Method and system for determining particle position information in a core stream of a flow cytometer

Abstract

Aspects of the present disclosure include methods for determining positional information of particles in a flow stream of a flow cytometer. In certain embodiments, the method includes propagating a composition containing particles through a flow stream including a core stream and a sheath flow stream, irradiating the particles of the composition with a light source, detecting light from the irradiated particles, detecting emitted light from the irradiated particles of the composition, and determining the position of the irradiated particles within the core stream of the flow stream based on the detected emitted light. In embodiments, the particles are stably attached (e.g., covalently attached) to an illumination power density-sensitive compound that emits light with an intensity determined by the illumination power density of the light source incident on the particles. Systems (e.g., flow cytometers) for implementing the subject methods are further described. Non-transitory computer-readable storage media are also provided.

Description

[Background technology]

[0001] Light detection is often used to characterize components of a sample (e.g., a biological sample), for example, when the sample is used to diagnose a disease or condition. When a sample is illuminated, light can be scattered by the sample, transmitted through the sample, as well as emitted by the sample (e.g., by fluorescence). Differences in sample components, such as morphology, absorbance, and the presence of fluorescent labels, can result in differences in the light scattered, transmitted, or emitted by the sample. These differences can be used to characterize and identify the presence of components in the sample. To quantify these differences, light is collected and directed to a detector surface.

[0002] One technique that utilizes optical detection to characterize components in a sample is flow cytometry. A flow cytometer includes an optical detection system consisting of optics, photodetectors, and electronics that allows for efficient detection of optical signals and conversion of the optical signals into corresponding electrical signals. The electronic signals are processed to obtain parameters that the user can use to perform the desired analysis. Flow cytometers include various types of photodetectors to detect optical signals, such as those from fluorescence, side-scattered light, or forward-scattered light. When an optical signal enters the photodetector, an electrical signal proportional to the incident optical signal is generated at the photodetector's output. Flow cytometry provides a rapid method for quantifying cellular characteristics. Summary of the Invention [Problem to be solved by the invention]

[0003] The light used to illuminate a flow stream in a flow cytometer can have a different intensity profile across the illumination spot; for example, the center of the laser beam has the highest power density, with the power density decreasing rapidly as one moves away from the center of the beam spot. Particles passing directly through the center of the beam spot receive greater laser power intensity than particles not passing through the center of the beam spot. In some cases, fluorophores present on particles passing around the periphery of the beam spot are not optimally excited, resulting in reduced brightness and greater noise in the data signal from the detected light. [Means for solving the problem]

[0004] Aspects of the present disclosure include methods for determining positional information of particles in a flow stream of a flow cytometer. In certain embodiments, the method includes propagating a composition containing particles through a flow stream including a core stream and a sheath flow stream, irradiating the particles of the composition with a light source, detecting light from the irradiated particles, detecting emitted light from the irradiated particles of the composition, and determining the position of the irradiated particles within the core stream of the flow stream based on the detected emitted light. In embodiments, the particles are stably attached (e.g., covalently attached) to an illumination power density-sensitive compound that emits light with an intensity determined by the illumination power density of the light source incident on the particles. Systems (e.g., flow cytometers) for implementing the subject methods are further described. Non-transitory computer-readable storage media are also provided.

[0005] In practicing the subject method, a composition including particles is irradiated with a light source within a flow stream, including a core stream and a sheath flow stream. In embodiments, the composition includes particles stably bound to an irradiance power density-sensitive compound, which emits light at an intensity determined by the irradiance power density of the light source incident on the particles. Optionally, the irradiance power density-sensitive compound is covalently attached to the particles in the composition via a linker. Optionally, the linker is a cleavable linker. Optionally, the linker is a non-cleavable linker. Optionally, the irradiance power density-sensitive compound is bound to the particles via a specific binding member, such as an antibody. In certain embodiments, the composition is a biological composition and the particles are cells. In some embodiments, the irradiance power density-sensitive compound is an upconversion nanoparticle (UCNP). Optionally, the nanoparticles are lanthanide-doped particles. Optionally, the nanoparticles are europium-doped or thulium-doped nanoparticles, such as NaYF4:Yb, Er nanoparticles, or NaYF4:YbTm nanoparticles.

[0006] In some embodiments, the irradiated particles of the composition have a spectral radiation intensity profile that varies with the power density of the light source. In some cases, the irradiated particles of the composition have two or more radiation peaks in response to irradiation by the light source. In some cases, the intensity of each radiation peak depends on the radiation power density of the light source. In some embodiments, one or more of the radiation peaks have a first intensity at a first radiation power density of the light source and a second intensity at a second radiation power density of the light source. In some cases, the irradiated particles of the composition have a first radiation peak having a first intensity at the first radiation power density and a second intensity at the second radiation power density, and a second radiation peak having the same intensity at both the first and second radiation power densities.

[0007] In some embodiments, the composition within the flow stream is irradiated using a light source (e.g., a laser) having a Gaussian beam power density profile across the horizontal axis of the core stream. In some cases, the core stream is irradiated with an elliptical beam spot having a major axis and a minor axis, e.g., the major axis of the beam spot is perpendicular to the horizontal axis of the core stream. In some cases, the laser is configured to irradiate the core stream such that the irradiated power density across the horizontal axis of the flow stream is higher at or near the center of the core stream and lower at or near the edges of the core stream.

[0008] In some embodiments, the method determines the location of the irradiated particle of the composition based on the difference between the radiation intensity of the first radiation peak and the radiation intensity of the second radiation peak. In some cases, when the first radiation peak and the second radiation peak have substantially the same intensity, the irradiated particle is determined to be propagating within the core stream at approximately the center of the light source irradiation location. In some cases, when the first radiation peak and the second radiation peak have different intensities, the irradiated particle is determined to be propagating within the core stream at an edge of the light source irradiation location. In some cases, when the first radiation peak and the second radiation peak have substantially the same intensity, the irradiated particle is determined to be propagating within the core stream at approximately the center of the core stream. In some cases, when the first radiation peak and the second radiation peak have different intensities, the irradiated particle is determined to be propagating within the core stream at an edge of the core stream.

[0009] In some cases, the method determines that the illuminated particles are propagating within the flow stream at or near the center of the core stream. In some cases, the method determines that the illuminated particles are propagating within the flow stream at or near the edge of the core stream. In some cases, the method generates one or more particle population clusters based on the determined locations of the illuminated particles within the core stream. In some embodiments, particles determined to be propagating within the flow stream at or near the center of the core stream are clustered together. In some embodiments, particles determined to be propagating within the flow stream at or near the edge of the core stream are clustered together. In some embodiments, data signals generated in response to light from illuminated particles of a composition determined to be propagating within the flow stream at or near the edge of the core stream are discarded.

[0010] Aspects of the present disclosure include a system for determining positional information of particles in a flowstream. In some embodiments, the system includes a flow cell configured to propagate a composition including particles in a flowstream, the flowstream including a core stream and a sheath flow stream, a light source for illuminating the particles of the composition, a light detection system for detecting light from the illuminated particles, and a processor operatively coupled to a memory, the memory storing instructions that, when executed by the processor, cause the processor to determine the position of the particles in the flowstream based on the detected emitted light from the illuminated particles.

[0011] In some embodiments, the light source comprises one or more lasers. Optionally, the light source is a laser configured to have a Gaussian beam power density profile across the horizontal axis of the flow stream. Optionally, the laser has an elliptical beam spot having a major axis and a minor axis. Optionally, the laser is configured to irradiate the core stream with an elliptical beam spot such that the major axis of the beam spot is perpendicular to the horizontal axis of the flow stream. Optionally, the laser is configured to irradiate the core stream such that the irradiating power density across the horizontal axis of the flow stream is higher at or near the center of the core stream and lower at or near the edges of the core stream. In some embodiments, the light source comprises an optical conditioning element configured to deliver light to the flow stream such that the major axis of the beam spot is perpendicular to the horizontal axis of the flow stream. Optionally, the optical conditioning element is configured to rotate the beam spot by 90 degrees. Optionally, the optical conditioning element comprises one or more lenses. In some embodiments, the light source comprises multiple lasers. In some cases, the light source includes at least one laser having an elliptical beam spot having a major axis and a minor axis, the major axis of the beam spot configured to illuminate the flow stream such that the major axis of the beam spot is perpendicular to the longitudinal axis of the flow stream, and another laser having an elliptical beam spot having a major axis and a minor axis, the major axis of the beam spot configured to illuminate the flow stream such that the major axis of the beam spot is perpendicular to the horizontal axis of the flow stream.

[0012] In some embodiments, the system includes a processor operatively coupled to a memory, the memory storing instructions that, when executed by the processor, cause the processor to determine a position of an irradiated particle of the composition based on a difference between the radiation intensity of the first radiation peak and the radiation intensity of the second radiation peak. In some cases, the memory includes instructions for determining that the irradiated particle is propagating in the core stream at approximately the center of the light source irradiation position when the first radiation peak and the second radiation peak have approximately the same intensity. In some cases, the memory includes instructions for determining that the irradiated particle is propagating in the core stream at an edge of the light source irradiation position when the first radiation peak and the second radiation peak have approximately the same intensity. In some cases, the memory includes instructions for determining that the irradiated particle is propagating in the core stream at approximately the center of the core stream when the first radiation peak and the second radiation peak have approximately the same intensity. In some cases, the memory includes instructions for determining that the irradiated particle is propagating in the core stream at an edge of the core stream when the first radiation peak and the second radiation peak have different intensities.

[0013] In some embodiments, the memory has instructions for determining that the illuminated particles are propagating within the flow stream at or near the center of the core stream. In some embodiments, the memory has instructions for determining that the illuminated particles are propagating within the flow stream at or near the edge of the core stream. In some embodiments, the memory has instructions for generating one or more particle population clusters based on the determined positions of the illuminated particles within the core stream. In some embodiments, the memory has instructions for clustering together particles determined to be propagating within the flow stream at or near the center of the core stream. In some embodiments, the memory has instructions for clustering together particles determined to be propagating within the flow stream at or near the edge of the core stream. In some embodiments, the memory has instructions for discarding data signals generated in response to light from illuminated particles of a composition determined to be propagating within the flow stream at or near the edge of the core stream.

[0014] Aspects of the present disclosure further include a non-transitory computer-readable storage medium for determining positional information of particles of a composition (e.g., cells of a biological sample) within a flow cytometer. In embodiments, the non-transitory computer-readable storage medium comprises an algorithm for propagating a composition including particles (e.g., cells) through a flow stream including a core stream and a sheath flow stream, an algorithm for irradiating particles of the composition with a light source, an algorithm for detecting light from the illuminated particles, an algorithm for determining the emission intensity of the illuminated particles of the composition, and an algorithm for determining the position of the illuminated particles within the core stream of the flow stream based on the detected emission light from the illuminated particles.

