Object type identification system that collects light from objects within a liquid column

Abstract

This disclosure relates to an object type discrimination system that collects light from an object within a liquid column. An optical device receives output light emitted from an object placed within a liquid column, the output light passing through an optical refractive boundary of the liquid column between the object and the optical device. The optical device modifies the output light such that the modified output light has a more uniform intensity than the original output light.

Description

[0001] This application is a divisional application of the original patent application No. 201980060330.0 (International Application No.: PCT / US2019 / 049742, Application Date: September 5, 2019, Invention Title: Collecting Light from an Object in a Liquid Column). Technical Field

[0002] This invention relates to collecting light from an object within a liquid column. Background Technology

[0003] Object discrimination techniques distinguish between different types of objects. These techniques are particularly useful for classifying biological cells based on cell type. Some cell classification methods rely on light emitted from the cell to determine its type. In some implementations, cells traveling in a liquid column are exposed to excitation light, and the light emitted from the cell in response to the excitation light is detected. The output light produced by a first type of cell differs from that of a second type of cell in some characteristics, such as wavelength and / or intensity. The difference in the output light emitted from the cell can serve as the basis for cell type discrimination and classification. Summary of the Invention

[0004] Some embodiments involve an optical device configured to receive output light emitted from an object disposed within a column of liquid. The output light passes through the optical refractive boundary of the liquid column between the object and the optical device. The optical device modifies the output light such that the intensity of the modified output light is more uniform than that of the original output light. For example, the intensity of the modified output light can be substantially uniform within the cross-section of the liquid column, regardless of the position of the object.

[0005] According to some embodiments, an optical device includes an optical element and also includes a detector that detects modified output light and provides an electrical signal in response to the modified output light.

[0006] According to some embodiments, a discrimination system includes an excitation source configured to generate excitation light and direct it toward an object in a liquid column. The object emits output light in response to the excitation light. The system includes an optical device configured to receive the output light. The output light passes through an optical refractive boundary of the liquid column between the object and the optical device. The optical device modifies the output light such that the intensity of the modified output light is more uniform than the intensity of the original output light; for example, the intensity of the modified output light is substantially uniform, regardless of the object's position in the cross-section of the liquid column. An optical detector is configured to detect the modified output light and provide an electrical signal in response to the modified output light. An object type discrimination circuit distinguishes between a first type of object and a second type of object based on the electrical signal. Attached Figure Description

[0007] Figure 1AIt is a diagram of a system incorporating optical equipment, based on some implementation schemes;

[0008] Figure 1B Show Figure 1A The xy-plane cross-section of the liquid column in the measurement area of ​​the system;

[0009] Figure 2A The image shows light emitted from an object located near the center of a liquid column, where there is essentially no refraction of light at the fluid-air interface of the liquid column.

[0010] Figure 2B The image shows light emitted from an object located in the upper part of an elliptical core of a liquid column, exhibiting refracted light at the fluid-air interface.

[0011] Figure 3 The development of an analytical formula for the angular dependence of in-plane ray density as a function of position x is shown.

[0012] Figure 4A A series of charts are provided to show the angular correlation of radiation at different object locations;

[0013] Figure 4B Provides a series of graphs showing the relative intensity of light collected from a liquid column relative to the object's position along the x-axis at different numerical apertures of the collecting optics.

[0014] Figure 5A A series of plots showing the angular correlation of radiation at different locations of the object are provided, and exclusion areas are shown.

[0015] Figure 5B The diagram shows the relative intensity of light collected from the liquid column relative to the object's position along the x-axis when no angle is excluded, when light rays with angles between -0.3 rad and +0.3 rad are excluded, and when light rays with angles between -0.4 rad and +0.4 rad are excluded.

[0016] Figure 6 It is a flowchart of a method for identifying objects traveling in a liquid column with a decrease in positional change of the detected output light, according to some implementation schemes;

[0017] Figure 7 It is a top view of a ray tracing simulation of an optical system including optical devices, based on some implementation schemes;

[0018] Figure 8 These are photographs of split-type objectives configured to reduce positional changes of the detected output light from objects in a liquid column, according to some implementation schemes.

[0019] Figure 9A and Figure 9B Show Figure 7The simulated performance of optical equipment;

[0020] Figure 10 It is a top view of a ray tracing simulation of an optical system including optical devices, based on some implementation schemes;

[0021] Figure 11 An optical device including an elongated mask feature is shown according to some embodiments;

[0022] Figure 12 An optical device, according to some embodiments, includes an elongated mask feature that can be used with a plate having holes;

[0023] Figure 13 An optical device comprising a plate and an elongated mask feature is shown according to some embodiments, wherein the optical transparency of the plate varies smoothly with position;

[0024] Figure 14 An optical device comprising a plate and an elongated mask feature is shown according to some embodiments, wherein the optical transparency of the plate varies with position;

[0025] Figure 15 The diagram is of an optical device according to some embodiments, the optical device including a plate and an elongated mask feature extending through a hole formed as an integral structure;

[0026] Figures 16 to 18 Various configurations of the longitudinal edges of the elongated mask feature according to several embodiments are shown;

[0027] Figure 19 These are photographs of optical devices, including wire masks, according to some implementation schemes; and

[0028] Figure 20 These are photographs of an optical device according to some embodiments, the optical device including a plate having elongated mask features extending through a hole formed as an integral structure.

