Light collection from objects in a liquid column

Abstract

To provide favorable light collection from objects within a liquid column.SOLUTION: An optical arrangement receives output light emanating from an object disposed within a liquid column that crosses an optical refraction boundary of the liquid column between the object and the optical arrangement. The optical arrangement modifies the output light such that the modified output light has intensity that is more uniform than the output light. Some embodiments are directed to an optical arrangement configured to receive output light emanating from an object disposed within a liquid column. The output light crosses an optical refraction boundary of the liquid column between the object and the optical arrangement. The optical arrangement modifies the output light such that the modified output light has intensity that is more uniform than intensity of the output light.SELECTED DRAWING: Figure 1A

Description

[Background technology]

[0001] Object discrimination techniques distinguish between different types of objects. These techniques are particularly useful for sorting biological cells according to cell type. Some cell sorting methods rely on light emitted from cells to determine the type of cell. In some embodiments, cells moving in a column of liquid are exposed to an excitation light, and light emitted from the cells in response to the excitation light is detected. A first type of cell produces an output light that differs in some characteristic, e.g., wavelength and / or intensity, from a second type of cell. The difference in the output light emitted from the cells can be used to determine the type of cell and to sort the cells. Summary of the Invention [Means for solving the problem]

[0002] Some embodiments are directed to an optical arrangement configured to receive output light emitted from an object disposed within a liquid column, the output light traversing an optically refractive boundary of the liquid column between the object and the optical arrangement, the optical arrangement conditioning the output light such that the conditioned output light has a more uniform intensity than the intensity of the output light, for example, within a cross-section of the liquid column, the intensity of the conditioned output light can be substantially uniform regardless of the position of the object.

[0003] According to some embodiments, the optical device includes an optical arrangement and further includes a detector that detects the conditioned output light and provides an electrical signal in response to the conditioned output light.