[0015] In some embodiments, the irradiated particles in the composition are stably bound to an irradiance power density-sensitive compound that emits light at an intensity determined by the irradiance power density of a light source incident on the particles. In some embodiments, the irradiated particles are configured to have a spectral radiation intensity profile that varies depending on the power density of the light source, for example, the particles of the composition are configured to have two or more radiation peaks in response to irradiation by the light source. In some cases, the intensity of each radiation peak depends on the irradiance power density of the light source. In some cases, one or more of the radiation peaks have a first intensity at a first irradiance power density of the light source and a second intensity at a second irradiance power density of the light source. In some embodiments, the particles of the composition have a first radiation peak that has a first intensity at a first irradiance power density and a second intensity at a second irradiance power density, and a second radiation peak that has the same intensity at both the first and second irradiance power densities.

[0016] In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for determining a position of an irradiated particle of the composition based on a difference between the radiation intensity of the first radiation peak and the radiation intensity of the second radiation peak. In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for determining that an irradiated particle is propagating in the core stream at approximately the center of the light source irradiation position when the first radiation peak and the second radiation peak have approximately the same intensity. In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for determining that an irradiated particle is propagating in the core stream at approximately the center of the light source irradiation position when the first radiation peak and the second radiation peak have different intensities. In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for determining that an irradiated particle is propagating in the core stream at approximately the center of the core stream when the first radiation peak and the second radiation peak have approximately the same intensity. In some cases, the non-transitory computer-readable storage medium has an algorithm for determining that the irradiated particle is propagating within the core stream at an edge of the core stream when the first emission peak and the second emission peak have different intensities.

[0017] In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for generating one or more particle population clusters based on the determined positions of the particles within the core stream. In some cases, the non-transitory computer-readable storage medium comprises an algorithm for clustering particles determined to be propagating within the flow stream at or near the center of the core stream. In some cases, the non-transitory computer-readable storage medium comprises an algorithm for clustering particles determined to be propagating within the flow stream at or near the edge of the core stream. In some cases, the non-transitory computer-readable storage medium comprises an algorithm for discarding data signals generated in response to light from illuminated particles determined to be propagating within the flow stream at or near the edge of the core stream. [Brief explanation of the drawings]

[0018] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which:

[0019] [Figure 1] FIG. 1 illustrates a process for determining positional information of particles of a composition within a core stream according to an embodiment. [Figure 2] 1 is a flowchart for characterizing positional information about particles of a composition according to an embodiment. [Figure 3] FIG. 1 illustrates a light source for irradiating a composition including particles in a flow stream, according to some embodiments. [Figure 4A] FIG. 1 is a functional block diagram illustrating a particle analysis system according to an embodiment. [Figure 4B] FIG. 1 illustrates a flow cytometer according to an embodiment. [Figure 5] FIG. 1 is a functional block diagram illustrating an example of a particle analysis control system according to an embodiment. [Figure 6A] FIG. 1 is a schematic diagram illustrating a particle sorting system according to an embodiment. [Figure 6B] FIG. 1 is a schematic diagram illustrating a particle sorting system according to an embodiment. [Figure 7] FIG. 1 is a block diagram illustrating a computing system according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0020] Aspects of the present disclosure include methods for determining positional information of particles in a flow stream of a flow cytometer. In certain embodiments, the method includes propagating a composition containing particles through a flow stream including a core stream and a sheath flow stream, irradiating the particles of the composition with a light source, detecting light from the irradiated particles, detecting emitted light from the irradiated particles of the composition, and determining the position of the irradiated particles within the core stream of the flow stream based on the detected emitted light. In embodiments, the particles are stably attached (e.g., covalently attached) to an illumination power density-sensitive compound that emits light with an intensity determined by the illumination power density of the light source incident on the particles. Systems (e.g., flow cytometers) for implementing the subject methods are further described. Non-transitory computer-readable storage media are also provided.

[0021] Before the present invention is described in more detail, it is to be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. The scope of the present invention will be limited only by the appended claims, and it is to be further understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0022] When a range of values ​​is given, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0023] In this specification, a range is presented with the term "about" before the numerical values. The term "about" is used herein to literally support the exact number that it precedes, as well as a number that is close to or approximately the number that it precedes. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately the number may be a number that, in the context in which the specifically stated number is presented, provides a substantial equivalent to the specifically stated number.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described.

[0025] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0026] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like, or for use of a "negative" limitation in connection with the recitation of claim elements.

[0027] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein comprises separate components and features which may be readily separated from or combined with any of the features of the other multiple embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0028] Although the apparatus and methods have been or will be described for grammatical fluidity with functional descriptions, it should be clearly understood that the claims, unless expressly recited under 35 U.S.C. 112, should not be construed as necessarily limited in any way by limitations of "means" or "step" construction, but should be accorded the full scope of the meaning and equivalents of the definition given by the claims under the judicial theory of equivalents, and that if a claim is expressly recited under 35 U.S.C. 112, it should be accorded the full statutory equivalents under 35 U.S.C. 112.

[0029] As summarized above, the present disclosure provides methods for determining positional information of particles in a flow stream within a flow cytometer. In further describing embodiments of the present disclosure, a method for determining positional information within a core stream of a flow stream for particles having an illumination power density-sensitive compound that emits light at an intensity that depends on the illumination power density of a light source incident on the particle is first described. A system (e.g., a flow cytometer) is then described that includes a light source and a light detection system for implementing the subject method. A non-transitory computer-readable storage medium is also provided.

[0030] Method for determining particle position information in a flow stream of a flow cytometer Aspects of the present disclosure include methods for determining positional information of particles in a flow stream of a flow cytometer. In some embodiments, the present disclosure provides methods for modifying or adjusting analysis of data signals related to particles in a sample composition based on positional information of particles in the flow stream. In some embodiments, the subject methods determine whether a particle is flowing in a core stream of a flow stream, including a core stream and a sheath flow stream. In some cases, the particle may be determined to be flowing at or near the center of the core stream. In other cases, the particle may be determined to be flowing at or near the edge of the core stream. In some embodiments, the methods described herein determine the position of one or more particles relative to the position of illumination of the core stream by a light source. In some cases, the particle in the composition is determined to be propagating in the core stream at a position at or near the center of illumination by the light source. In other cases, the particle in the composition is determined to be propagating in the core stream at a position at or near the edge of illumination by the light source.

[0031] The light used to illuminate a flow stream in a flow cytometer can have a different intensity profile across the illumination spot, e.g., the center of the laser beam has the highest power density, with the power density decreasing as one moves away from the center of the beam spot. Particles passing directly through the center of the beam spot can receive a greater laser power intensity than particles not passing through the center of the beam spot. In some cases, fluorophores present on particles passing through the periphery of the beam spot are not optimally excited, resulting in reduced brightness and greater noise in the data signal from the detected light. In some cases, the methods for determining particle positional information in a core stream as described herein provide reduced variation in illumination of particles in the core stream, e.g., the illumination intensity incident on particles of a composition varies by 10% or less across the core stream, e.g., 9% or less, e.g., 8% or less, e.g., 7% or less, e.g., 6% or less, e.g., 5% or less, e.g., 4% or less, e.g., 3% or less, e.g., 2% or less, e.g., 1% or less, e.g., 0.5% or less, e.g., 0.1% or less, e.g., 0.01% or less, e.g., 0.001% or less. In certain embodiments, the subject methods determine which particles in the sample composition are not optimally excited and adjust or discard data signals from particles that are not optimally excited by illumination with the light source. In certain embodiments, the subject methods increase the signal-to-noise ratio of data signals generated in response to light from illuminated particles of the sample composition, e.g., by 5% or more, e.g., by 10% or more, e.g., by 25% or more, e.g., by 50% or more, e.g., by 75% or more, e.g., by 90% or more, e.g., by 99% or more.

[0032] In practicing the subject methods, a composition containing particles propagating within the core stream is illuminated with a light source. In some embodiments, the light source is a continuous wave light source, e.g., the light source provides an uninterrupted beam of light, maintaining illumination of the sample particles in the flow stream with little or no undesirable changes in light intensity. In some embodiments, the continuous light source provides non-pulsed or non-stroboscopic illumination. In certain embodiments, the continuous light source provides a substantially constant luminous intensity. For example, the method may involve illuminating the sample in the flowstream with a continuous light source that provides an luminescence intensity during a time interval of illumination that varies by 10% or less, such as 9% or less, for example 8% or less, such as 7% or less, for example 6% or less, such as 5% or less, for example 4% or less, such as 3% or less, for example 2% or less, such as 1% or less, for example 0.5% or less, such as 0.1% or less, for example 0.01% or less, such as 0.001% or less, for example 0.0001% or less, for example 0.00001% or less, e.g., the luminescence intensity during a time interval of illumination varies by 0.000001% or less. The intensity of the light output may be measured by any convenient protocol, including, but not limited to, scanning slit profilers, charge coupled devices (CCDs, e.g., intensified charge coupled devices (ICCDs)), position sensors, power sensors (e.g., thermopile power sensors), optical power sensors, energy meters, digital laser photometers, laser diode detectors, among other types of photodetectors.

[0033] In other embodiments, a pulsed light source is used to illuminate the compositions in the flowstream, e.g., light is emitted at predetermined time intervals, each time interval having a predetermined illumination duration (i.e., pulse width). In certain embodiments, a method uses a pulsed light source to illuminate the sample in each interrogation region of the flowstream with periodic flashes of light. For example, the frequency of each light pulse can be 0.0001 kHz or more, such as 0.0005 kHz or more, for example 0.001 kHz or more, such as 0.005 kHz or more, for example 0.01 kHz or more, such as 0.05 kHz or more, for example 0.1 kHz or more, such as 0.5 kHz or more, for example 1 kHz or more, such as 2.5 kHz or more, for example 5 kHz or more, for example 10 kHz or more, for example 25 kHz or more, for example 50 kHz or more, for example 100 kHz or more. In some cases, the frequency of the pulsed irradiation by the light source is in the range of 0.00001 kHz to 1000 kHz, such as 0.00005 kHz to 900 kHz, for example 0.0001 kHz to 800 kHz, for example 0.0005 kHz to 700 kHz, for example 0.001 kHz to 600 kHz, for example 0.005 kHz to 500 kHz, for example 0.01 kHz to 400 kHz, for example 0.05 kHz to 300 kHz, for example 0.1 kHz to 200 kHz, for example 1 kHz to 100 kHz. The duration of light irradiation of each light pulse (i.e. pulse width) may vary and may be 0.000001 milliseconds or more, such as 0.000005 milliseconds or more, for example 0.00001 milliseconds or more, such as 0.00005 milliseconds or more, for example 0.0001 milliseconds or more, for example 0.0005 milliseconds or more, such as ...1 milliseconds or more, for example 0.005 milliseconds or more, such as 0.01 milliseconds or more, for example 0.05 milliseconds or more, such as 0.1 milliseconds or more, for example 0.5 milliseconds or more, such as 1 millisecond or more, for example 2 milliseconds or more, for example 3 milliseconds or more, such as 4 milliseconds or more, for example 5 milliseconds or more, such as 10 milliseconds or more, for example 25 milliseconds or more, for example 50 milliseconds or more, such as 100 milliseconds or more, for example 500 milliseconds or more.For example, the duration of light irradiation may be in the range of 0.000001 milliseconds to 1000 milliseconds, for example, 0.000005 milliseconds to 950 milliseconds, for example, 0.00001 milliseconds to 900 milliseconds, for example, 0.00005 milliseconds to 850 milliseconds, for example, 0.0001 milliseconds to 800 milliseconds, for example, 0.0005 milliseconds to 750 milliseconds, for example, 0.001 milliseconds to 700 milliseconds, for example, 0.005 milliseconds to 650 milliseconds, for example, 0.01 milliseconds to 600 milliseconds, for example, 0.05 milliseconds to 550 milliseconds, for example, 0.1 milliseconds to 500 milliseconds, for example, 0.5 milliseconds to 450 milliseconds, for example, 1 millisecond to 400 milliseconds, for example, 5 milliseconds to 350 milliseconds, or for example, 10 milliseconds to 300 milliseconds.