[0029] The accompanying drawings are not necessarily drawn to scale. The same numbers used in the drawings refer to the same components. However, it will be understood that the use of numbers to refer to components in a given drawing is not intended to limit the components labeled with the same numbers in another drawing. Detailed Implementation

[0030] The embodiments described herein relate to apparatus, systems, and methods for distinguishing different types of objects. In response to excitation light directed at an object in a liquid column (e.g., a flow), the object emits output light. In some embodiments, cell types are distinguished based on the intensity of the output light emitted from the object. Specific embodiments discussed herein relate to distinguishing X-chromosome sperm cells from Y-chromosome sperm cells. It should be understood that the methods of this disclosure can be applied more generally to distinguishing any type of object, provided that the output light emitted from one object type has a identifiable difference in at least one characteristic compared to light emitted from another object type. In some of the examples provided, the liquid column is a flow with a curved boundary or interface where light refraction may occur. For example, the cross-section of the curved boundary of the liquid column may typically be circular. The liquid column may be confined by a solid wall or may be ejected into the air. The object may move along the liquid column, which may include a central core formed of a sheath fluid that at least partially surrounds the central core. Light emitted from the object encounters at least one optically refractive boundary between the object and other materials, such as at the interface between the liquid column and air.

[0031] At least in part due to refraction at the fluid-air interface, the light collection efficiency outside the liquid column from light emitted from an object within the column depends on the position of the object in the prior art system. This position-dependent light collection efficiency is disadvantageous in applications where precise quantification of light emitted from the object is required, and such precision is limited by random (not directly observable) positional fluctuations of the object. The method disclosed herein improves the precision of systems potentially limited by these fluctuations (e.g., air jet flow cytometers). As discussed in more detail below, the positional variability of light intensity collected from an object within the liquid column can be addressed by selectively masking light rays on one or more planes of the optical system (e.g., aperture stops, field stops) to reduce the intensity's positional dependence.

[0032] The methods outlined in this paper are particularly applicable to flow cytometry. However, these methods can be applied to any system that collects light from an object on one side of an interface, which emits light from the other side of the interface, where the interface causes variations in the emitted light path in a manner dependent on the object's position relative to the detector. The methods in this paper modify the light collection efficiency of the output light emitted from the object to compensate for positional variations within the liquid column.

[0033] Figure 1AThe "air-jet" flow cytometer system 100 schematically illustrated is one type of flow cytometer used to discuss the concepts of this disclosure. The "air-jet" flow cytometer system 100 pumps fluid into a chamber 110 under high pressure, causing a flow stream 150 comprising a liquid column to be ejected from an outlet nozzle 160 of the chamber 110 at a high speed, for example, about 20 m / s. The cross-section of the liquid column 150 exiting from the outlet nozzle 160 may be generally circular, and in some embodiments may have a diameter of about 10 μm to about 100 μm. The flow stream 150 consists of a core stream 151 within a sheath stream 152, wherein... Figure 1A The arrows in the diagram indicate the flow directions of the core flow 151 and the sheath flow 152.

[0034] Within chamber 110, sample output nozzle 111 ejects a core stream 151 containing objects 171, 172 of various types. The core stream 151 is defined and shaped by a sheath fluid stream 152 ejected from a sheath fluid nozzle (not shown) into chamber 110. The sheath fluid stream 152 at least partially surrounds the core stream 151, and the sheath fluid stream 152 and core stream 151 are substantially non-mixed. Before and after the flow stream 150 is ejected from outlet nozzle 160 of chamber 110, the inclined or angled walls 115 of chamber 110 cause the sheath fluid stream 152 to narrow and / or maintain the cross-sectional dimensions of the core stream 151 within the flow stream 150. As the fluid column 150 is ejected from chamber 110, the movement of the sheath fluid stream 152 constrains the objects 171, 172 in the core stream 151 to move toward the center of the flow stream 150. The flow stream 150 transfers objects 171, 172 to the measurement area 175 of the flow stream 150, for example, as a single file.

[0035] As the object passes through the measurement region 175 of the flow 150, light from the excitation source 180 provides excitation light to the objects 171 and 172. The excitation source 180 can provide light with a wide or narrow wavelength band. For example, the excitation source 180 can be a laser. In some configurations, the excitation light can be modified by optical elements 181. For example, the excitation light can be focused onto the measurement region 175 by a lens 181. In response to the excitation source 180, the object in the measurement region 175 emits light, such as scattered light or fluorescence.