[0004] According to some embodiments, the discrimination system includes an excitation light source configured to generate an excitation light and direct the excitation light towards an object in the liquid column. The object emits an output light in response to the excitation light. The system includes an optical arrangement configured to receive the output light. The output light crosses an optically refractive boundary of the liquid column between the object and the optical arrangement. The optical arrangement adjusts the output light such that the adjusted output light has a more uniform intensity than the intensity of the output light. For example, the intensity of the adjusted output light is substantially uniform regardless of the position of the object within the cross section of the liquid column. The optical detector is configured to detect the adjusted output light and provide an electrical signal in response to the adjusted output light. The object type discrimination circuit discriminates between a first type of object and a second type of object based on the electrical signal. The present specification also provides, for example, the following items: (Item 1) an optical arrangement configured to receive output light emitted from an object disposed in a liquid column, the output light crossing an optically refractive boundary of the liquid column between the object and the optical arrangement, the optical arrangement configured to condition the output light such that the conditioned output light has a more uniform intensity than the output light; a detector configured to detect the conditioned output light and provide an electrical signal in response to the conditioned output light; An optical device comprising: (Item 2) 2. The optical device of claim 1, wherein the intensity of the adjusted output light is substantially uniform across a cross-section of the liquid column. (Item 3) an excitation light source positioned to provide excitation light to the object within the liquid column, the object emitting the output light in response to the excitation light; an optical filter configured to substantially block the excitation light from reaching the detector; a focusing lens positioned to collect the conditioned output light and focus the conditioned output light towards the detector; Item 1 , further comprising one or more of: (Item 4) 2. The apparatus of claim 1, further comprising an electrical circuit configured to receive the electrical signal from the detector and distinguish between a first type of object and a second type of object based on the electrical signal. (Item 5) an amplitude of the electrical signal responsive to an intensity of the adjusted output light; 5. The apparatus of claim 4, wherein the electrical circuit is configured to distinguish between the first type of object and the second type of object by comparing an amplitude of the electrical signal to a threshold. (Item 6) 1. An optical arrangement configured to receive output light emitted from an object disposed in a liquid column, the output light crossing an optically refractive boundary between the object and the optical arrangement, and configured to condition the output light such that the conditioned output light has a substantially uniform intensity regardless of a position of the object. (Item 7) the optical arrangement is configured to preferentially attenuate light received by the optical arrangement from a first location within the liquid column compared to light received from a second location within the liquid column; 7. The optical arrangement of claim 6, wherein the second position is further from a center of the liquid column than the first position along an axis perpendicular to an optical axis of the optical arrangement. (Item 8) 8. The optical arrangement of claim 7, wherein the optical arrangement includes a mask configured to attenuate light from the first position in the liquid column. (Item 9) 9. The optical arrangement of claim 8, wherein the mask comprises wires. (Item 10) 8. The optical arrangement of claim 7, wherein the optical arrangement is configured to preferentially collect a first portion of the output light emitted from the object at a larger angle relative to the optical axis compared to a second portion of the output light emitted from the object at a smaller angle relative to the optical axis. (Item 11) Item 11. The optical arrangement of item 10, wherein the optical arrangement includes a split objective lens configured to preferentially collect the first portion of the output light. (Item 12) The first portion of light is output light emitted from the object at a positive angle relative to the optical axis of the optical arrangement; output light emitted from the object at a negative angle relative to the optical axis of the optical arrangement; Including, The split objective lens is a first lens configured to receive the light emitted from the object at a positive angle relative to the optical axis of the optical arrangement; and a second lens configured to receive the light emitted from the object at a negative angle relative to the optical axis of the optical arrangement; and 12. The optical arrangement according to item 11, comprising: (Item 13) Item 10. The optical arrangement of item 9, further comprising a mask configured to attenuate a second portion of the output light. (Item 14) an excitation light source configured to generate excitation light and direct the excitation light towards an object in a liquid column, the object emitting an output light in response to the excitation light; an optical arrangement configured to receive output light that crosses an optically refractive boundary of the liquid column between the object and the optical arrangement, the optical arrangement configured to condition the output light such that the conditioned output light has a more uniform intensity than the output light; an optical detector configured to detect the conditioned output light and provide an electrical signal in response to the conditioned output light; and an object type discrimination circuit configured to discriminate between a first type of object and a second type of object based on the electrical signal. (Item 15) Item 15. The system of item 14, wherein the intensity of the conditioned output light is substantially uniform regardless of the position of the object. (Item 16) Item 15. The system of item 14, wherein the circuitry is configured to perform X / Y sperm cell discrimination. (Item 17) Item 15. The system of item 14, wherein the optical arrangement includes a split objective lens configured to preferentially collect a first portion of the output light emitted from the object at a higher angle relative to the optical axis compared to collecting a second portion of the output light emitted from the object at a lower angle relative to the optical axis. (Item 18) the optical arrangement including a mask configured to preferentially attenuate output light received from a first location within the liquid column compared to output light received from a second location within the liquid column; Item 15. The system of item 14, wherein the second position is farther from a center of the liquid column than the first position along an axis perpendicular to the optical axis of the optical arrangement. (Item 19) Item 15. The system of item 14, wherein the optical arrangement includes an elongated mask feature that attenuates the output light received from the first location, the length of the elongated mask feature being much greater than the width of the elongated mask feature, and the elongated mask feature being positioned adjacent to an active area of ​​the detector such that a longitudinal axis of the elongated mask feature is substantially parallel to a direction of flow of the object in the liquid column. (Item 20) 20. The system of claim 19, wherein the elongated mask feature has a round cross-sectional area. (Item 21) 20. The system of claim 19, wherein the elongated mask feature has a rectangular cross-sectional area. (Item 22) 20. The system of claim 19, wherein the elongated mask feature is a metal wire. (Item 23) 20. The system of claim 19, wherein the elongated mask features are traces deposited on glass. (Item 24) 20. The system of claim 19, wherein the elongated mask feature is a filament. (Item 25) 20. The system of claim 19, wherein the elongated mask features are ink lines printed on a transparency. (Item 26) 20. The system of claim 19, wherein the elongated mask feature is positioned approximately at the center of an active area of ​​the detector. (Item 27) receiving, with an optical device, output light from an object within the liquid column that has crossed an optically refractive boundary of the liquid column; conditioning the output light such that the conditioned output light has a substantially uniform intensity regardless of the position of the object within the liquid column; detecting the conditioned output light; and generating an electrical signal in response to the conditioned output light; A method comprising: (Item 28) adjusting the output light preferentially collecting a first portion of the output light emitted from the object at a higher angle relative to the optical axis compared to collecting a second portion of the output light emitted from the object at a lower angle relative to the optical axis; attenuating a first portion of the output light received from a first location in the liquid column relative to a second portion of the output light received from a second location in the liquid column, the second location being farther from a center of the liquid column than the first location along an axis perpendicular to an optical axis of the optical device; 28. The method according to item 27, comprising at least one of the following: (Item 29) an excitation light source configured to generate excitation light and direct the excitation light towards an object in a liquid column, the object emitting an output light in response to the excitation light; an optical arrangement configured to receive output light that crosses an optically refractive boundary of the liquid column between the object and the optical arrangement, the optical arrangement configured to condition the output light such that a conditioned output light has an intensity that is more uniform than an intensity of the output light, the optical arrangement including a plate including an aperture and an elongated mask feature disposed at least partially across the aperture, the elongated mask feature disposed such that a longitudinal axis of the elongated mask feature is substantially parallel to a direction of flow of the object in the liquid column, the plate and the elongated mask feature being less transmissive to the output light than the opening; an optical detector disposed adjacent to the plate and positioned to receive the conditioned output light transmitted through the aperture, the optical detector configured to detect the conditioned output light and provide an electrical signal in response to the conditioned output light; an object type discrimination circuit configured to discriminate between a first type of object and a second type of object based on the electrical signal; A flow cytometer system comprising: (Item 30) 30. The system of claim 29, wherein the plate and the elongated mask feature are one unitary structure. (Item 31) 30. The system of claim 29, wherein the elongated mask feature is attached to the plate. (Item 32) 30. The system of claim 29, wherein the optical transparency of the plate to the output light varies as a function of position across the plate. (Item 33) 30. The system of claim 29, wherein opposing longitudinal ends of the elongated mask profile are substantially parallel to one another. (Item 34) 30. The system of claim 29, wherein opposing longitudinal ends of the elongated mask feature are not parallel to one another. (Item 35) 30. The system of claim 29, wherein opposing longitudinal ends of the elongated mask profile are tapered along a longitudinal axis of the elongated mask profile. [Brief description of the drawings]