[0034] The flow stream may be irradiated using any convenient light source, including laser and non-laser light sources (e.g., light emitting diodes). In certain embodiments, the method irradiates the particles using a laser, such as a pulsed or continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the subject systems include a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, the lasers of interest include metal vapor lasers, such as helium cadmium (HeCd), helium mercury (HeHg), helium selenium (HeSe), helium silver (HeAg), strontium, neon copper (NeCu), copper, or gold lasers, and combinations thereof. In still other cases, the subject systems include solid-state lasers, such as ruby, Nd:YAG, NdCrYAG, Er:YAG, Nd:YLF, Nd:YVO, Nd:YCaO(BO), Nd:YCOB, titanium sapphire, thulium YAG, ytterbium YAG, YbO, or cerium-doped lasers, and combinations thereof.

[0035] In some embodiments, the light source outputs a particular wavelength, e.g., between 200 nm and 1500 nm, e.g., between 250 nm and 1250 nm, e.g., between 300 nm and 1000 nm, e.g., between 350 nm and 900 nm, e.g., between 400 nm and 800 nm. In certain embodiments, the continuous wave light source emits light having a wavelength of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm, or 850 nm.

[0036] The flow stream may be illuminated by the light source from any suitable distance, for example, from a distance of 0.001 mm or more, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, for example, 100 mm or more. Additionally, illumination of the flow stream may be at any suitable angle, for example, from 10° to 90°, for example, from 15° to 85°, for example, from 20° to 80°, for example, from 25° to 75°, for example, from 30° to 60°, for example, at an angle of 90°.

[0037] In some embodiments, the method further conditions the light from the flow stream before detecting the light. For example, the light may pass through one or more lenses, mirrors, pinholes, slits, gratings, optical refractors, and any combination thereof. In some cases, the collected light passes through one or more focusing lenses, for example, to reduce the light profile. In other cases, the light emitted from the sample passes through one or more collimators to reduce the divergence of the light beam.

[0038] In certain embodiments, the methods irradiate the flowstream with two or more beams of frequency-shifted light. A light beam generator having a laser and an acousto-optical device for frequency-shifting the laser light may be used. In these embodiments, the methods irradiate the acousto-optical device using a laser. Depending on the desired wavelength of light generated in the output laser beam (e.g., for use in irradiating samples in the flowstream), the laser may have a particular wavelength within a range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. The acousto-optical device may be irradiated with one or more lasers, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers. The lasers may include any combination of laser types. For example, in some embodiments, the methods irradiate the acousto-optical device using an array of lasers, e.g., an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0039] When two or more lasers are used, the lasers may be used to illuminate the acousto-optic device simultaneously, sequentially, or a combination thereof. For example, each of the lasers may be used to illuminate the acousto-optic device simultaneously. In other embodiments, each of the lasers may be used to illuminate the acousto-optic device sequentially. When two or more lasers are used to illuminate the acousto-optic device sequentially, the time for which each laser illuminates the acousto-optic device may independently be 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 30 microseconds or more, e.g., 60 microseconds or more. For example, the method may include irradiating the acousto-optic device with a laser for a duration in the range of 0.001 microseconds to 100 microseconds, e.g., 0.01 microseconds to 75 microseconds, e.g., 0.1 microseconds to 50 microseconds, e.g., 1 microsecond to 25 microseconds, e.g., 5 microseconds to 10 microseconds. In embodiments in which the acousto-optic device is illuminated sequentially with two or more lasers, the duration for which the acousto-optic device is illuminated by each laser may be the same or different.

[0040] In an embodiment, a method applies high frequency drive signals to an acousto-optic device to generate angularly deflected laser beams. Two or more high frequency drive signals, such as three or more high frequency drive signals, for example four or more high frequency drive signals, for example five or more high frequency drive signals, for example six or more high frequency drive signals, for example seven or more high frequency drive signals, for example eight or more high frequency drive signals, for example nine or more high frequency drive signals, for example ten or more high frequency drive signals, for example fifteen or more high frequency drive signals, for example twenty-five or more high frequency drive signals, for example fifty or more high frequency drive signals, for example one hundred or more high frequency drive signals may be applied to the acousto-optic device to generate an output laser beam comprising a desired number of angularly deflected laser beams.

[0041] The angularly deflected laser beams generated by the high frequency drive signals each have an intensity based on the amplitude of the applied high frequency drive signal. In some embodiments, the methods apply high frequency drive signals having amplitudes sufficient to generate angularly deflected laser beams of a desired intensity. In some cases, the applied high frequency drive signals independently each have an amplitude within a range of about 0.001 V to about 500 V, e.g., about 0.005 V to about 400 V, e.g., about 0.01 V to about 300 V, e.g., about 0.05 V to about 200 V, e.g., about 0.1 V to about 100 V, e.g., about 0.5 V to about 75 V, e.g., about 1 V to about 50 V, e.g., about 2 V to about 40 V, e.g., about 3 V to about 30 V, or e.g., about 5 V to about 25 V. The applied high frequency drive signal, in some embodiments, has a frequency within the range of about 0.001 MHz to about 500 MHz, for example, about 0.005 MHz to about 400 MHz, for example, about 0.01 MHz to about 300 MHz, for example, about 0.05 MHz to about 200 MHz, for example, about 0.1 MHz to about 100 MHz, for example, about 0.5 MHz to about 90 MHz, for example, about 1 MHz to about 75 MHz, for example, about 2 MHz to about 70 MHz, for example, about 3 MHz to about 65 MHz, for example, about 4 MHz to about 60 MHz, for example, about 5 MHz to about 50 MHz.

[0042] In these embodiments, the angularly deflected laser beams of the output laser beam are spatially separated. Depending on the applied high frequency drive signal and the desired illumination profile of the output laser beam, the angularly deflected laser beams may be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more, such as 5000 μm or more. In some embodiments, the angularly deflected laser beams overlap, for example, with adjacent angularly deflected laser beams along the horizontal axis of the output laser beam. The overlap of adjacent angularly deflected laser beams (e.g., overlap of beam spots) may be 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more.

[0043] In some cases, multiple beams of frequency-shifted light are irradiated onto the flow stream, as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, and U.S. Patent Application Publication No. Cells in a flow stream are imaged by fluorescence imaging using radio frequency tag emission (FIRE) to generate a frequency-encoded image, as described in U.S. Patent Application Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894, the disclosures of which are incorporated herein by reference.

[0044] In some cases, the core stream is irradiated with an elliptical beam spot having a major axis and a minor axis, e.g., the major axis of the beam spot is perpendicular to the horizontal axis of the core stream. In some embodiments, the flow stream is irradiated with a light source (as described in more detail below) having a Gaussian beam power density profile across the horizontal axis of the core stream. In some cases, the laser is configured to irradiate the core stream such that the irradiated power density across the horizontal axis of the flow stream is higher at or near the center of the core stream and lower at or near the edge of the core stream. In some cases, the intensity profile of the output laser beam has an intensity that increases from the edge to the center of the output laser beam along the horizontal axis (i.e., the minor axis of the elliptical beam spot). In these cases, the intensity of the output laser beam at the edge of the output laser beam may be in the range of 0.1% to about 99% of the intensity of the output laser beam at the center of the output laser beam along the horizontal axis, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., about 2% to about 85%, e.g., about 3% to about 80%, e.g., about 4% to about 75%, e.g., about 5% to about 70%, e.g., about 6% to about 65%, e.g., about 7% to about 60%, e.g., about 8% to about 55%; e.g., the intensity of the output laser beam at the edge of the output laser beam may be in the range of about 10% to about 50% of the intensity of the output laser beam at the center of the output laser beam along the horizontal axis. In still other cases, the intensity profile of the output laser beam has a Gaussian distribution along the horizontal axis of the output laser beam. In still other cases, the intensity profile of the output laser beam has a super-Gaussian distribution along the horizontal axis of the output laser beam.

[0045] In some embodiments, the laser has optical conditioning components (e.g., one or more lenses) configured to rotate the output laser beam 90° so that the major axis of the elliptical beam spot is parallel to the longitudinal axis of the flow stream (i.e., irradiating across the horizontal axis of the flow stream along the minor axis of the elliptical beam spot). In some cases, the optical conditioning components are configured to produce the output laser beam such that the irradiating power density across the horizontal axis of the flow stream is higher at or near the center of the core stream and lower at or near the edge of the core stream. In some cases, the light source has optical conditioning components that provide an intensity profile of the output laser beam that increases in intensity from the edge to the center of the output laser beam along the horizontal axis (i.e., the minor axis of the elliptical beam spot). In these cases, the optical adjustment component generates an output laser beam with an intensity at its edge within a range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., about 2% to about 85%, e.g., about 3% to about 80%, e.g., about 4% to about 75%, e.g., about 5% to about 70%, e.g., about 6% to about 65%, e.g., about 7% to about 60%, e.g., about 8% to about 55%, of the intensity of the output laser beam at the center of the output laser beam along the horizontal axis; e.g., the intensity of the output laser beam at the edge of the output laser beam is within a range of about 10% to about 50% of the intensity of the output laser beam at the center of the output laser beam along the horizontal axis. In still other cases, the optical adjustment component generates an output laser beam with an intensity profile along the horizontal axis that has a Gaussian distribution. In still other cases, the optical adjustment component generates an output laser beam with an intensity profile along the horizontal axis that has a super-Gaussian distribution.

[0046] Light from the illuminated flowstream is detected by a light detection system having a photodetector. The photodetector may be any convenient light detection protocol, including, but not limited to, optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes (APD), quad-segment photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, among other photodetectors. In one embodiment, the photodetector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10 cm 2 , e.g., 0.05 cm 2 ~9cm 2 , e.g. 0.1cm 2 ~8cm 2 , e.g. 0.5cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .times. ...

[0047] The photodetector may measure light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., twenty-five or more different wavelengths, e.g., fifty or more different wavelengths, e.g., one hundred or more different wavelengths, e.g., two or more different wavelengths, e.g., two or more different wavelengths, e.g., three or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., two ... For example, depending on the number of different fluorescence photodetectors in the subject optical detection system, fluorescence may be detected at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, or any combination thereof. In certain embodiments, the methods detect light at wavelengths corresponding to the fluorescence peak wavelengths of certain fluorescent dyes present in the sample.

