrangement and ii) detects the position of sperm cells in the fluid column.; and
an analyzer (<NUM>) having instructions stored thereon i) to normalize the intensity of the output electromagnetic radiation represented in the electrical signal based on the position of the sperm cells in the fluid column, and ii) to discriminate a first type of sperm from other sperm cells.