[0005] [Figure 1A] FIG. 1 is a diagram of a system incorporating an optical device, according to some embodiments. [Figure 1B] 1B shows an xy cross-section of a liquid column within the measurement region of the system of FIG. 1A. [Figure 2A] 1 shows light emitted from an object located near the center of a liquid column where substantially no light is refracted at the liquid-air interface of the liquid column. [Figure 2B] 1 shows light emitted from an object located above the elliptical core of a liquid column that exhibits light refraction at the liquid-air interface. [Diagram 3] The analytical development of the expression for the angular dependence of the in-plane ray density as a function of position x is shown. [Figure 4A] A series of graphs showing the angular dependence of radiance for different object positions is provided. [Figure 4B] 1 provides a series of graphs of relative intensity of light collected from a liquid column versus object position along the x-axis for different numerical apertures of the collection optics. [Figure 5A] A series of graphs showing the angular dependence of radiance for different positions on the object and the region of exclusion are provided. [Figure 5B] FIG. 14 shows the relative intensity of light collected from the liquid 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 6] FIG. 1 is a flow diagram of a scheme for identifying a moving object in a liquid column with reduced positional change of a detected output light, according to some embodiments. [Figure 7] 1 is a top view of a ray tracing simulation of an optical system including an optical device, according to some embodiments. [Figure 8] 1 is a photograph of a split objective lens configured to reduce positional change of detected output light for an object in a liquid column, according to some embodiments. [Figure 9A] 8 shows simulated performance of the optical arrangement of FIG. 7. [Figure 9B] 8 shows simulated performance of the optical arrangement of FIG. 7. [Figure 10] 1 is a top view of a ray tracing simulation of an optical system including an optical device, according to some embodiments. [Figure 11] 1 illustrates an optical device including an elongated mask feature, according to some embodiments. [Figure 12] 1 illustrates an optical device including an elongated mask feature that can be used in conjunction with a plate having an aperture, according to some embodiments. [Figure 13] 1 illustrates an optical device including a plate and an elongated mask feature, where the optical transparency of the plate varies smoothly with position, according to some embodiments. [Figure 14] 1 illustrates an optical device including a plate and an elongated mask feature, where the optical transparency of the plate varies with position, according to some embodiments. [Figure 15] 1 is a diagram of an optical device including a plate formed as a unitary structure and an elongated mask feature extending across an aperture, according to some embodiments. [Figure 16] 13A-13C illustrate various configurations of longitudinal ends of elongated mask features according to some embodiments. [Figure 17] 13A-13C illustrate various configurations of longitudinal ends of elongated mask features according to some embodiments. [Figure 18] 13A-13C illustrate various configurations of longitudinal ends of elongated mask features according to some embodiments. [Figure 19] 1 is a photograph of an optical apparatus including a wire mask according to some embodiments. [Figure 20]1 is a photograph of an optical device including a plate having elongated mask features extending across an aperture formed as a unitary structure, according to some embodiments.

[0006] The drawings are not necessarily to scale. Like numbers used in the drawings indicate like components. However, it will be understood that the use of a number to refer to a component in a given drawing is not intended to limit the component in another drawing labeled with the same number. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The embodiments described herein relate to devices, systems and methods for distinguishing between different types of objects. The objects emit output light in response to an excitation light directed towards the objects in a liquid column, such as a flow stream. In some implementations, cell types are differentiated based on the intensity of the output light emitted from the objects. Certain embodiments discussed herein are directed to distinguishing between X-chromosome sperm cells and Y-chromosome sperm cells. It will be recognized that the disclosed approach is more generally applicable to distinguishing between any objects of different types, so long as the output light emitted from one object type has a distinguishable difference in at least one characteristic when compared to the light emitted from another object type. In some examples provided, the liquid column is a flow stream with a curved boundary or interface where refraction of light may occur. For example, the curved boundary of the liquid column may generally be of circular cross section. The liquid column may be bounded by a solid wall or may be jetted into air. The object may travel along the liquid column, which may include a central core shaped by a sheath liquid at least partially surrounding the central core. Light emitted from the object encounters at least one optically refractive boundary between the object and another material, such as the interface between a liquid column and air.

[0008] Due at least in part to refraction at the liquid-air interface, light collection efficiency outside the liquid column of light emitted from an object in the column depends on the position of the object in prior art systems. Light collection efficiency that varies with position is undesirable 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. The approaches disclosed herein increase the precision of systems that may be limited by such variations, such as jet-in-air flow cytometers. As discussed in more detail below, the positional variability of the light intensity collected from an object in the liquid column can be addressed by selectively masking the light beam at one or more surfaces of the optical system (e.g., aperture stop, field stop) to reduce the dependence of intensity on position.

[0009] The approach outlined herein is particularly applicable to flow cytometry. However, it can be applied to any system in which light is collected on one side of an interface from an object that emits light from the other side of the interface, where the interface causes a change in the emitted light path depending on the object's position relative to the detector. The approach herein adjusts the light collection efficiency of the output light emitted from the object to compensate for position changes within the liquid column.

[0010] A "jet-in-air" flow cytometer system 100, shown diagrammatically in FIG. 1A, is one type of flow cytometer that can be used to discuss the concepts of the present disclosure. The "jet-in-air" flow cytometer system 100 pumps liquid at high pressure into a chamber 110, causing a flow stream 150, including a column of liquid, to emerge from an outlet nozzle 160 of the chamber 110 at high velocity, e.g., about 20 m / s. The liquid column 150 emerging from the outlet nozzle 160 can be approximately circular in cross-section and, in some embodiments, can have a diameter of about 10 μm to about 100 μm. The flow stream 150 is comprised of a core stream 151 within a sheath stream 152, where the arrows in FIG. 1A indicate the direction of flow of the core stream and the sheath streams 151, 152.

[0011] Within the chamber 110, the sample output nozzle 111 discharges a core stream 151 containing objects 171, 172, which may be of multiple types. The core stream 151 is bounded and shaped by a stream of sheath liquid 152 that is discharged into the chamber 110 from a sheath liquid nozzle (not shown). The sheath stream 152 at least partially surrounds the core stream 151, and the sheath stream 152 and the core stream 151 do not substantially mix. The inclined or angled walls 115 of the chamber 110 narrow the sheath stream 152 before and after the flow stream 150 exits the exit nozzle 160 of the chamber 110 and / or maintain the cross-sectional size of the core stream 151 within the flow stream 150. The movement of the sheath stream 152 is constrained such that the objects 171, 172 in the core stream 151 move toward the center of the flow stream 150 as the liquid column 150 exits the chamber 110. Flow stream 150 delivers objects 171, 172, for example, in single file, into a measurement region 175 of flow stream 150.

[0012] As objects pass through the measurement region 175 of the flow stream 150, light from the excitation light source 180 provides excitation light to the objects 171, 172. The excitation light source 180 can provide light in a broad wavelength band or a narrow wavelength band. For example, the excitation light source 180 can be a laser. In some configurations, the excitation light may be conditioned by an optical element 181. For example, the excitation light may be focused by a lens 181 into the measurement region 175. Objects in the measurement region 175 emit light, e.g., scattered light or fluorescent light, in response to the excitation light source 180.