[0048] Light may be measured continuously or at discrete intervals. In some cases, the detector of interest is configured to measure light continuously. In other cases, the detector of interest is configured to measure light at discrete intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, e.g., every 1000 milliseconds, or other intervals. Measurements of light from the flow stream may be taken one or more times, e.g., two or more times, e.g., three or more times, e.g., five or more times, e.g., ten or more times, during each discrete time interval. In some embodiments, light from the flow stream is measured by the photodetector two or more times, and the data is optionally averaged.

[0049] Each optical detector may be positioned at any suitable distance from the flow stream so long as an available optical signal is detectable. For example, detectors of the subject systems may be positioned at 1 mm or more from the flow stream, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, such as 50 mm or more, such as 100 mm or more, such as 150 mm or more, such as 250 mm or more, such as 500 mm or more from the flow stream. Detectors may be positioned at any angle from the flow stream. For example, detectors may be positioned at an angle between 10° and 90°, such as between 15° and 85°, such as between 20° and 80°, such as between 25° and 75°, such as between 30° and 60°, relative to the vertical axis of the flow stream. In some cases, one or more detectors are positioned at an angle between 30° and 60° relative to the vertical axis of the flow stream.

[0050] In embodiments, the light detection system is configured to detect forward scattered light, side scattered light, emitted light, transmitted light, or a combination thereof. In certain embodiments, the light signal from the illuminated flow stream may be detected by one or more detectors configured as forward scatter detectors. In these embodiments, the forward scatter detector is positioned on the opposite side of the flow stream from the light source and is configured to collect and detect forward-propagating (e.g., scattered) light. In some embodiments, light from the illuminated sample in the flow stream is detected in one or more fluorescence detector channels. In some embodiments, light from the illuminated flow stream is detected in one or more scattered light detector channels.

[0051] In some embodiments, a method determines a spectral emission intensity profile of particles of a composition propagating within a core stream of a flow stream. In some embodiments, the flow stream includes a core stream and a sheath flow stream that flows laminarly along the core stream. In some embodiments, the composition includes particles having an emission power density sensitive compound that emits light at an intensity determined by the emission power density of a light source incident on the particles. In other words, irradiated particles of the composition exhibit a change in properties based on the intensity of illumination by the light source. In some cases, irradiated particles exhibit a change in emission intensity based on the intensity of illumination by the light source incident on the particles. In some embodiments, irradiated particles exhibit an increase in emission intensity as the emission power density of the light source incident on the particles increases. In some embodiments, irradiated particles exhibit a decrease in emission intensity as the emission power density of the light source incident on the particles increases.

[0052] In some cases, the compositions described herein are biological samples, and the particles are cells. The term "biological sample" is used in its conventional sense to refer to a whole organism, a plant, a fungus, or a subset of animal tissues, cells, or components, as may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, umbilical cord blood, urine, vaginal fluid, or semen. Thus, "biological sample" refers to both a naturally occurring organism or a subset of its tissues, and homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. Biological samples may be any type of organismal tissue, including both healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or a derivative thereof, e.g., plasma, tears, urine, semen, etc., and in some cases, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger stick (which may or may not be combined with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0053] In some embodiments, the source of the composition is a "mammal" or "mammalian," which terms are used broadly to describe organisms belonging to the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The methods may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., newborn, infant, juvenile, adolescent, adult), and in some embodiments, the human subject is a juvenile, adolescent, or adult. While the present invention may be applied to samples from human subjects, it should be understood that the methods may also be performed on samples from other animal subjects (i.e., "non-human subjects"), including, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0054] In embodiments, each irradiance power density sensitive compound is stably bound to the particle. By stably bound, it is meant that the irradiance power density sensitive compound does not easily separate from the particle and come into contact with a liquid medium, such as an aqueous medium. In some embodiments, one or more of the irradiance power density sensitive compounds are covalently coupled to the particle. In other embodiments, one or more of the irradiance power density sensitive compounds are physically bound (i.e., non-covalently bound) to the particle. In other embodiments, one or more irradiance power density sensitive compounds are covalently coupled to the particle and one or more irradiance power density sensitive compounds are physically bound to the particle.

[0055] In some embodiments, the irradiation power density sensitive compound is bound to particles in the composition. In some cases, the irradiation power density sensitive compound is bound to particles in the composition by one or more hydrogen bonds, dipole-dipole interactions, or ionic bonds. In some cases, the irradiation power density sensitive compound is covalently bound to particles in the composition. In some cases, the irradiation power density sensitive compound is directly covalently bound to particles. In some cases, the irradiation power density sensitive compound is covalently bound to particles via a linker. The linker may be any convenient covalent bonding protocol, such as a zero-length crosslinker, a homobifunctional linker, a heterobifunctional linker, or a trifunctional crosslinker. In some cases, the linker is a cleavable linker. In some cases, the linker is a non-cleavable linker. The linker may include one or more functional groups, such as an amide, alkylamine, carbamate, carbonate, thiol ether, alkyl, cycloalkyl, or aryl moiety, as desired. In some embodiments, the linker includes a carbamate moiety. In some cases, the linker comprises a chemoselective functional group for conjugation, e.g., to a comonomer or biomolecule. In some embodiments, the linker is an amine-containing linker. In some cases, the linker is an N-hydroxysuccinimide (NHS) ester with one or more maleimide groups that allow for conjugation with sulfhydryl groups.In certain embodiments, the irradiance power density sensitive compound is a compound as described in U.S. Pat. No. 11,333,666, U.S. Pat. Appl. Pub. No. 2004 / 0142344, U.S. Pat. Appl. Pub. No. 2008 / 0293164, U.S. Pat. Appl. Pub. No. 2008 / 0064042, U.S. Pat. Appl. Pub. No. 2010 / 0136702, U.S. Pat. Appl. Pub. No. 2011 / 0256549, U.S. Pat. Appl. Pub. No. 2011 / 0257374, U.S. Pat. Appl. Pub. No. 2012 / 0028828, U.S. Pat. Appl. Pub. No. 2013 / 0136702, U.S. Pat. Appl. Pub. No. 2014 / 014234 ... and the linker is covalently attached to the particle in the composition via a linker as described in U.S. Patent Application Publication Nos. 12 / 0252986, 2013 / 0190193, 2016 / 0264737, 2016 / 0266131, 2018 / 0231530, 2018 / 0009990, 2018 / 0009989, 2018 / 0163054, and 2022 / 0089877.

[0056] In some embodiments, the irradiation power density sensitive compound is part of a labeled specific binding member. The labeled specific binding member is a conjugate of an irradiation power density sensitive compound (e.g., as described herein) and a specific binding member. Any of the irradiation power density sensitive compounds described herein may be conjugated to a specific binding member. The specific binding member and the irradiation power density sensitive compound may be conjugated (e.g., covalently bonded) to each other via any linker at any convenient position on the two molecules. In some embodiments, the labeled specific binding member is aggregation-resistant. As used herein, "aggregation-resistant" means that the labelled specific binding member is capable of forming a homogeneous aqueous composition without forming a flocculation precipitate at a concentration of 1 mg / ml or greater in the aqueous buffer of interest, for example, the labelled specific binding member is capable of forming a homogeneous aqueous composition without forming a flocculation precipitate at a concentration of 2 mg / ml or greater, 3 mg / ml or greater, 4 mg / ml or greater, 5 mg / ml or greater, 6 mg / ml or greater, 7 mg / ml or greater, 8 mg / ml or greater, 9 mg / ml or greater, 10 mg / ml or greater, or greater.

[0057] As used herein, the term "specific binding member" refers to one member of a pair of molecules that have binding specificity for one another. One member of the pair of molecules may have a surface or cavity region that specifically binds to a surface or cavity region of the other member of the pair of molecules. Thus, the members of the pair have the property of specifically binding to one another to form a binding complex. In some embodiments, the affinity between the specific binding members in the binding complex is greater than or equal to 10. -6 M or less, e.g. 10 -7 M or less, e.g. 10 -8 M or less, e.g. 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, e.g. 10 -15 K below M dIn some embodiments, a specific binding member specifically binds with high avidity. High avidity means that the binding member specifically binds with a dissociation constant greater than 10×10 -9 M or less, e.g., 1×10 -9 M or less, 3×10 -10 M or less, 1×10 -10 M or less, 3×10 -11 M or less, 1×10 -11 M or less, 3×10 -12 M or less, or 1 x 10 -12 Apparent K below M d This means that the antibody specifically binds with an apparent affinity characterized by:

[0058] A specific binding member can be proteinaceous. As used herein, the term "proteinaceous" refers to a moiety consisting of amino acid residues. A proteinaceous moiety can be a polypeptide. In some cases, a proteinaceous specific binding member is an antibody. In some embodiments, a proteinaceous specific binding member is an antibody fragment, e.g., a binding fragment of an antibody that specifically binds to a polymeric dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides substantially encoded by all or part of a recognized immunoglobulin gene. For example, the recognized human immunoglobulin genes include the kappa (k), lambda (l), and heavy chain gene loci, which in turn contain numerous variable region genes as well as the constant region genes μ(u), δ(d), γ(g), σ(e), and α(a), which encode the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. The variable region of an immunoglobulin light or heavy chain consists of a "framework" region (FR) interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs has been precisely defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., US Department of Health and Human Services, (1991)). All antibody amino acid sequences described herein are numbered according to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope on an antigen. The term "antibody" is meant to include full-length antibodies and may refer to natural antibodies from any organism, artificial antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes as further defined below.

[0059] Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, ScFv, or other antigen-binding subsequences of antibodies, either produced by modification of whole antibodies or synthesized de novo using recombinant DNA technology. The antibodies may be monoclonal or polyclonal and may have other specific activities on cells (e.g., antagonist, agonist, neutralizing, inhibitory, or stimulatory). It is understood that the antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen-binding function or other antibody functions.

[0060] In some embodiments, the specific binding member is a Fab fragment, a F(ab')2 fragment, an ScFv, a diabody, or a triabody. In some embodiments, the specific binding member is an antibody. Optionally, the specific binding member is a murine antibody or binding fragment thereof. Optionally, the specific binding member is a recombinant antibody or binding fragment thereof.

[0061] In some embodiments, the radiation power density-sensitive compound is bound to the particles of the composition via a specific binding member. For example, the radiation power density-sensitive compound may be covalently bound to a proteinaceous binding member, such as an antibody or antibody fragment, which is bound to the particles of the composition. In some embodiments, the radiation power density-sensitive compound is covalently bound (e.g., directly or via a linker) to a binding member that has binding specificity for a receptor on the surface of the particle, such as a surface-bound protein or antigen.

[0062] The irradiance power density sensitive compound may be any compound that exhibits a change in a characteristic, such as a characteristic of an emission spectrum profile or intensity, in response to a change in the power density of irradiation by a light source. In some embodiments, the irradiance power density sensitive compound is an upconversion nanoparticle (UCNP). In some cases, the nanoparticle is a lanthanide-doped particle. In some cases, the nanoparticle is a europium-doped nanoparticle or a thulium-doped nanoparticle, such as NaYF4:Yb, Er nanoparticle, or NaYF4:YbTm nanoparticle.