[0036] Compared to the light emitted from the second type of object 172, the first type of object 171 will emit light that differs in at least one characteristic. For example, in some cases, the intensity of the light emitted by the first type of object 171 is higher than the intensity of the light emitted from the second type of object 172.

[0037] An optical collecting device 190 is arranged to collect output light 161 emitted from an object within the measurement region 175, the output light intersecting the optical refractive boundary of the flow 150 at the fluid-air interface. The optical device 190 is configured to modify the output light 161 to provide a modified output light 162, which compensates for the position dependence of light emitted from the object 172a in the measurement region 175, as discussed in more detail below. A detector 185 receives the modified output light 162 and, in response, generates an electrical signal. In some cases, the amplitude of the electrical signal may differ for different object types. A discrimination circuit 187 uses the electrical signal to distinguish between different types of objects 171, 172. For example, the discrimination circuit 187 may be configured to compare the amplitude of the electrical signal with a threshold to distinguish between a first type of object 171 and a second type of object 172.

[0038] Figure 1B A cross-section of the flow 150 in the measurement region 175 in the xy plane is shown. In the xy cross-section of the measurement region 175, the core flow 151 is elliptical, and the fluid of the core flow 151 includes at least one object 172a suspended in a buffer solution. The sheath flow 152 substantially surrounds the core flow 151. In the specific example used for this discussion in this disclosure, the objects 171, 172 are sperm cells, and the system 100 is implemented to distinguish X chromosome sperm from Y chromosome sperm.

[0039] A focused laser beam generated by excitation source 180 irradiates sperm cells 172a within measurement area 175. Cells 171 and 172 are stained with fluorescent dye, and the excitation light causes cells 172a within the measurement area to emit fluorescent output light. The purpose of the elliptical core 151 is to orient sperm cells 172a so that their flat sides face left and right, as shown. Figure 1B As shown in the diagram. In this orientation, the flat sides of the sperm cell 172a face the laser 180 and the optical collecting device 190, respectively.

[0040] When the core flow 151 is elliptical, sperm cells 172a can occupy any number of positions along the x-axis within the core flow 151. Figure 1B Three possible locations of sperm cells 172a within the oval core 151 are shown. Figure 1B In the orientation shown, the first possible position of the sperm cell 172a in the core 151 is approximately at the center of the elliptical core 151 (on the optical axis 199 of the optical collecting device 190), the second possible position is at the top of the core 151 (above the optical axis 199), and the third possible position is at the bottom of the core 151 (below the optical axis 199). Position-dependent refraction of the output light emitted from the sperm cell 172a occurs at the fluid-air interface 153 at different locations within the core 151.

[0041] When Figure 1B As shown, when sperm cell 172a is located in the first position and flow 150 has a circular cross-section, in-plane light rays emanating from sperm cell 172a are incident approximately perpendicularly onto the fluid-air interface 153. Light rays emanating from points far from the center of sperm cell 172a, or from the graphic plane, are not incident perfectly normally onto interface 153; these rays are not considered in this simplified discussion, but those skilled in the art will see how the discussion can be generalized to include them. Therefore, no refraction of light occurs at the fluid-air interface 153.

[0042] Figure 2A The diagram shows when sperm cell 172a is in Figure 1B At the first position within the elliptical core 151 shown, there is no light refraction of the output light 298 emanating from the sperm cell 172a and intersecting with the interface 153. Correspondingly, leaving... Figure 2A The in-plane density of the ray 298 of the flow 150 is uniform with respect to the ray angle. The uniform angular density of the ray corresponds to uniform radiation as a function of the ray angle.

[0043] Conversely, when sperm cell 172a leaves the optical axis 199 and is closer to the top or bottom of the elliptical core 151, for example in Figure 1B At the second and third positions of the elliptical core 151 shown, at least some of the output light rays emitted from the sperm cell 172a encounter the fluid-air interface 153 at an angle. Contrary to the normal incidence case discussed above, these output light rays are refracted at the fluid-air interface 153. The most angled rays are refracted most severely. This refraction causes the radiation distribution of the fluorescence leaving the flow stream 150 through the fluid-air interface 153 to become non-uniform and varies with the position of the cell 172a along the x-axis. In other words, this refraction alters the radiation distribution of the output light emitted from the sperm cell 172a outside the flow stream 150.

[0044] For example, when cell 172a is located outside the optical axis 199, for example, located at Figure 1B At the second or third position shown, the light density and therefore the radiation on the air side of interface 153 at angles parallel to the optical axis 199, or negative or positive ray angles, are higher than the radiation at positive or negative ray angles, respectively, relative to the optical axis 199, on the air side of interface 153. Positive and negative refer to... Figure 3 The sign of the ray angle γ in the diagram. Figure 2BThis diagram illustrates the light ray 299 emitted from cell 172a and exiting the flow stream 150 via the fluid-air interface 153 when cell 172a is located in the second position of the elliptical core 151. In this case, the density of light rays or radiation at a positive ray angle is greater than the density of light rays parallel to the optical axis 199 or at a negative ray angle. For an optical system with a predetermined numerical aperture (NA), the amount of light collected by the system from the same type of cell (e.g., collection efficiency) may vary depending on whether the cell is in the first or second position. The position dependence of the system's collection efficiency leads to inaccuracy in determining the cell type.