[0013] The first type of object 171 emits light that differs in at least one characteristic as compared to the light emitted from the second type of object 172. For example, in some scenarios, the first type of object 171 emits light that has a higher intensity than the light emitted from the second type of object 172.

[0014] The optical collection arrangement 190 is arranged to collect output light 161 emitted from objects in the measurement region 175 that cross the optically refractive boundary of the flow stream 150 at the liquid-air interface. The optical arrangement 190 is configured to condition the output light 161 to provide a conditioned output light 162 that compensates for position-dependence of light emitted from the objects 172a in the measurement region 175, as discussed in more detail below. The detector 185 receives the conditioned output light 162 and, in response, generates an electrical signal. In some scenarios, the amplitude of the electrical signal may differ for each type of object. The electrical signal is used by the discrimination circuitry 187 to distinguish between the different types of objects 171, 172. For example, the discrimination circuitry 187 may be configured to compare the amplitude of the electrical signal to a threshold value to discriminate between a first type of object 171 and a second type of object 172.

[0015] 1B shows an xy cross-section of flow stream 150 within measurement region 175. In the xy cross-section of measurement region 175, core stream 151 is elliptical in shape and the liquid in core stream 151 includes at least one object 172a suspended in a buffer solution. Sheath stream 152 substantially surrounds core stream 151. In the particular example used for this discussion in this disclosure, objects 171, 172 are sperm cells and system 100 is implemented to distinguish between X-chromosome and Y-chromosome sperm.

[0016] A focused laser beam generated by excitation source 180 illuminates sperm cells 172a within measurement region 175. Cells 171, 172 are stained with a fluorescent dye, and the excitation light causes cells 172a within the measurement region to emit fluorescent output light. The purpose of elliptical core 151 is to orient sperm cell 172a so that the flat sides of the sperm cell face left and right, as shown in FIG. 1B. In this orientation, the flat sides of sperm cell 172a face laser 180 and optical collection arrangement 190, respectively.

[0017] When the core stream 151 is elliptical, the sperm cell 172a can take any number of positions along the x-axis within the core stream 151. FIG. 1B shows three possible positions of the sperm cell 172a within the elliptical core 151. In the orientation shown in FIG. 1B, the first possible position of the sperm cell 172a within the core stream 151 is approximately at the center of the elliptical core 151 (on the optical axis 199 of the optical collection arrangement 190), the second possible position is at the top of the core stream 151 (above the optical axis 199), and the third possible position is at the bottom of 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 the liquid-air interface 153 at different positions within the core stream 151.

[0018] When sperm cell 172a is located in a first position and flow stream 150 has a circular cross section as shown in FIG. 1B, in-plane light rays emitted from sperm cell 172a are nearly normally incident on liquid-air interface 153. Light rays emitted from points on sperm cell 172a away from the center of the core, or out of the plane of the figure, are not normally incident exactly on interface 153. These light rays are not considered in this simplified discussion, but one skilled in the art can understand how the discussion can be generalized to include them. Thus, no refraction of light occurs at liquid-air interface 153.

[0019] The diagram of Figure 2A shows that there is no optical refraction of output light 298 emitted from sperm cell 172a across interface 153 when sperm cell 172a is in a first position within elliptical core 151 as shown in Figure 1B. Correspondingly, the in-plane density of light rays 298 exiting flow stream 150 of Figure 2A is uniform with respect to ray angle. A uniform angular density of rays corresponds to a uniform radiance as a function of ray angle.

[0020] In contrast, when the sperm cell 172a is off the optical axis 199 and closer to the top or bottom of the elliptical core 151, e.g., in the second and third positions of the elliptical core 151 shown in FIG. 1B, at least some of the output light rays emitted from the sperm cell 172a strike the liquid-air interface 153 at an oblique angle. These output light rays are refracted at the liquid-air interface 153, in contrast to the normal incidence scenario discussed above. The most oblique light rays are refracted most severely. The refraction of the light rays causes the radiance distribution of the fluorescent light exiting the flow stream 150 across the liquid-air interface 153 to be non-uniform and varies with the position of the cell 172a along the x-axis. That is, this refraction changes the radiance distribution of the output light emitted from the sperm cell 172a outside of the flow stream 150.

[0021] For example, when the cell 172a is positioned off the optical axis 199, e.g., in the second or third position shown in FIG. 1B, the density of rays and therefore the radiance on the air side of the interface 153 is higher at positive or negative ray angles with respect to the optical axis 199, respectively, 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. 3. FIG. 2B illustrates rays 299 emanating from the cell 172a when the cell 172a is in a second position of the elliptical core 151 and exiting the flow stream 150 through the liquid-air interface 153. In this scenario, the density of rays, i.e., the radiance, at positive ray angles is greater 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 errors in the determination of cell type.

[0022] Referring to FIG. 3, the light angle γ and the sperm position [ka] An analytical expression for the ray density as a function of is determined using Snell's law, where γ is the angle of the ray emitted from the object after refraction at the liquid-air interface with respect to the optical axis. Only rays within or tangent to a two-dimensional cross-section of the flow stream are considered in this analysis.