[0063] In some embodiments, the irradiated particles in the composition have a spectral radiation intensity profile that varies with the power density of the light source. In some embodiments, the irradiated particles in the composition have two or more radiation peaks in response to irradiation by the light source, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more radiation peaks. In some cases, one or more peaks in the spectral profile exhibit a change in intensity with the power density of the light source. In some cases, the intensity of each radiation peak depends on the irradiation power density of the light source. In one example, one or more of the radiation peaks have a first radiation intensity at a first irradiation power density of the light source and a second radiation intensity at a second irradiation power density of the light source. In some cases, the radiation peaks have a higher radiation intensity at a higher irradiation power density of the light source and a lower radiation intensity at a lower irradiation power density. In some cases, one or more of the radiation peaks have a first radiation intensity when the particles propagate within the core stream at or near the center of the irradiation by the light source and a second radiation intensity when the particles propagate within the core stream at or near the edge of the irradiation by the light source. In these examples, when the center of the core stream is aligned with the center of illumination by the light source (e.g., the center of the beam spot is at the center of the core stream), one or more of the emission peaks have a first emission intensity when the particle propagates at or near the center of the core stream and a second emission intensity when the particle propagates at or near the edge of the core stream.

[0064] In some embodiments, the method determines location information of the irradiated particle of the composition based on the radiation intensity of one or more radiation peaks. In one example, if the radiation peak indicates a high radiation intensity, the particle is determined to be propagating at or near the center of the core stream, and if the radiation peak indicates a low radiation intensity, the particle is determined to be propagating at or near the edge of the core stream. In another example, if the radiation peak indicates a low radiation intensity, the particle is determined to be propagating at or near the center of the core stream, and if the radiation peak indicates a high radiation intensity, the particle is determined to be propagating at or near the edge of the core stream.

[0065] In some embodiments, the irradiated particles of the composition have a first radiation peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density, and a second radiation peak having the same intensity at the first irradiation power density and the second irradiation power density. In these embodiments, the intensity of the first radiation peak is determined by the irradiation power density of the light source, while the intensity of the second radiation peak remains the same. In one example, the first radiation peak has a higher radiation intensity at a higher irradiation power density of the light source and a lower radiation intensity at a lower irradiation power density. The second radiation peak has the same radiation intensity at higher and lower irradiation power densities of the light source. In some cases, the first radiation peak has a first radiation intensity when the particles propagate within the core stream at or near the center of the irradiation by the light source and a second radiation intensity when the particles propagate within the core stream at or near the edge of the irradiation by the light source. The second emission peak has the same emission intensity when the particle propagates within the core stream at or near the center of illumination by the light source and when the particle propagates within the core stream at or near the edge of illumination by the light source. In these examples, if the center of the core stream is aligned with the center of illumination by the light source (e.g., the center of the beam spot is at the center of the core stream), the first emission peak has a first emission intensity when the particle propagates within the core stream at or near the center and a second emission intensity when the particle propagates within the core stream at or near the edge. The second emission peak has the same emission intensity when the particle propagates within the core stream at or near the center and when the particle propagates within the core stream at or near the edge.

[0066] In some embodiments, the method determines location information of the irradiated particle of the composition based on the radiation intensities of the first radiation peak and the second radiation peak. In one example, if the first radiation peak exhibits a high radiation intensity, the particle is determined to be propagating at or near the center of the core stream, and if the first radiation peak exhibits a low radiation intensity, the particle is determined to be propagating at or near the edge of the core stream. In another example, if the first radiation peak exhibits a low radiation intensity, the particle is determined to be propagating at or near the center of the core stream, and if the first radiation peak exhibits a high radiation intensity, the particle is determined to be propagating at or near the edge of the core stream.

[0067] In other cases, the method determines the location of the particle based on the ratio of the intensity of the first radiation peak to the intensity of the second radiation peak. In some cases, the ratio of the intensity of the first radiation peak to the intensity of the second radiation peak is in the range of 1:100 to 100:1, such as 1:75 to 75:1, for example 1:50 to 50:1, for example 1:25 to 25:1, for example 1:10 to 10:1, such as 1:9 to 9:1, for example 1:8 to 8:1, for example 1:7 to 7:1, for example 1:6 to 6:1, for example 1:5 to 5:1, for example 1:4 to 4:1. In some embodiments, a particle is determined to be propagating at or near the center of the core stream when the ratio of the intensity of the first emission peak to the intensity of the second emission peak is within a range of 0.7:1 to 1:0.7, e.g., 0.8:1 to 1:0.8, e.g., 0.9:1 to 1:0.9, e.g., 0.95:1 to 1:0.95. In some cases, a particle is determined to be propagating at or near the center of the core stream when the ratio of the intensity of the first emission peak to the intensity of the second emission peak is approximately 1:1 (i.e., a particle is propagating at the center of the core stream (or at the center of illumination by the light source) when the first emission peak and the second emission peak have approximately the same emission intensity). In some embodiments, the greater the ratio of the intensity of the first emission peak to the intensity of the second emission peak, the greater the distance from the center of illumination (or the center of the core stream when the light source and core stream are aligned) that the particle is propagating.

[0068] FIG. 1 illustrates a process for determining particle positional information of a composition within a core stream according to one embodiment. A light source exhibiting an elliptical beam spot is used to irradiate particles (e.g., cells) in a flow stream 100 that are bound (e.g., covalently bound) to an irradiance power density-sensitive compound. The minor axis of the elliptical beam spot is perpendicular to the longitudinal axis of the flow stream (i.e., the major axis of the beam spot is parallel to the longitudinal axis of the flow stream). In an embodiment, a beam spot that crosses the horizontal axis exhibits an irradiance power density in which the intensity at the center is higher than the intensity at or near the edge of the beam spot. As shown in FIG. 1, an output laser beam having a Gaussian beam profile 110 irradiates the flow stream. A light source having a Gaussian beam profile 110 exhibits an irradiance power density that is higher at or near the center of the irradiance and lower away from the center. The irradiance power density of a light source having a Gaussian beam profile 110 is lowest at or near the edge of the irradiance. Particle 100a exhibits a spectral emission profile with two emission peaks at 400 nm and 800 nm. Particle 100a propagating along the left edge 101 of the light source illumination exhibits spectral profile 101a in which the emission peak at 400 nm is greater than the emission peak at 800 nm (i.e., the ratio of the emission intensity at 400 nm to the emission intensity at 800 nm is greater than 1). Particle 100a propagating along the center 102 of the light source illumination exhibits spectral profile 102a in which the emission peak at 400 nm is approximately the same as the emission peak at 800 nm (i.e., the ratio of the emission intensity at 400 nm to the emission intensity at 800 nm is approximately 1). Particle 100a propagating along the right edge 103 of the light source illumination exhibits spectral profile 103a in which the emission peak at 400 nm is greater than the emission peak at 800 nm (i.e., the ratio of the emission intensity at 400 nm to the emission intensity at 800 nm is greater than 1). Based on the intensity difference between the 400 nm and 800 nm emission peaks, the position of the particle can be determined. When a light source with a Gaussian beam profile 110 is aligned with the core stream of the flow stream 100, the position of the particle within the core stream can be determined.If the 400 nm and 800 nm emission peaks have approximately the same intensity, the particle is determined to be propagating at or near the center of the core stream. If the 400 nm and 800 nm emission peaks have different intensities, the particle is determined to be propagating at or near the edge of the core stream. In some cases, the distance from the center of the core stream is determined based on the intensity ratio of the 400 nm and 800 nm emission peaks.

[0069] In some embodiments, one or more particle population clusters are generated based on the determined locations of the irradiated particles within the core stream. In some cases, the particles are classified as being irradiated at or near the center of the core stream based on the determined spectral radiation intensity profile. In other cases, the particles are classified as being irradiated at or near the edge of the core stream based on the determined spectral radiation intensity profile. In some cases, data signals from particles classified as being irradiated at or near the center of the core stream are clustered together. In some cases, data signals from particles classified as being irradiated at or near the edge of the core stream are clustered together. In some embodiments, data signals from particles of the sample composition classified as being irradiated at or near the edge of the core stream are discarded.

[0070] FIG. 2 is a flowchart for characterizing positional information for particles of a composition according to one embodiment. In step 201, particles bound to an irradiance power density-sensitive compound of the composition (e.g., as shown in FIG. 1) are illuminated in the core stream using a light source. In step 202, light from the illuminated particles is detected using a light detection system, and in step 203, data signals for the particles of the composition are generated. In step 204, the generated data signals for the particles of the composition are used to determine positional information for the particles of the sample composition. Based on the determined positions of the particles in the core stream, data signals from the particles of the composition may be clustered together. In some cases, in step 205a, particles propagating at or near the center of the core stream are clustered together, while in step 205b, particles propagating at or near the edge of the core stream are clustered together. In one embodiment, data signals from particles of the composition determined to be propagating at or near the edge of the core stream are excluded from further analysis (step 210).

[0071] In certain embodiments, the particles of the composition further comprise one or more stably bound fluorophores (e.g., by covalent bonds or via specific binding members, as described above). In some embodiments, each particle comprises two or more different types of fluorophores. Any two fluorophores are considered to be different and distinct fluorophores if they differ from each other by one or more of their molecular formulas, excitation maxima, and emission maxima. Thus, different, i.e., distinct fluorophores may differ from each other in chemical composition or in one or more properties of the fluorophores. For example, different fluorophores may differ from each other by at least one of their excitation maxima and emission maxima. In some cases, different fluorophores differ from each other by their respective excitation maxima. In some cases, different fluorophores differ from each other by their respective emission maxima. In some cases, different fluorophores differ from each other by both their respective excitation maxima and emission maxima. Thus, in embodiments comprising a first fluorophore and a second fluorophore, the first fluorophore and the second fluorophore may differ from each other by at least one of their excitation maxima and emission maxima. For example, the first and second fluorophores may differ from each other by an excitation maximum, by an emission maximum, or by both an excitation maximum and an emission maximum. A given set of fluorophores may be considered distinct if they differ from each other in terms of their excitation or emission maxima, where the magnitude of such difference is optionally 5 nm or more, such as 10 nm or more, for example 15 nm or more, and optionally the magnitude of the difference is in the range of 5 to 400 nm, such as 10 to 200 nm, for example 15 to 100 nm, for example 25 to 50 nm.