[0045] refer to Figure 3 Snell's law was used to determine the ray angle γ and sperm position. x The analytical formula for the ray density as a function of γ is given, where γ is the angle of the ray emitted from the object relative to the optical axis after refraction at the fluid-air interface. This analysis considers only rays within or tangential to the two-dimensional cross-section of the flow.

[0046] We want to determine the light density relative to angle γ, which we can use to determine the location of each sperm cell. x The light density at the entrance pupil of the optical collection system. This can be written as:

[0047] (1)

[0048] For this purpose, we can assume that sperm cells emit light uniformly in all directions, and therefore the density of light emitted relative to angle θ is:

[0049] (2)

[0050] That is, from arrive Uniform distribution. Through geometric analysis:

[0051] (3)

[0052] ; and (4)

[0053] (5)

[0054] Among the angles and distance exist Figure 3 As shown in the figure. Because the flow has a refractive index... Therefore, Snell's law produces another relationship between angles:

[0055] (6)

[0056] Light density outside the interface The following formula is used to determine the light density within the interface. Related, among which This represents the average transmittance across the interface spanning two polarizations:

[0057] (7)

[0058] Transmittance and s-polarization and p-polarization It is related to the Fresnel reflection coefficient, and its formula is as follows:

[0059]

[0060] Use equation (7) with the above and the following additional relationships:

[0061]

[0062] The expression for the density of gamma rays is:

[0063] (15)

[0064] Now, through the maximum ray angle The sine of the optical collection device is used to derive NA, therefore we can solve for this angle based on NA:

[0065] (16)

[0066] Finally, by changing equation (15) from... Points to and through The integral value at the location is normalized to obtain the sperm location. The function of relative collected light intensity:

[0067] Relative strength = (17)

[0068] Using the formula for ray density distribution from equation (15), it can be seen that... Figure 4A The angle dependence of ray density (radiation) for different sperm locations was plotted. Figure 4A In the context of sperm location, Each line represents the ray density as a function of angle γ, where angle γ is expressed in radians. This figure corresponds to the area about A series of positions within a symmetrical range (corresponding to) Figure 4A In curve 404), the ray density (radiation) is uniform as a function of angle within the range. When When positive (e.g., Figure 1B The second position in the graph (corresponding to curve 402) shows that for a positive ray angle γ, the relative radiation is higher, while for a negative ray angle γ, the relative radiation is lower. When it is negative (for example, in Figure 1B When it is in the third position (corresponding to curve 403), the situation is exactly the opposite.

[0069] If optical devices are collected ( Figure 1A and Figure 1B In optical collecting devices (190) with a large numerical aperture, such as close to 1, the collected light intensity varies relatively little with respect to the position of light emitted from an object within the elliptical core. This is because essentially all light emitted from the object and directed to the right will be collected by the collecting optics, regardless of the exact ray direction, and the total amount of emitted light remains constant with respect to the object's position (given uniform excitation). Conversely, a smaller numerical aperture results in a relatively large variation in collected intensity with respect to the object's position, because variations in the object's position affect the radiation distribution, and a smaller numerical aperture means that only a portion of this varied radiation distribution is collected. Practical systems can have a numerical aperture (NA) significantly smaller than 1 (e.g., less than 0.5 or less than 0.3). Figure 4B The family of curves provided in the diagram shows the location of the object. The function is the relative intensity of light collected from an object by collecting optics with different NAs. Figure 4A Shown by Figure 4B The range of angles γ captured by different numerical apertures.

[0070] exist Figure 4B In the family of graphs, graph 412 shows the collection optics relative to a numerical aperture (NA) of 0.2 (e.g., Figure 1A and Figure 1B The relative intensity of the position of the optical collecting device 190 (shown) along the x-axis; graph 414 shows the relative intensity relative to the position of the collecting optics along the x-axis with NA of 0.4; graph 416 shows the relative intensity relative to the position of the collecting optics along the x-axis with NA of 0.6; graph 418 shows the relative intensity relative to the position of the collecting optics along the x-axis with NA of 0.8; and graph 419 shows the relative intensity relative to the position of the collecting optics along the x-axis with NA of 0.9. From Figure 4A and Figure 4B It is clearly evident that, compared to collecting optics with a larger NA, collecting optics with a smaller NA exhibit a greater variation in the intensity of collected light relative to the object's position. Furthermore, compared to collecting optics with a smaller NA, collecting optics with a larger NA collect light with a wider range of refraction angles, thus achieving higher overall collection efficiency.