[0023] We want to solve for the density of rays with respect to the angle γ. We can use this to find the location of each sperm. [ka] We can determine the density of rays at the entrance pupil of the optical collection system for , which can be written as: [ka]

[0024] For our purposes, we can assume that the sperm cell radiates light uniformly in all directions. Therefore, the density of the emitted light with respect to the angle θ is: [ka]

[0025] That is, [ka] By geometric analysis, [ka]

[0026] Here, the angle [ka] and distance [ka] is shown in Figure 3. The flow stream has a refractive index [ka] , Snell's Law gives another relationship between the angles: [ka]

[0027] Density of rays outside the interface [ka] is the density of rays inside the interface according to the following formula: [ka] It is related to [ka] represents the average transmission through the interface over both polarizations. [ka]

[0028] The transmittance is calculated by the Fresnel reflection coefficients for s-polarized light and p-polarized light according to the following formula: [ka] It is related to. [ka]

[0029] Using equation (7) together with the above and the following additional relations: [ka]

[0030] The expression for the density of rays in terms of γ is obtained. [ka]

[0031] where the NA of the optical collection arrangement is the maximum ray angle [ka] We can solve this angle for NA since it is given by the sine of [ka]

[0032] Finally, sperm location [ka] The relative collected light intensity as a function of is given by [ka] Integrate up to and use the integral value [ka] is given by normalizing with [ka]

[0033] Using the equation for the light density distribution in Eq. (15), the angular dependence of light density (radiance) for different sperm positions can be plotted as in Figure 4A. In Figure 4A, each of the lines represents the angular dependence of the light density (radiance) for a given sperm position. [ka] This plot represents the density of rays as a function of angle γ for a given point, where γ is in radians. This plot is for a point where the ray density (radiance) is uniform as a function of angle, [ka] 4A (corresponding to graph 404 in FIG. 4A). [ka] When is positive (e.g., corresponding to the second position in FIG. 1B , graph 402), the relative radiance is high at positive ray angles γ and low at negative ray angles γ. [ka] The opposite is true when is negative (eg, corresponding to the third position in FIG. 1B, graph 403).

[0034] If the numerical aperture of the collection optics (optical collection arrangement 190 in Figs. 1A and 1B) is large, e.g., close to 1, the variation in collected light intensity with respect to the position of the light emitted from the object within the elliptical core is relatively small. This is because substantially all light emitted from the object and directed to the right will be collected by the collection optics, regardless of the exact ray direction, and the total amount of emitted light is invariant with respect to the object position (as a uniform excitation). In contrast, with a small numerical aperture, the variation in collected intensity with respect to the object position is relatively large, since the radiance distribution is affected as the object position changes, and a small numerical aperture means that only a portion of the radiance distribution that is thus changing is collected. Practical systems may have an NA significantly smaller than 1, e.g., less than 0.5 or less than 0.3. The series of graphs provided in Fig. 4B shows that ... [ka] 4A shows the relative intensity of light collected from an object through collection optics with different NA as a function of γ. FIG. 4A shows the range of angles γ obtained by different numerical apertures in FIG. 4B.

[0035] In the series of graphs in FIG. 4B, 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 (e.g., the optical collection arrangement 190 shown in FIGS. 1A and 1B), 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. 4A and 4B, a collection optic with a smaller NA experiences a larger change in collected light intensity with respect to object position when compared to a collection optic with a larger NA. In addition, a collection optic with a larger NA has a higher overall collection efficiency because it collects light rays with a wider range of refractive angles than a collection optic with a smaller NA.

[0036] Various embodiments disclosed herein are directed to collection devices (e.g., arrangement 190 shown in Figs. 1A and 1B) that reduce the variation of collected light intensity with respect to object position in the flow stream. Some embodiments discussed herein can provide conditioned output light with a change in measured intensity of less than about 3%, or less than about 2%, or even less than about 1% for deviations in object position that are less than 60% of the flow stream 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 that can result from a variety of causes. Because it is difficult to reduce the intensity variation by precisely controlling the object position in the flow stream, it is useful instead to reduce the variation of collected light intensity with respect to object position by carefully designing the optical collection arrangement. For applications such as X / Y sperm sorting, two or more cell populations are often separated based on the difference between the populations in measured fluorescence intensity. If random positional variations cause the magnitude of the variation in collected light intensity to be greater than the nominal difference in the fluorescence intensity of the two populations, it is not possible to distinguish between them while still achieving high yield and high purity. The difference in fluorescence intensity between X and Y sperm cells is typically only a few percent (e.g., approximately 4% for bovine sperm). Current sperm sorter systems could theoretically achieve higher throughput by increasing the flow rate of the core stream, but the effect of this is to increase the width of the core stream. This results in greater uncertainty in the position of the sperm within the core of the flow stream. This position uncertainty and the resulting variability in collected fluorescence intensity limit the maximum throughput of current sperm sorter systems to the extent that the small fluorescence intensity difference between X and Y sperm is noticeable.

[0037] In sperm sorter applications, sperm cells may be stained with the cell permeable dye Hoechst33342 (Ho33342). This dye enters the cell nucleus and selectively binds to AT base pairs in the minor groove of double-stranded DNA in the sperm heads of live cells. Typically, a UV laser is used to excite the stained sperm cells. When optically excited (at or near 350 nm), Y chromosome-bearing (male) sperm and X chromosome-bearing (female) sperm stained by Ho33342 can be separated by measuring the small difference in total fluorescence from each cell. The difference in total fluorescence is proportional to the amount of staining in the sperm cell, which is proportional to chromosome content. This difference varies between mammalian species, but is on the order of 4% in livestock.

[0038] One scheme for intensity-position compensation is evident in Figures 4A and 4B. The brackets in Figure 4A highlight the integrated area corresponding to the fluorescence collection optics with a given NA. A graph of the change in collected intensity with respect to object position for the NA of Figure 4A is provided in Figure 4B. In Figure 4B, an integration over the fluorescence collection area is performed so that, for a given NA, the intensity of collected light can be plotted as a function of each sperm position. From Figure 4B, it is evident 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.