[0072] Fluorophores of interest in certain embodiments have excitation maxima in the range of 100 nm to 800 nm, such as 150 nm to 750 nm, for example 200 nm to 700 nm, for example 250 nm to 650 nm, for example 300 nm to 600 nm, for example 400 nm to 500 nm. Fluorophores of interest in certain embodiments have emission maxima in the range of 400 nm to 1000 nm, for example 450 nm to 950 nm, for example 500 nm to 900 nm, for example 550 nm to 850 nm, for example 600 nm to 800 nm. In some cases, the fluorophore is a luminescent dye, e.g., a fluorescent dye having a peak emission wavelength of 200 nm or more, e.g., 250 nm or more, e.g., 300 nm or more, e.g., 350 nm or more, e.g., 400 nm or more, e.g., 450 nm or more, e.g., 500 nm or more, e.g., 550 nm or more, e.g., 600 nm or more, e.g., 650 nm or more, e.g., 700 nm or more, e.g., 750 nm or more, e.g., 800 nm or more, e.g., 850 nm or more, e.g., 900 nm or more, e.g., 950 nm or more, e.g., 1000 nm or more, e.g., 1050 nm or more. For example, the fluorophore may be a fluorescent dye having a peak emission wavelength in the range of 200 nm to 1200 nm, e.g., 300 nm to 1100 nm, e.g., 400 nm to 1000 nm, e.g., 500 nm to 900 nm, or a fluorescent dye having a peak emission wavelength in the range of 600 nm to 800 nm. In certain embodiments, multispectral particles of interest provide stable excitation with lasers emitting at wavelengths of 349 nm or about 349 nm (UV laser), 488 nm or about 488 nm (blue laser), 532 nm or about 532 nm (Nd:YAG solid-state laser), 640 nm or about 640 nm (red laser), and 405 nm or about 405 nm (violet laser). In some cases, multispectral particles of interest provide stable excitation with light sources across the entire spectral detection band, e.g., from 350 nm to 850 nm.

[0073] In some cases, each particle includes a fluorophore that emits fluorescence in response to illumination by a light source. In some embodiments, fluorophores of interest may include dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.), such as, but not limited to, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurodine dyes, safranine dyes, indamines, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronine dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, and combinations of two or more of the foregoing dyes (e.g., tandem dyes), polymeric dyes having one or more monomeric dye units, and mixtures of two or more of the foregoing dyes. Many dyes are commercially available from a variety of sources, such as Molecular Probes (Eugene, OR), Dyomics GmbH (Jena, Germany), Sigma-Aldrich (St. Louis, MO), Sirigen, Inc. (Santa Barbara, CA), and Exciton (Dayton, OH). For example, fluorophores include 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and its derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin, phycoerythrin, peridinin - chlorophyll proteins, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; brilliant yellow;Coumarin and its derivatives, such as 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine and its derivatives, such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7; 4',6-diamidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine; Amine pentaacetate; 4,4'-Diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); Eosin and its derivatives, such as eosin and eosin isothiocyanate; Erythrosin, and erythrosin B, erythrosin isothiocyanate derivatives; ethidium; fluorescein and its derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC ​​(XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); and coral reef-derived fluorescent protein RCFP; Lissamine™; Lissamine rhodamine, Lucifer Yellow; Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Oregon Green; phenol red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A);Rhodamine, and 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine Lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), ... These dyes may include derivatives such as methylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), and the like; riboflavin; rosolic acid and terbium chelate derivatives; xanthenes; dye-conjugated polymers (i.e., dyes bound to polymers) such as fluorescein isothiocyanate dextran, and dyes in which two or more dyes are combined (e.g., tandem dyes), polymeric dyes having one or more monomeric dye units, and mixtures of two or more of the foregoing dyes, or combinations thereof;

[0074] In some cases, the fluorophore is a polymeric dye. In some example methods, the polymeric dye comprises a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure, including a backbone of alternating unsaturated bonds (e.g., double and / or triple bonds) and saturated bonds (e.g., single bonds), allowing π electrons to move from one bond to another. Therefore, the conjugated backbone may impart an extended linear structure to the polymeric dye, with restricted bond angles between the repeating units of the polymer. For example, proteins and nucleic acids are also macromolecules, but in some cases, they do not form extended rod structures but rather fold into higher-order three-dimensional shapes. In addition, CPs may form "rigid rod"-like polymer backbones, exhibiting restricted twist (e.g., torsion) angles between the repeating monomer units along the polymer backbone chain. In some cases, the polymeric dye comprises a CP with a rigid rod-like structure. The structural characteristics of the polymeric dye may affect the fluorescent properties of the molecule.

[0075] Polymeric dyes of interest include those disclosed in U.S. Patent Application Publication Nos. 2004 / 0142344, 2008 / 0293164, 2008 / 0064042, 2010 / 0136702, 2011 / 0256549, 2011 / 0257374, 2012 / 0028828, 2013 / 0136702, 2014 / 0142344, 2015 / 0142344, 2016 / 0142344, 2017 / 01423164, 2018 / 0293164, 2018 / 0064042, 2010 / 0136702, 2011 / 0256549, 2011 / 0257374, 2012 / 0028828, 2013 / 0136702, 2014 / 0136702, 2015 / 0136702, 2016 / 0136702, 2017 / 0136549 ... Nos. 2013 / 0190193, 2016 / 0264737, 2016 / 0266131, 2018 / 0231530, 2018 / 0009990, 2018 / 0009989, and 2018 / 0163054, the entire disclosures of which are incorporated herein by reference. et al., J. Am. Chem. Soc., 2001, 123 (26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010,39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133 (32), pp 12600-12607.

[0076] The polymer dye may have one or more desirable spectral properties, such as a specific absorption maximum wavelength, a specific emission maximum wavelength, an extinction coefficient, a quantum yield, etc. (See, for example, Chattopadhyay et al., "Brilliant violet fluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments," Cytometry Part A, 81A(6), 456-466, 2012). In some embodiments, the polymer dye has an absorption curve in the range of 280 nm to 475 nm. In certain embodiments, the polymer dye has an absorption maximum (excitation maximum) in the range of 280 nm to 475 nm. In some embodiments, the polymer dye absorbs incident light having a wavelength in the range of 280 nm to 475 nm. In some embodiments, the polymeric dye has an emission maximum wavelength within the range of 400 nm to 850 nm, e.g., 415 nm to 800 nm. Specific examples of emission maxima of interest include, but are not limited to, 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm. In some embodiments, the polymeric dye has an emission maximum wavelength within a range selected from the group consisting of 410 nm to 430 nm, 500 nm to 520 nm, 560 nm to 580 nm, 590 nm to 610 nm, 640 nm to 660 nm, 700 nm to 720 nm, and 775 nm to 795 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 421 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 510 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 570 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 602 nm. In some cases, the polymeric dye has an emission maximum wavelength of 650 nm. In some cases, the polymeric dye has an emission maximum wavelength of 711 nm. In some cases, the polymeric dye has an emission maximum wavelength of 786 nm. In some cases, the polymeric dye has an emission maximum wavelength of 421 nm ± 5 nm.In some embodiments, the polymeric dye has an emission maximum wavelength of 510 nm ± 5 nm. In some cases, the polymeric dye has an emission maximum wavelength of 570 nm ± 5 nm. In some cases, the polymeric dye has an emission maximum wavelength of 602 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 650 nm ± 5 nm. In some cases, the polymeric dye has an emission maximum wavelength of 711 nm ± 5 nm. In some cases, the polymeric dye has an emission maximum wavelength of 786 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum selected from the group consisting of 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm.

[0077] Specific polymer dyes that may be used include, but are not limited to, BD Horizon Brilliant™ Dyes, such as BD Horizon Brilliant™ Violet Dye (e.g., BV421, BV510, BV605, BV650, BV711, BV786); BD Horizon Brilliant™ Ultraviolet Dye (e.g., BUV395, BUV496, BUV737, BUV805); and BD Horizon Brilliant™ Blue Dye (e.g., BB515) (BD Biosciences, San Jose, CA).

[0078] System configured to determine positional information for particles in a flow stream Aspects of the present disclosure include a system for determining positional information of particles in a flowstream. In some embodiments, the system includes a flow cell configured to propagate a composition including particles in a flowstream, the flowstream including a core stream and a sheath flowstream; a light source for irradiating particles of the composition; a light detection system for detecting light from the irradiated particles; and a processor operatively coupled to a memory, the memory storing instructions that, when executed by the processor, cause the processor to determine the position of the particles in the flowstream based on the detected emitted light from the irradiated particles. In some embodiments, the irradiated particles in the composition are stably bound to an irradiance power density-sensitive compound that emits light at an intensity determined by the irradiance power density of the light source incident on the particles.

[0079] In embodiments, the system includes a light source for illuminating the flow stream. In embodiments, the light source may be any suitable broadband or narrowband light source. Depending on the components in the sample (e.g., cells, beads, non-cellular particles, etc.), the light source may be configured to emit light at various wavelengths within a range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. For example, the light source may include a broadband light source that emits light having a wavelength within a range of 200 nm to 900 nm. In other cases, the light source may include a narrowband light source that emits at a wavelength within a range of 200 nm to 900 nm. For example, the light source may be a narrowband LED (1 nm to 25 nm) that emits light having a wavelength within a range of 200 nm to 900 nm.

[0080] In some embodiments, the light source is a laser. Lasers of interest may include pulsed lasers or continuous wave lasers. For example, the laser may be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, a CO laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser; a metal vapor laser, such as a helium cadmium (HeCd) laser, a helium mercury (HeHg) laser, a helium selenium (HeSe) laser, a helium silver (HeAg) laser, a strontium laser, a neon copper (NeCu) laser, a copper laser, or a gold laser, or a combination thereof; a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO4 laser, Nd:YCa4O(BO3)3 laser, Nd:YCOB laser, Ti:sapphire laser, Thulium YAG laser, Ytterbium YAG laser, Yb2O3 laser or Cerium doped laser and combinations thereof; semiconductor diode laser, optically pumped semiconductor laser (OPSL), or frequency doubled or tripled of any of the above lasers.

[0081] In other embodiments, the light source is a non-laser light source, such as, but not limited to, a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a light emitting diode, such as a broadband LED with a continuous spectrum, a superluminescent diode, a semiconductor light emitting diode, a broadband LED white light source, a multi-LED integrated, etc. In some cases, the non-laser light source is a stabilized fiber coupled broadband light source, a white light source, or any combination thereof, among other light sources.

[0082] In some embodiments, the light source is an optical beam generator configured to generate two or more beams of frequency-shifted light. In some cases, the optical beam generator comprises a laser, a radio frequency generator configured to apply a radio frequency drive signal to an acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous wave laser. For example, the laser of the light beam generator of interest may be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, a CO laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof; a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser; a metal vapor laser, such as a helium cadmium (HeCd) laser, a helium mercury (HeHg) laser, a helium selenium (HeSe) laser, a helium silver (HeAg) laser, a strontium laser, a neon copper (NeCu) laser, a copper laser, or a gold laser, or a combination thereof; a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG The laser may be an Er:YAG laser, an Nd:YLF laser, an Nd:YVO4 laser, an Nd:YCa4O(BO3)3 laser, an Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, an Yb2O3 laser or a cerium doped laser and combinations thereof.

[0083] The acousto-optic device may be any convenient acousto-optic protocol configured to frequency-shift laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device of the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal may be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0084] In an embodiment, the controller is configured to apply high frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams of the output laser beam, for example configured to apply 3 or more high frequency drive signals, for example 4 or more high frequency drive signals, for example 5 or more high frequency drive signals, for example 6 or more high frequency drive signals, for example 7 or more high frequency drive signals, for example 8 or more high frequency drive signals, for example 9 or more high frequency drive signals, for example 10 or more high frequency drive signals, for example 15 or more high frequency drive signals, for example 25 or more high frequency drive signals, for example 50 or more high frequency drive signals, for example configured to apply 100 or more high frequency drive signals.