[0071] The various embodiments disclosed herein relate to collection devices (e.g., Figure 1A and Figure 1B The device 190 shown herein reduces the variation in collected light intensity relative to the position of an object in the flow stream. Some embodiments discussed herein provide modified output light with a measured intensity variation of less than about 3%, or less than about 2%, or even less than about 1%, for objects whose positional deviation from the center of the flow stream along an axis perpendicular to the optical axis is less than 60% of the flow stream radius. Many applications are sensitive to intensity measurement errors that can be caused by a variety of sources. Since it is difficult to reduce intensity fluctuations by precisely controlling the position of objects within the flow stream, it is useful instead to reduce the variation in collected light intensity relative to the position of objects by carefully designing the optical collection device. For applications such as X / Y sperm classification, two or more cell populations are typically separated based on the measured difference in fluorescence intensity between the two populations. If random positional fluctuations cause the amplitude of the collected light intensity fluctuations to be greater than the nominal difference in fluorescence intensity between the two populations, it is impossible to distinguish them simultaneously with high yield and high purity. The fluorescence intensity difference between X and Y sperm cells is typically only a few percent (e.g., ~4% for bovine sperm). Theoretically, current sperm classifier systems can achieve high throughput by increasing the flow rate of the core stream, but this increases the width of the core stream. Therefore, there is significant uncertainty regarding the position of sperm within the core of the flowing stream. This positional uncertainty, along with the eventual fluctuations in the collected fluorescence intensity, limits the maximum throughput of current sperm classifier systems to a level that does not mask small differences in fluorescence intensity between X and Y sperm.

[0072] In sperm classifier applications, Hoechst 33342 (Ho33342) can be used to stain sperm cells. Hoechst 33342 is a cell-permeable dye that enters the cell nucleus and selectively binds to the AT base pairs in the minor groove of the double-stranded DNA within the head of live sperm cells. Typically, stained sperm cells are excited using a UV laser. When optically excited (at or near 350 nm), Ho33342-stained Y-chromosome-bearing (male) and X-chromosome-bearing (female) sperm can be distinguished by measuring the minute differences in total fluorescence between each cell. The difference in total fluorescence is proportional to the amount of staining within the sperm cell, and the amount of staining is proportional to the chromosome content. This difference varies between mammalian species, but in livestock, the difference is approximately 4%.

[0073] A method for strength-position compensation, such as Figure 4A and Figure 4B As shown. Figure 4A The bracketed range highlights the integration region corresponding to a fluorescence collecting optics with a given NA. Figure 4B China provides relative to Figure 4A A graph showing the intensity variation collected at the object location of the NA. Figure 4B In this context, for a given NA, integration is performed over the fluorescence collection region such that the intensity of the collected light can be plotted as a function of each sperm location. Figure 4B It can be seen that increasing the NA of the collecting optics helps to reduce the influence of the object position on the fluorescence intensity collected by the collecting optics.

[0074] The embodiments described herein relate to the collection of optical devices (e.g., Figure 1A and Figure 1B The optical collecting device 190 in the image reduces the variation in collected light intensity relative to the object's position, as described above. According to some embodiments, the collecting optics operate by masking rays in "angle space," i.e., selectively collecting, attenuating, and / or blocking rays from different angles γ to achieve the desired intensity and positional distribution. In practice, "angle space" masking can be applied at the pupil of an optical system (e.g., entrance pupil, exit pupil, or aperture stop), where the intersection of the ray and the pupil plane corresponds to angle γ. In some embodiments, the collecting optics achieves the desired (e.g., a flatter) intensity and positional distribution by preferentially collecting rays at larger angles (pointing away from the optical axis) over rays at smaller angles.

[0075] Figure 5A and Figure 5B This illustrates how excluding low-angle refracted rays at a given NA flattens the intensity-position curve. Excluding low-angle rays removes the rays that produce the greatest variation in intensity and position distribution, while the angular variation of radiation at high positive angles often cancels out the corresponding variation at high negative angles. Figure 5A A graph showing the relative radiation versus ray angle γ at different object positions along the x-axis is presented, where angle γ is expressed in radians. Figure 5A In the diagram, each curve corresponds to the object position x within the core of the flow, such as... Figure 3 As indicated in the document. Figure 5A The bracketed range indicates the exclusion of rays with an angle smaller than 0.3 rad. Figure 5A The range within the underlined brackets) and when excluding rays with an angle less than 0.4 rad ( Figure 5A The portion of light rays excluded by the collecting optics for each position x (within the parentheses) will be the portion excluded by the collecting optics for each position x.

[0076] Figure 5BThe graph shows the relative collected light intensity relative to the object's position along the x-axis when no excluding angle is used (Graph 500), when the excluding angle is between -0.3 rad and +0.3 rad (Graph 503), and when the excluding angle is between -0.4 rad and +0.4 rad (Graph 504). Figure 5B The graph shows that when lower-angle rays are excluded, the relative intensity versus position curve exhibits a small intensity variation relative to position.