[0039] The embodiments described herein relate to collection optics (e.g., optical collection arrangement 190 of FIGS. 1A and 1B) that reduce variation in collected light intensity with respect to object position as described above. According to some embodiments, the collection optics operates by masking rays in "angle space", i.e., the collection optics selectively collects, attenuates, and / or blocks rays from various angles γ to achieve a desired intensity vs. position profile. In practice, the "angle space" masking function can be applied at the pupil (e.g., entrance pupil, exit pupil, or aperture stop) of the optical system, where the location of the ray intersection with the pupil plane corresponds to the angle γ. In some embodiments, the collection optics arrangement achieves a desired, e.g., flatter, intensity vs. position profile by preferentially collecting higher angle rays (pointing away from the optical axis) compared to lower angle rays.

[0040] 5A and 5B show how to flatten the intensity versus position curve by excluding low angle refracted rays at a given NA. excluding low angle rays excludes rays that cause the largest changes in the intensity versus position profile, while the angular change in radiance at large positive angles tends to cancel the corresponding changes at large negative angles. FIG. 5A shows a plot of relative radiance versus ray angle γ for different positions of the object along the x-axis, where angle γ is in radians. In FIG. 5A, each graph corresponds to an object position x within the core of the flow stream as shown in FIG. 3. The brackets in FIG. 5A indicate the portion of the rays that are excluded by the collection optics for each position x when rays with angular magnitudes less than 0.3 rad are excluded (lower brackets in FIG. 5A) and when rays with angular magnitudes less than 0.4 rad are excluded (upper brackets in FIG. 5A).

[0041] 5B shows the relative collected light intensity versus object position along the x-axis when no angles are excluded (graph 500), when rays having angles between -0.3 rad and +0.3 rad are excluded (graph 503), and when rays having angles between -0.4 rad and +0.4 rad are excluded (graph 504). Graph 5B shows that when lower angle rays are excluded, the graph of relative intensity versus position exhibits smaller intensity variations with position.

[0042] A scheme for identifying an object moving within a liquid column in the presence of positional changes is shown in the flow diagram of FIG. 6. The process includes adjusting 620 an output light emitted from an object passing through a cross section of the flow stream such that the intensity of the adjusted output light is more uniform than the intensity of the unadjusted output light. In some embodiments, the adjusted output light is substantially uniform regardless of the position of the object. The adjusted output light is detected 630, and an electrical signal is generated 640 in response to the detected adjusted output light. A processor or other circuitry may use the electrical signal to discriminate 650 between different types of objects. For example, the circuitry may compare the amplitude of the electrical signal (corresponding to the intensity of the detected light) to a threshold to discriminate between a first type of object and a second type of object. Optionally, in some implementations, an excitation light may be generated 610 by an excitation source and directed at the cross section of the flow stream. The object then emits output light in response to the excitation light.

[0043] FIG. 7 is a top view of a ray tracing simulation of an optical system 700 including an optical device 710, according to some embodiments. The device 710 effectively expands the NA of the collection optics in the plane of the cross section of the liquid column to preferentially collect higher angle rays that are more balanced in terms of position vs. intensity compared to lower angle rays that tend to introduce variations in the position vs. intensity profile, as discussed in the discussion of FIGS. 5A and 5B. The optical collection arrangement 710 conditions the light emitted from the object in the cross section of the flow stream such that the intensity of the conditioned output light is more uniform than the output light emitted from the object. The conditioned output light can be substantially uniform regardless of the position of the object in the cross section. In this particular embodiment, the intensity of the conditioned output light is substantially uniform regardless of the position of the object along an axis perpendicular to the optical axis of the collection arrangement. In other embodiments, the intensity of the conditioned output light can be substantially uniform regardless of the position of the object along another axis, such as the optical axis of the collection arrangement.

[0044] The light collecting arrangement 710 preferentially collects light rays emitted from an object at higher angles relative to the optical axis 799 of the optical arrangement compared to light rays emitted from the object at lower angles relative to the optical axis 799. In some implementations, the optical collecting arrangement 710 is a split objective lens. A first portion 711 of the split objective lens 710 collects a first portion 751 of the higher angle light emitted from the object (object not shown in FIG. 7). A second portion 712 of the split objective lens 710 collects a second portion 752 of the higher angle light emitted from the object. As shown in FIG. 7, in some embodiments, a mask that further blocks collection of the lower angle light rays may be placed anywhere the lower angle light rays are blocked, for example, near the aperture stop or pupil plane where the light is collimated. For example, the mask 786 may be placed between the two lenses 711, 712 as shown in FIG. 7.

[0045] As shown in FIG. 7, the system 700 may be implemented as a folded optical system using mirrors 721, 722, 723, 724 to redirect portions of the collected light along an optical axis 799 of the system 700 and toward a detector 785. The mirrors 721, 722 redirect a first portion 751 of the light toward and along the optical axis, and the mirrors 723, 724 redirect a second portion 752 of the light toward and along the optical axis 799. As shown in FIG. 7, the system 700 may optionally include a filter 730, such as an optical bandpass or longpass filter, configured to substantially attenuate the excitation light. The system 700 may include a lens 740 configured to focus the first and second portions 751, 752 of the light toward the detector 785.

[0046] FIG. 8 is a photograph of a split objective lens configured to reduce intensity variation with object position. It should be noted that the split objective design allows the fluorescence collection optics to be placed closer to the flow stream than would otherwise be possible due to spatial obstructions caused by the nozzle that generates the flow stream. A single lens with the same effective NA as the split objective would be too large to place its focus directly beneath the nozzle that generates the flow stream. Optical detection of objects in the flow stream is best performed just after the flow stream exits the nozzle, where the stream is most stable, so it is important to have high NA optics that do not interfere with the nozzle.

[0047] FIG. 9A shows the simulated performance of a split objective based on the model of FIG. 7 versus the comparative arrangement shown in FIG. 10 without the spatial mask 1010. Graph 901 provides intensity with respect to object position for a system including the split objective discussed above. Graph 902 is intensity versus position for the comparative arrangement and is provided in FIG. 9A for comparison. Note the higher overall collection efficiency of the split objective arrangement in addition to the reduced effect of position changes on the collected light intensity.