[0085] In some cases, to generate an intensity profile of the angularly deflected laser beam of the output laser beam, the controller is configured to apply a high frequency drive signal having a varying amplitude within a range, for example, from about 0.001 V to about 500 V, for example, from about 0.005 V to about 400 V, for example, from about 0.01 V to about 300 V, for example, from about 0.05 V to about 200 V, for example, from about 0.1 V to about 100 V, for example, from about 0.5 V to about 75 V, for example, from about 1 V to about 50 V, for example, from about 2 V to about 40 V, for example, from about 3 V to about 30 V, or for example, from about 5 V to about 25 V. The applied high frequency drive signal in some embodiments has a frequency within the range of about 0.001 MHz to about 500 MHz, for example, about 0.005 MHz to about 400 MHz, for example, about 0.01 MHz to about 300 MHz, for example, about 0.05 MHz to about 200 MHz, for example, about 0.1 MHz to about 100 MHz, for example, about 0.5 MHz to about 90 MHz, for example, about 1 MHz to about 75 MHz, for example, about 2 MHz to about 70 MHz, for example, about 3 MHz to about 65 MHz, for example, about 4 MHz to about 60 MHz, for example, about 5 MHz to about 50 MHz.

[0086] In one embodiment, the controller includes a processor operatively coupled to a memory, the memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam including an angularly deflected laser beam having a desired intensity profile. For example, the memory may include instructions for generating two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more angularly deflected laser beams of the same intensity, e.g., the memory may include instructions for generating one hundred or more angularly deflected laser beams of the same intensity. In other embodiments, the memory may include instructions for generating two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more angularly deflected laser beams of different intensities, e.g., the memory may include instructions for generating one hundred or more angularly deflected laser beams of different intensities.

[0087] In some embodiments, the controller includes a processor operatively coupled to a memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam that increases in intensity from the center to the edges of the output laser beam along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam may be in a range of 0.1% to about 99%, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, or may be in a range of about 10% to about 50% of the intensity of the angularly deflected laser beam at the edges of the output laser beam along the horizontal axis. In other embodiments, the controller includes a processor operatively coupled to a memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam that increases in intensity from the edge to the center of the output laser beam along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam may be in a range of 0.1% to about 99%, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, or may be in a range of about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In yet another embodiment, the controller includes a processor operatively coupled to a memory such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam having a Gaussian intensity profile along a horizontal axis.In yet another embodiment, the controller includes a processor to which a memory is operatively coupled such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0088] In embodiments, the optical beam generator of interest may be configured to generate spatially separated angularly deflected laser beams of the output laser beam. Depending on the applied high frequency drive signal and the desired irradiance profile of the output laser beam, the angularly deflected laser beams may be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more, such as 5000 μm or more. In some embodiments, the system is configured to generate angularly deflected laser beams of the output laser beam that overlap, for example, adjacent angularly deflected laser beams along a horizontal axis of the output laser beam. The overlap of adjacent angularly deflected laser beams (e.g., overlap of beam spots) may be 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more.

[0089] In some cases, the light beam generator configured to generate two or more beams of frequency-shifted light comprises a laser excitation module as described in U.S. Pat. Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Application Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0090] The light source may be positioned at any suitable distance from the flow stream, for example, at a distance of 0.001 mm or more, such as 0.005 mm or more, for example 0.01 mm or more, such as 0.05 mm or more, for example 0.1 mm or more, such as 0.5 mm or more, for example 1 mm or more, such as 5 mm or more, for example 10 mm or more, such as 25 mm or more, for example 100 mm or more. Additionally, the light source may illuminate the flow stream at any suitable angle (e.g., relative to the vertical axis of the flow stream), for example, at an angle in the range of 10° to 90°, for example 15° to 85°, for example 20° to 80°, for example 25° to 75°, for example 30° to 60°, for example 90°.

[0091] The light source may be configured to illuminate the composition in the flow stream continuously or at discrete intervals. In some cases, the system includes a light source configured to continuously illuminate the sample, e.g., having a continuous wave laser that continuously illuminates the flow stream at the interrogation point of the flow cytometer. In other cases, systems of interest include a light source configured to illuminate the sample at discrete intervals, e.g., every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, e.g., every 1000 milliseconds, or other intervals. When the light source is configured to illuminate the sample at discrete intervals, the system may include one or more additional components for intermittently illuminating the sample with the light source. For example, the subject systems in these embodiments may include one or more laser beam choppers, manual or computer-controlled beam stops, for blocking and exposing the sample to the light source.

[0092] In some cases, the light source is configured to irradiate the core stream with an elliptical beam spot having a major axis and a minor axis, e.g., the major axis of the beam spot is perpendicular to the horizontal axis of the core stream. In certain embodiments, the flow stream is irradiated with a light source (as described in more detail below) having a Gaussian beam power density profile across the horizontal axis of the core stream. In some cases, the laser is configured to irradiate the core stream such that the irradiated power density across the horizontal axis of the flow stream is higher at or near the center of the core stream and lower at or near the edges of the core stream. In some cases, the intensity profile of the output laser beam has an intensity that increases from the edge to the center of the output laser beam along the horizontal axis (i.e., the minor axis of the elliptical beam spot). In these cases, the intensity of the output laser beam at the edge of the output laser beam may be in the range of 0.1% to about 99% of the intensity of the output at the center of the output laser beam along the horizontal axis, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., about 2% to about 85%, e.g., about 3% to about 80%, e.g., about 4% to about 75%, e.g., about 5% to about 70%, e.g., about 6% to about 65%, e.g., about 7% to about 60%, e.g., about 8% to about 55%; e.g., the intensity of the output laser beam at the edge of the output laser beam may be in the range of about 10% to about 50% of the intensity of the output laser beam at the center of the output laser beam along the horizontal axis. In still other cases, the intensity profile of the output laser beam has a Gaussian distribution along the horizontal axis of the output laser beam. In still other cases, the intensity profile of the output laser beam has a super-Gaussian distribution along the horizontal axis of the output laser beam.

[0093] In some embodiments, the laser has optical conditioning components (e.g., one or more lenses) configured to rotate the output laser beam 90° so that the major axis of the elliptical beam spot is parallel to the longitudinal axis of the flow stream (i.e., irradiating across the horizontal axis of the flow stream along the minor axis of the elliptical beam spot). In some cases, the optical conditioning components are configured to produce the output laser beam such that the irradiating power density across the horizontal axis of the flow stream is higher at or near the center of the core stream and lower at or near the edge of the core stream. In some cases, the light source has optical conditioning components that provide an intensity profile of the output laser beam that has increasing intensity from the edge to the center of the output laser beam along the horizontal axis (i.e., the minor axis of the elliptical beam spot). In these cases, the optical adjustment component generates an output laser beam with an intensity at its edge within a range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., about 2% to about 85%, e.g., about 3% to about 80%, e.g., about 4% to about 75%, e.g., about 5% to about 70%, e.g., about 6% to about 65%, e.g., about 7% to about 60%, e.g., about 8% to about 55%, of the intensity of the output laser beam at the center of the output laser beam along the horizontal axis; e.g., the intensity of the output laser beam at the edge of the output laser beam is within a range of about 10% to about 50% of the intensity of the output laser beam at the center of the output laser beam along the horizontal axis. In still other cases, the optical adjustment component generates an output laser beam with an intensity profile along the horizontal axis that has a Gaussian distribution. In still other cases, the optical adjustment component generates an output laser beam with an intensity profile along the horizontal axis that has a super-Gaussian distribution.

[0094] In some embodiments, the light source comprises multiple lasers. In some cases, the light source comprises multiple lasers, where a first laser is positioned to irradiate the flow stream at a first location, a second laser is positioned to irradiate the flow stream at a second location, a third laser is positioned to irradiate the flow stream at a third location, and a fourth laser is positioned to irradiate the flow stream at a fourth location, the second location being downstream of the first location, the third location being downstream of the second location, and the fourth location being downstream of the third location. In certain embodiments, the first laser is a trigger laser, e.g., the system is configured to trigger after light emitted from a particle irradiated by the second laser is detected by the light detection system. In some cases, the light source includes at least one laser having an elliptical beam spot having a major axis and a minor axis, the major axis of the beam spot configured to illuminate the flow stream such that the major axis of the beam spot is perpendicular to the longitudinal axis of the flow stream, and another laser having an elliptical beam spot having a major axis and a minor axis, the major axis of the beam spot configured to illuminate the flow stream such that the major axis of the beam spot is perpendicular to the horizontal axis of the flow stream.

[0095] The distance between each laser's illumination of the flow stream may vary, with the inter-illumination spacing independently being 0.0001 μm or more, such as 0.0005 μm or more, for example 0.001 μm or more, such as 0.005 μm or more, for example 0.01 μm or more, such as 0.05 μm or more, for example 0.1 μm or more, such as 0.5 μm or more, for example 1 μm or more, such as 2 μm or more, for example 3 μm or more, such as 4 μm or more, for example 5 μm or more, for example 6 μm or more, such as 7 μm or more, for example 8 μm or more, for example 9 μm or more, for example 10 μm or more. In some cases, the lasers of a given light source are configured to illuminate locations on the flow stream that are immediately adjacent to each other (i.e., there is no inter-illumination spacing).

[0096] Depending on the desired wavelength of light to be generated in the output laser beam (e.g., for use in illuminating a sample in a flow stream), each laser may have a particular wavelength within a range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. In certain embodiments, the lasers of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 628 nm laser. In some embodiments, each laser outputs light of a different wavelength. In some cases, the lasers in a system are arranged such that each downstream laser outputs light of a longer wavelength. For example, if a system includes four lasers, the wavelength of light from the second laser is longer than the wavelength of light from the first laser, the wavelength of light from the third laser is longer than the wavelength of light from the second laser, and the wavelength of light from the fourth laser is longer than the wavelength of light from the third laser. In some embodiments, the system includes four lasers, where the wavelength of light from a first laser is 488 nm, the wavelength of light from a second laser is 405 nm, the wavelength of light from a third laser is 561 nm, and the wavelength of light from a fourth laser is 628 nm. In some embodiments, the light from the second laser is configured to have a Gaussian beam power density profile across a horizontal axis of the flow stream. In some embodiments, the second laser has an elliptical beam spot having a major axis and a minor axis. In some embodiments, the second laser is coupled to an optical conditioning element configured to propagate light into the flow stream such that the major axis of the beam spot is perpendicular to the horizontal axis of the flow stream. In some embodiments, the optical conditioning element is configured to rotate the beam spot generated by the second laser by 90 degrees. In some embodiments, the optical conditioning element includes one or more lenses. In some cases, the first, third, and fourth lasers are configured to have major and minor axes and are coupled to optics that transmit light from the first, third, and fourth lasers into the flow stream so that the major axes of their beam spots are perpendicular to the horizontal axis of the flow stream.