[0077] exist Figure 6 The flowchart illustrates a method for identifying an object traveling in a liquid column in the presence of a change in position. The process includes modifying 620 the output light emitted from an object across a cross-section of the flow stream such that the intensity of the modified output light is more uniform than that of the unmodified output light. In some embodiments, the modified output light is substantially uniform regardless of the object's position. 630 The modified output light is detected, and 640 an electrical signal is generated in response to the detected modified output light. A processor or other circuitry can use the electrical signal to distinguish 650 different types of objects. For example, the circuitry can compare the amplitude of the electrical signal (corresponding to the intensity of the detected light) with a threshold to distinguish between a first type of object and a second type of object. Optionally, in some embodiments, the excitation light may be generated 610 by an excitation source and directed towards a cross-section of the flow stream, wherein the object emits output light in response to the excitation light.

[0078] Figure 7 This is a top view of a ray tracing simulation of an optical system 700 including an optical device 710 according to some embodiments. Device 710 effectively extends the NA (receiving optical field) within the cross-sectional plane of the liquid column, thereby preferentially collecting larger-angle rays that are more balanced in position and intensity than smaller-angle rays, which tend to inject variations into the position and intensity distribution, such as in... Figure 5A and Figure 5B As explained in the discussion. The optical collecting device 710 modifies the light emitted from an object in the cross-section of the flow so that the intensity of the modified output light is more uniform than the intensity of the output light emitted from the object. The modified output light can be substantially uniform regardless of the position of the object within the cross-section. In this particular embodiment, the intensity of the modified output light is substantially uniform, regardless of the position of the object along an axis perpendicular to the optical axis of the collecting device. Other embodiments may make the intensity of the modified output light substantially uniform, regardless of the position of the object along another axis (e.g., the optical axis of the collecting device).

[0079] The optical collecting device 710 preferentially collects light emitted from the object at a larger angle relative to the optical axis 799 than light emitted from the object at a smaller angle relative to the optical axis 799. In some embodiments, the optical collecting device 710 is a detachable objective lens. The first segment 711 of the detachable objective lens 710 collects light emitted from the object (… Figure 7 The first portion 751 of the light emitted at a larger angle from the object (not shown). The second section 712 of the split objective lens 710 collects the second portion 752 of the light emitted at a larger angle from the object. Figure 7 As shown, in some embodiments, a mask that further prevents the collection of smaller angle rays can be placed anywhere that blocks smaller angle rays, for example, near the aperture stop or pupil plane where the light is collimated. For example, as Figure 7 As shown, the mask 786 can be placed between the two lenses 711 and 712.

[0080] like Figure 7 As indicated, system 700 can be implemented as a folding optical system that uses mirrors 721, 722, 723, and 724 to redirect a portion of the collected light along the optical axis 799 of system 700 and toward detector 785. Mirrors 721 and 722 redirect a first portion 751 of the light toward and along the optical axis, and mirrors 723 and 724 redirect a second portion 752 of the light toward and along the optical axis 799. Figure 7 As shown, system 700 may optionally include a filter 730, such as an optical bandpass or longpass filter, configured to substantially attenuate the excitation light. System 700 may include a lens 740 configured to focus a first portion 751 and a second portion 752 of light toward detector 785.

[0081] Figure 8 This is a photograph taken with a detachable objective lens configured to reduce intensity variations relative to the object's position. It should be noted that the detachable objective lens design allows for the placement of fluorescence collecting optics closer to the flow stream than would normally be due to the spatial obstruction caused by the flow stream generated by the nozzle. A single lens with the same effective NA as the detachable objective lens would be too large to focus directly below the nozzle generating the flow stream. Optical detection of the object within the flow stream is optimally performed immediately after the flow stream leaves the nozzle (where the flow is most stable), therefore, high NA optics that do not interfere with the nozzle are crucial.

[0082] Figure 9A It shows the basis Figure 7 The simulation performance of the split objective lens in the model is comparable to that without using a spatial mask 1010. Figure 10The simulation performance of the comparison device is shown. Graph 901 provides the intensity relative to the object position in a system including the detachable objectives discussed above. Graph 902 compares the intensity of the arrangement with the position. Figure 9A The following information is provided for comparison. It should be noted that, in addition to the reduced effect of positional changes on the intensity of collected light, the overall collection efficiency of the split objective arrangement is also higher.

[0083] To illustrate this comparison more clearly, Figure 9B In the diagram, each curve 901a and 902a is normalized to 100%. Curve 901a provides the collection efficiency relative to the center position of the split objective arrangement. Curve 902a shows the collection efficiency relative to the center position for a comparison system with only a single objective. The deviation of the split objective from the center collection efficiency at an object position of 20 μm is less than 1%, while the deviation of the comparison arrangement from the center collection efficiency at the same object position is greater than 10%.