[0048] To show this comparison more clearly, in Fig. 9B, each graph 901a, 902a is normalized to 100%. Graph 901a provides the collection efficiency versus the central position for a split objective lens arrangement. Graph 902a shows the collection efficiency versus the central position for a comparison system with only a single objective lens. The split objective lens results in less than 1% deviation from the central collection efficiency at an object position of 20 μm, while the comparison arrangement results in more than 10% deviation from the central collection efficiency at the same object position.

[0049] Other approaches can also be used to reduce the change in the intensity profile versus position, all of which are considered novel aspects of the present disclosure. For example, rather than a collection arrangement that selects rays in angle space (e.g., close to the pupil of the optical system), as exemplified by a split objective, it is possible to select rays in image or position space (e.g., close to the image plane of the optical system). One example of an optical collection arrangement that selects rays in position space is a spatial mask near the image plane of the optical system. To make such masks easier to align and more stable to alignment, it is useful to increase the magnification of the optical system, allowing the use of larger mask feature sizes.

[0050] FIG. 10 is a top view of a ray tracing simulation of an optical system 1000 including an optical collection arrangement 1010, e.g., a spatial mask, that attenuates light rays emitted from objects near the center of the flow stream cross section, while not attenuating light rays emitted from objects at the top and bottom of the flow stream cross section. The term "attenuate" as used herein encompasses partially blocking or completely blocking a light ray. For example, an attenuated light ray may have a reduction in intensity of 25%, 50%, 75%, or even 100% when compared to its original intensity, where 25%, 50%, and 75% attenuation corresponds to a partially blocked light ray and 100% attenuation corresponds to a completely blocked light ray. The system 1000 shown in FIG. 10 includes a single objective lens 1070 that collimates light emitted from an object (object not shown in FIG. 10). The system 1000 optionally includes a filter 1030, such as a bandpass or longpass filter, configured to block the excitation light from reaching the detector 1085. A lens 1040 can be used to focus the collected light towards a sensitive area 1086 of the detector 1085. The spatial mask 1010 attenuates or blocks light emitted from the center of the flow stream cross section 1050 from reaching the detector 1085, while allowing light emitted from the upper and lower regions 1051, 1052 of the flow stream cross section 1050 to reach the detector 1085 unattenuated or blocked. In FIG. 10, the upper region 1051 of the flow stream cross section 1050 refers to the portion of the flow stream cross section that is above the optical axis 1099 of FIG. 10. The lower region 1052 of the flow stream cross section 1050 is below the optical axis 1099 of FIG. The mask 1010 may be opaque or semi-transparent to the emitted light. In some embodiments, the optical transparency of the mask 1010 may vary with position, for example, such that an image at the center of the flow stream cross section is more attenuated than an image at the top and / or bottom of the flow stream cross section.

[0051] A simple spatial mask that mitigates variations in collected intensity with object position is a thin wire (e.g., having a diameter in the range of about 100 microns to 300 microns, e.g., about 200 microns) in front of the optical detector 1085 and near the image of the flow stream, with the wire axis oriented parallel to the flow stream and nominally centered about the optical axis 1099. The effect of the wire can be varied by moving it in or out of the image plane 1087, which is where the image of the flow stream appears (a magnified image in this embodiment).

[0052] A spatial mask that reduces variation in collected intensity with object position is shown in Figure 11. The spatial mask includes elongated features 1110, which may be implemented as thin wires with a circular cross-sectional area, bars with a rectangular cross-sectional area, or other mask features positioned at least partially across the active area 1185 of the optical detector and near the image of the flow stream. The elongated mask features 1110 may be implemented in many ways, including extruded metal wires, etched metal features, human or animal hair, traces placed on a glass slide, or ink lines printed on a transparent medium.

[0053] Generally, the length L of a mask feature is much greater than its width W. The mask feature 1110 may be oriented such that the length of the mask feature 1110 extends parallel to the flow stream and the mask feature 1110 is nominally centered with respect to the optical axis of the system (see FIG. 10). In some embodiments, the mask feature 1110 may have a width of about 100 microns to about 300 microns, e.g., about 200 microns.

[0054] As shown in Figure 12, the elongated mask feature 1210 can be used in conjunction with a plate 1220 having an aperture 1222, where the elongated mask feature 1210 is disposed at least partially across the aperture 1222 as shown in Figure 12. The plate 1220 can be particularly useful for alignment of the system optics to achieve optimal intensity differential between two types of objects that have slightly different intensities.

[0055] In some embodiments, the plate may be made of a material that partially blocks (blocks more than 25% to less than 75% of the light), substantially blocks (blocks more than 75% of the light), or completely blocks (blocks 100% of the light) the light emitted from the inspected object from reaching the active area 1230 of the detector. The aperture 1222 in the plate 1220 transmits substantially all of the light emitted from the object to the active area 1230 of the detector. The plate 1220 and aperture 1222 facilitate alignment of the system optics, allowing an operator to align the mask feature 1210 to achieve optimal contrast between the lower light intensity emitted from the first type of object and the slightly higher light intensity emitted from the second type of object.

[0056] 13 shows another embodiment in which the optical transparency of the plate varies across its length and width. In this example, the plate 1320 is more optically transparent closer to the aperture 1322 and less transmissive further from the aperture. However, the opposite scenario is also possible, where the plate is less transmissive near the aperture and more transmissive further away.

[0057] Figure 14 shows another version of a plate 1420 with a step-like gradient in optical transparency. The plate 1420 becomes more optically transmissive in different steps in regions 1421a, 1421b, 1421c, 1421d closer to the aperture 1422. Comparing the plate 1320 of Figure 13 with the plate 1420 of Figure 14, the optical transparency of the plate 1320 makes a gradual transition from the outer edges of the plate 1320, which have a lower optical transparency, to a higher optical transparency closer to the aperture 1322 in the center of the plate 1320.

[0058] In some embodiments, the aperture plate and the elongated mask features extending across the aperture are formed as a single structure as shown in Figure 15. Figure 15 shows a plate 1520 including an elongated feature 1510 that bisects an aperture 1522. In some embodiments, the single aperture plate 1520 can have a gradient of optical transparency as discussed and shown above with reference to Figures 13 and 14. A single aperture plate such as that of Figure 15 can be formed, for example, by photoetching a metal plate.

[0059] The longitudinal ends 1510a, 1510b of the elongated mask feature 1510 need not be parallel as they are shown in Figure 15. In some embodiments, the alignment process may be enhanced by having the elongated mask features 1610, 1710 with non-parallel longitudinal ends 1610a, 1610b, 1710a, 1710b as shown in Figures 16 and 17. In some embodiments, the longitudinal ends 1810a, 1810b of the elongated mask feature 1810 may be curved as shown in Figure 18.

[0060] 19 is a photograph showing an optical arrangement including a plate 1920 with an aperture 1922 according to some embodiments. An elongated mask feature 1910 including thin wires is positioned across the aperture 1922 in front of the entrance to a photomultiplier detector. The wires condition the output light emitted from objects in the flow stream by preferentially attenuating light emitted from objects in the center of the flow stream cross section, as discussed above. The preferential attenuation of light provides a more uniform light intensity versus object position profile when compared to the unconditioned output light.

[0061] 20 is a photograph showing an optical arrangement according to some embodiments, including a plate 2020 formed as one unitary structure and an elongated mask feature 2010 extending across an aperture 2022. For example, the plate and elongated mask feature 2010 may be formed by photoetching the (split) aperture 2022.

[0062] 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 possible implementations to the disclosed embodiments. Many modifications and variations are possible in light of the above teachings.

Claims

1. A discrimination system, comprising: a measurement region configured to receive a flow stream having a core stream containing the object, the core stream being surrounded by a sheath stream defining an optically refractive boundary; an excitation source configured to generate an excitation light directed towards the object in the measurement volume, the object emitting an output light in response to the excitation light; an optical arrangement having an optical axis configured to receive and condition the output light emitted from the object disposed within a flow stream, the optical arrangement further comprising a mask for attenuating the output light emitted from an object at a center of the flow stream relative to the optical axis, the mask configured to preferentially collect a first portion of the output light emitted from the object at a larger angle relative to the optical axis compared to a second portion of the output light emitted from the object at a smaller angle relative to the optical axis; an optical detector disposed to receive the conditioned output light, the optical detector configured to detect the conditioned output light and provide an electrical signal in response to the conditioned output light; an object type discrimination circuit configured to discriminate between a first type of object and a second type of object based on the electrical signal; A discrimination system comprising:

2. A discrimination system, comprising: a measurement region configured to receive a flow stream having a core stream containing the object, the core stream being surrounded by a sheath stream defining an optically refractive boundary; an excitation source configured to generate an excitation light directed towards the object in the measurement volume, the object emitting an output light in response to the excitation light; an optical arrangement having an optical axis configured to receive and condition the output light emitted from the object disposed within a flow stream, the optical arrangement further comprising a mask for attenuating the output light emitted from an object central to the flow stream relative to the optical axis, the mask including a wire; an optical detector disposed to receive the conditioned output light, the optical detector configured to detect the conditioned output light and provide an electrical signal in response to the conditioned output light; an object type discrimination circuit configured to discriminate between a first type of object and a second type of object based on the electrical signal; A discrimination system comprising:

3. 2. The discrimination system of claim 1, wherein the mask further comprises a plate including an aperture and an elongated mask feature disposed at least partially across the aperture, the elongated mask feature being positioned such that a longitudinal axis of the elongated mask feature is substantially parallel to a direction of flow of the objects in the flow stream, and the plate and the elongated mask feature are less transmissive to the output light than the aperture.

4. The discrimination system of claim 3 , wherein the plate and the elongated mask feature are one unitary structure.

5. The discrimination system of claim 3 , wherein the elongated mask feature is attached to the plate.

6. 4. The discrimination system of claim 3, wherein the optical transparency of the plate to the output light varies as a function of position across the plate.

7. The discrimination system of claim 3 , wherein opposing longitudinal ends of the elongated mask feature are substantially parallel to one another.

8. The discrimination system of claim 3 , wherein opposing longitudinal ends of the elongated mask feature are not parallel to one another.

9. The discrimination system of claim 3 , wherein opposing longitudinal ends of the elongated mask profile are tapered along a longitudinal axis of the elongated mask profile.

10. 2. The discrimination system of claim 1, wherein the mask is configured to preferentially attenuate output light received from a first location in the flow stream compared to output light received from a second location in the flow stream, the second location being farther from a center of the flow stream than the first location along an axis perpendicular to an optical axis of the optical arrangement.

11. 11. The discrimination system of claim 10, wherein the mask includes an elongated mask feature that attenuates output light received from the first location, the elongated mask feature being positioned adjacent to an active area of ​​the optical detector such that a longitudinal axis of the elongated mask feature is substantially parallel to a direction of flow of the objects in the flow stream.

12. The discrimination system of claim 11 , wherein the elongated mask feature has a round cross-sectional area.

13. The discrimination system of claim 11 , wherein the elongated mask feature has a rectangular cross-sectional area.

14. The discrimination system according to claim 11 , wherein the elongated mask shape is a metal wire.

15. The discrimination system of claim 11 , wherein the elongated mask features are traces deposited on glass.

16. The discrimination system according to claim 11 , wherein the elongated mask feature is a filament.

17. The discrimination system of claim 11 , wherein the elongated mask features are ink lines printed on a transparency.

18. The discrimination system of claim 11 , wherein the elongated mask feature is located approximately at the center of an active area of ​​the detector.

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