[0084] Other methods can also be used to reduce variations in position and intensity distribution, all of which are considered novel aspects of this disclosure. For example, in addition to the ray-collecting device exemplified by a split objective lens that selects rays in angular space (e.g., near the pupil of the optical system), rays can be selected in image or position space (e.g., near the image plane of the optical system). An example of an optical ray-collecting device that selects rays in position space is a spatial mask near the image plane of the optical system. To make such a mask easier and more securely aligned, this helps to increase the magnification of the optical system, thereby allowing the use of a larger mask feature size.

[0085] Figure 10 This is a top view of a ray tracing simulation of an optical system 1000, which includes an optical collection device 1010, such as a spatial mask, that attenuates light rays emitted from objects near the center of the flow cross-section, but not from objects at the top and bottom of the flow cross-section. As used herein, the term "attenuation" encompasses partially or completely blocking light rays. For example, attenuating light reduces its intensity by 25%, 50%, 75%, or even 100% compared to its original intensity, where 25%, 50%, and 75% attenuation correspond to partially blocked light rays, while 100% attenuation corresponds to completely blocked light rays. Figure 10 The system 1000 shown includes a single objective lens 1070, which enables the lens to focus on the object (…). Figure 10The light emitted from the object (not shown) is collimated. System 1000 optionally includes a filter 1030, such as a bandpass filter or a longpass filter, configured to block the excitation light from reaching detector 1085. Lens 1040 can be used to focus the collected light toward the sensitive region 1086 of detector 1085. Spatial mask 1010 attenuates or blocks light emitted from the center of flow cross section 1050 from reaching detector 1085 without attenuating or blocking light emitted from the top region 1051 and bottom region 1052 of flow cross section 1050 from reaching detector 1085. Figure 10 In the context of the flow cross-section 1050, the top region 1051 refers to the area of ​​the flow cross-section within the cross-section. Figure 10 The portion above the optical axis 1099. The bottom region 1052 of the flow cross-section 1050. Figure 10 Below the optical axis 1099. 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 the image at the center of the flow cross-section is attenuated more than the image at the top / or bottom of the flow cross-section.

[0086] A simple spatial mask to mitigate the variation in collected intensity with object position is a fine line (e.g., in the range of approximately 100 to 300 micrometers in diameter, such as approximately 200 micrometers in diameter) in front of the optical detector 1085 and close to the image of the flow stream, wherein the line axis is oriented parallel to the flow stream and nominally centered relative to the optical axis 1099. The role of the line can be altered by moving the line in and out of the image plane 1087, in which the image of the flow stream (a magnified view in the current embodiment) appears.

[0087] exist Figure 11 The diagram illustrates a spatial mask that reduces the variation of collected intensity with object location. The spatial mask includes an elongated feature 1110, which can be implemented as a thin line with a circular cross-sectional area, a strip with a rectangular cross-sectional area, or other mask features that at least partially span the active region 1185 of the optical detector and are close to the image placement of the flow stream. The elongated mask feature 1110 can be implemented in various ways, including extruded wires, etched metallic features, human or animal hair, traces deposited on a glass slide, or ink lines printed on a transparent medium.

[0088] Typically, the length L of the mask feature is much larger than its width W. Mask feature 1110 can be oriented such that the length of mask feature 1110 is parallel to the flow, and mask feature 1110 is relative to the optical axis of the system (see [link to documentation]). Figure 10It is nominally centered. In some embodiments, mask feature 1110 may have a width of about 100 micrometers to about 300 micrometers, for example about 200 micrometers.

[0089] like Figure 12 As shown, the elongated mask feature 1210 can be used in conjunction with a plate 1220 having a hole 1222, wherein, as Figure 12 As shown, the elongated mask feature 1210 is positioned at least partially across the aperture 1222. The plate 1220 is particularly suitable for alignment system optics to achieve optimal intensity difference between two types of objects with slightly different intensities.

[0090] In some embodiments, the plate may be made of a material that partially blocks (blocks more than 25% and less than 75% of light), substantially blocks (blocks more than 75% of light), or completely blocks (blocks 100% of light) light emitted from the object under test from reaching the active area 1230 of the detector. A hole 1222 in the plate 1220 transmits substantially all light emitted from the object into the active area 1230 of the detector. The plate 1220 and the hole 1222 facilitate the alignment of the system optics, thereby allowing the operator to align the mask feature 1210 to achieve optimal contrast between the lower light intensity emitted from a first type of object and the slightly higher light intensity emitted from a second type of object.

[0091] Figure 13 Another embodiment is shown in which the optical transparency of the plate varies along its length and width. In this example, the plate 1320 is more optically transparent closer to the aperture 1322, and the further away from the aperture, the lower the transmittance of the plate. However, the opposite is also possible, where the plate has lower transmittance near the aperture and higher transmittance further away from the aperture.

[0092] Figure 14 Another version of plate 1420 with a gradual optical transparency gradient is shown. In different steps within regions 1421a, 1421b, 1421c, and 1421d, plate 1420 becomes more transparent closer to aperture 1422. Figure 13 Board 1320 and Figure 14 Compared to plate 1420, the optical transparency of plate 1320 gradually transitions from lower optical transparency at the outer edge of plate 1320 to higher optical transparency near the hole 1322 at the center of plate 1320.

[0093] In some implementations, the perforated plate and the elongated mask features extending through the perforation are formed as follows: Figure 15 The overall structure shown. Figure 15 A plate 1520 is shown, which includes an elongated feature 1510 that divides a hole 1522 in two. In some embodiments, the integral hole plate 1520 may have the features previously referenced. Figure 13and Figure 14 The optical transparency gradient is discussed and illustrated. (See figure.) Figure 15 The monolithic perforated plate shown is formed, for example, by photolithography on a metal plate.

[0094] The longitudinal edges 1510a and 1510b of the elongated mask feature 1510 do not need to be as Figure 15 The parallelism shown is illustrated. In some embodiments, it can be achieved by having, as shown in the example... Figure 16 and Figure 17 The elongated mask features 1610 and 1710, shown with non-parallel longitudinal edges 1610a, 1610b, 1710a, 1710b, are used to enhance the alignment process. In some embodiments, the longitudinal edges 1810a and 1810b of the elongated mask feature 1810 can be as follows: Figure 18 The bend shown in the image.

[0095] Figure 19 This is a photograph illustrating an optical device according to some embodiments, including a plate 1920 with an aperture 1922. An elongated mask feature 1910, comprising fine lines, is positioned across the aperture 1922 in front of the inlet of a photomultiplier tube detector. As previously discussed, the wires modify the output light emitted from an object in the flow stream by preferentially attenuating the light emitted from the object at the center of the flow stream cross-section. This preferential attenuation of light provides a more uniform distribution of light intensity and object position compared to unmodified output light.

[0096] Figure 20 The image shows an optical device according to some embodiments, the optical device including a plate 2020 and an elongated mask feature 2010 extending through an aperture 2022 formed as an integral structure. For example, the plate and the elongated mask feature 2010 can be formed by photolithography of the (divided) aperture 2022.

[0097] The foregoing description of various embodiments has been presented for illustrative and descriptive purposes and is not intended to be limiting. 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 foregoing teachings.

Claims

1. A discrimination system, the discrimination system comprising: A measurement region configured to receive a flow including a core stream containing an object, the core stream being surrounded by a sheath stream defining an optical refractive boundary; An excitation source configured to generate excitation light directed at the object in the measurement region, the object emitting output light in response to the excitation light; An optical device having an optical axis, the optical device being configured to receive and modify the output light emitted from the object disposed within a flow stream, the optical device further comprising a mask for attenuating excitation light emitted from the object near the center of the flow stream, wherein the mask 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, relative to a second portion of the output light emitted from the object at a smaller angle relative to the optical axis; An optical detector, arranged to receive modified output light, configured to detect the modified output light and provide an electrical signal in response to the modified output light; and An object type discrimination circuit is configured to distinguish between a first type of object and a second type of object based on the electrical signal.

2. The discrimination system according to claim 1, wherein, The mask includes wires.

3. The discrimination system according to claim 1, wherein, The mask further includes a plate comprising an aperture and an elongated mask feature disposed at least partially across the aperture, the elongated mask feature being arranged such that the longitudinal axis of the elongated mask feature is substantially parallel to the flow direction of the object in the flow stream, and the plate and the elongated mask feature have lower transmittance for the output light compared to the aperture.

4. The discrimination system according to claim 3, wherein, The plate and the elongated mask feature are a single, integral structure.

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

6. The discrimination system according to claim 3, wherein, The optical transparency of the plate to the output light varies with the position across the plate.

7. The discrimination system according to claim 3, wherein, The opposing longitudinal edges of the elongated mask features are substantially parallel to each other.

8. The discrimination system according to claim 3, wherein, The opposing longitudinal edges of the elongated mask features are not parallel to each other.

9. The discrimination system according to claim 3, wherein, The opposing longitudinal edges of the elongated mask feature gradually taper along the longitudinal axis of the elongated mask feature.

10. The discrimination system according to claim 1, wherein, The mask is configured to preferentially attenuate output light received from a first position in the flow stream compared to output light received from a second position in the flow stream, wherein the second position is further away from the center of the flow stream than the first position along an axis perpendicular to the optical axis of the optical device.

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

12. The discrimination system according to claim 11, wherein, The elongated mask feature has a circular cross-sectional area.

13. The discrimination system according to 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 feature is a metal line.

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

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

17. The discrimination system according to claim 11, wherein, The elongated mask features are ink lines printed on transparent paper.

18. The discrimination system according to claim 11, wherein, The elongated mask feature is positioned approximately at the center of the detector's active area.

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