[0097] The light source may further include one or more optical conditioning components. The term "optical conditioning" is used herein in its conventional sense to refer to any device capable of changing the spatial width or other illumination characteristics of the illumination from one or more lasers, such as illumination direction, wavelength, beam width, beam intensity, focus, and pulse width. The optical conditioning protocol may be any convenient device that adjusts one or more characteristics of the laser, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof. In some embodiments, the system of interest includes one or more focusing lenses. In one example, the focusing lens may be a demagnifying lens. In another example, the focusing lens is a magnifying lens. In other embodiments, the system of interest includes one or more mirrors. In yet other embodiments, the system of interest includes optical fibers. In some embodiments, the beams of light from each laser are combined by a beam combiner, such as a dichroic mirror beam combiner. In these embodiments, the beam combiner combines the light beams from each laser and transmits the light to the beam shaping component.

[0098] In some embodiments, light from each laser is transmitted to the flow stream via a mirror assembly. In some cases, the mirror assembly may include a first mirror and a second mirror positioned to transmit light from the first mirror to the flow stream. In some embodiments, the second mirror is positioned to transmit light from the first mirror at an angle relative to the first mirror, e.g., between 1° and 90°, e.g., between 5° and 85°, e.g., between 10° and 80°, e.g., between 15° and 75°, e.g., between 20° and 70°, e.g., between 25° and 65°, e.g., between 30° and 60°. In some cases, the second mirror is positioned to transmit light from the first mirror orthogonally. In other embodiments, the second mirror is positioned to propagate light from the first mirror at an angle between 1° and 90°, e.g., between 5° and 85°, e.g., between 10° and 80°, e.g., between 15° and 75°, e.g., between 20° and 70°, e.g., between 25° and 65°, e.g., between 30° and 60°, relative to the laser. In some embodiments, the second mirror is positioned to propagate light orthogonally relative to the laser. In some embodiments, the second mirror is further a beam combiner configured to combine beams of light from two or more lasers. In these embodiments, the second mirror may be a dichroic mirror that selectively passes light of desired wavelengths.

[0099] FIG. 3 illustrates a light source for irradiating a composition containing particles in a flowstream, according to one embodiment. Light source 300 includes laser module 301 and laser module 302. Laser module 301 includes a first laser 305 having a wavelength of 488 nm, a second laser 306 having a wavelength of 405 nm, a third laser 307 having a wavelength of 561 nm, and a fourth laser 308 having a wavelength of 628 nm. A long-wavelength (e.g., 980 nm) laser 302a is output from laser module 302. Laser 302a is configured to irradiate particles 310 in the flowstream with an elliptical beam spot (shown at 304), the major axis of which is perpendicular to the horizontal axis of the flowstream. In some cases, this beam spot configuration provides a Gaussian beam power density profile across the horizontal axis of the flowstream. Laser module 301 is in optical communication with first optical conditioning element 303a, which transmits light from lasers 305, 306, 307, and 308 to the flowstream. In some embodiments, the output laser beams from lasers 305, 306, 307, and 308 are configured so that the major axis of the elliptical beam spot is perpendicular to the longitudinal axis of the flowstream (i.e., perpendicular to the beam spot from laser 302a). Laser module 302 is in optical communication with second optical conditioning element 303b, which is configured to rotate the beam spot from laser 302a by 90 degrees to produce the beam profile shown at 304, providing a Gaussian beam power density profile across the horizontal axis of the flowstream. An example of second optical conditioning element 303b is shown in FIG. 3.

[0100] The disclosed system further includes a light detection system having a photodetector for detecting light from the illuminated core stream. The photodetector may be any convenient light detection protocol, including, but not limited to, optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes (APD), quad-segment photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, among other photodetectors. In one embodiment, the photodetector is a photomultiplier tube, e.g., having a resolution of 0.01 cm. 2 ~10 cm 2 , e.g., 0.05 cm 2 ~9cm 2 , e.g. 0.1cm 2 ~8cm 2 , e.g. 0.5cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .times. ...

[0101] In some embodiments, the light detection system of interest comprises a plurality of light detectors. In some cases, the light detection system comprises a plurality of solid-state detectors, e.g., photodiodes. In some cases, the light detection system comprises a light detector array, e.g., an array of photodiodes. In these embodiments, the light detector array may comprise 4 or more light detectors, e.g., 10 or more light detectors, e.g., 25 or more light detectors, e.g., 50 or more light detectors, e.g., 100 or more light detectors, e.g., 250 or more light detectors, e.g., 500 or more light detectors, e.g., 750 or more light detectors, e.g., 1000 or more light detectors. For example, the detector may be a photodiode array having 4 or more photodiodes, e.g., 10 or more photodiodes, e.g., 25 or more photodiodes, e.g., 50 or more photodiodes, e.g., 100 or more photodiodes, e.g., 250 or more photodiodes, e.g., 500 or more photodiodes, e.g., 750 or more photodiodes, e.g., 1000 or more photodiodes.

[0102] The photodetectors may be arranged in any geometric configuration as desired, including, but not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The photodetectors of the photodetector array may be oriented at angles (relative to the XZ plane) relative to the other photodetectors within a range of 10° to 180°, e.g., 15° to 170°, e.g., 20° to 160°, e.g., 25° to 150°, e.g., 30° to 120°, e.g., 45° to 90°. The photodetector array may have any suitable shape, including rectilinear shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curvilinear shapes such as circular and elliptical; and irregular shapes such as a parabolic base joined to a planar top. In some embodiments, the photodetector array has a rectangular active surface.

[0103] Each photodetector (e.g., photodiode) in the array may have an active surface with a width in the range of 5 μm to 250 μm, such as 10 μm to 225 μm, for example 15 μm to 200 μm, for example 20 μm to 175 μm, for example 25 μm to 150 μm, for example 30 μm to 125 μm, for example 50 μm to 100 μm, and a length in the range of 5 μm to 250 μm, for example 10 μm to 225 μm, for example 15 μm to 200 μm, for example 20 μm to 175 μm, for example 25 μm to 150 μm, for example 30 μm to 125 μm, for example 50 μm to 100 μm, and the surface area of ​​each photodetector (e.g., photodiode) in the array may be in the range of 25 μm to 250 μm. 2 ~10000 μm 2 , e.g., 50 μm 2 ~9000μm 2 , e.g., 75 μm 2 ~8000μm 2 , e.g., 100 μm 2 ~7000μm 2 , e.g., 150 μm 2 ~6000μm 2 , e.g., 200 μm 2 ~5000μm 2 is within the range.

[0104] The size of the photodetector array may vary depending on the amount and intensity of light, the number of photodetectors, and the desired sensitivity, and may have a length in the range of 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, for example 0.1 mm to 80 mm, for example 0.5 mm to 70 mm, for example 1 mm to 60 mm, for example 2 mm to 50 mm, for example 3 mm to 40 mm, for example 4 mm to 30 mm, for example 5 mm to 25 mm. The width of the photodetector array may also vary in the range of 0.01 mm to 100 mm, for example 0.05 mm to 90 mm, for example 0.1 mm to 80 mm, for example 0.5 mm to 70 mm, for example 1 mm to 60 mm, for example 2 mm to 50 mm, for example 3 mm to 40 mm, for example 4 mm to 30 mm, for example 5 mm to 25 mm. Thus, the active surface of the photodetector array may be 0.1 mm to 100 mm, for example 0.05 mm to 90 mm, for example 0.1 mm to 80 mm, for example 0.5 mm to 70 mm, for example 1 mm to 60 mm, for example 2 mm to 50 mm, for example 3 mm to 40 mm, for example 4 mm to 30 mm, for example 5 mm to 25 mm. 2 ~10000 mm 2 , e.g., 0.5 mm 2~5000mm 2 , e.g. 1 mm 2 ~1000mm 2 , e.g. 5mm 2 ~500 mm 2 , e.g. 10mm 2 ~100 mm 2 may be in the range of

[0105] The photodetector may measure light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., twenty-five or more different wavelengths, e.g., fifty or more different wavelengths, e.g., one hundred or more different wavelengths, e.g., two or more different wavelengths, e.g., two or more different wavelengths, e.g., three or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., four or more different wavelengths, e.g., two ... For example, fluorescence may be detected at one or more of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, or any combination thereof, depending on the number of different fluorescence photodetectors in the subject optical detection system.

[0106] Light may be measured continuously or at discrete intervals. In some cases, the detector of interest is configured to measure light continuously. In other cases, the detector of interest is configured to measure at discrete intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, e.g., every 1000 milliseconds, or other intervals. Measurements of light from the flow stream may be taken one or more times, such as two or more times, such as three or more times, such as five or more times, e.g., ten or more times, during each discrete time interval. In some embodiments, light from the flow stream is measured by the photodetector two or more times, and the data is optionally averaged.

[0107] Each optical detector may be positioned at any suitable distance from the flow stream so long as an available optical signal is detectable. For example, detectors of the subject systems may be positioned at 1 mm or more from the flow stream, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, such as 50 mm or more, such as 100 mm or more, such as 150 mm or more, such as 250 mm or more, such as 500 mm or more from the flow stream. Detectors may also be positioned at any angle from the flow stream. For example, detectors may be positioned at an angle between 10° and 90°, such as between 15° and 85°, such as between 20° and 80°, such as between 25° and 75°, such as between 30° and 60°, relative to the vertical axis of the flow stream. In some cases, one or more detectors are positioned at an angle between 30° and 60° relative to the vertical axis of the flow stream.

[0108] In embodiments, the light detection system is configured to detect forward scattered light, side scattered light, emitted light, transmitted light, or a combination thereof. In certain embodiments, the light signal from the illuminated flow stream may be detected by one or more detectors configured as forward scatter detectors. In these embodiments, the forward scatter detector is positioned on the opposite side of the flow stream from the light source and is configured to collect and detect forward-propagating (e.g., scattered) light. In some embodiments, light from the illuminated sample in the flow stream is detected in one or more fluorescence detector channels. In some embodiments, light from the illuminated sample in the flow stream is detected in one or more scattered light detector channels.

[0109] In some embodiments, the system includes a light collection system for collecting light from the flow stream and directing it to the light detection system. The light collection system may be physically coupled to the light detection system, such as with an adhesive, or may be molded together or integrated into the light detection system. In certain embodiments, the light collection system and the light detection system are integrated into a single unit. In other embodiments, the light collection system is coupled to the light detection system using connectors, such as hook and loop fasteners, magnets, latches, notches, countersinks, counterbores, grooves, pins, tethers, hinges, Velcro®, non-permanent adhesives, or combinations thereof.

[0110] In other embodiments, the light detection system and the light collection system are in optical communication but not physically adjacent, for example, the light collection system may be positioned 0.001 mm or more from the light detection system, such as 0.005 mm or more, such as 0.01 mm or more, for example 0.05 mm or more, such as 0.1 mm or more, for example 0.5 mm or more, such as 1 mm or more, for example 10 mm or more, such as 25 mm or more, for example 50 mm or more, for example 100 mm or more from the light detection system.

[0111] In some embodiments, the light collection system includes optical fibers. For example, the light collection system may be a fiber optic relay bundle, and light from the flow stream is transmitted through the fiber optic relay bundle to the first set of linear variable optical filters of the light detection system. Any fiber optic relay system may be used to transmit light, and in some embodiments, suitable fiber optic relay systems include, but are not limited to, fiber optic relay systems such as those described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0112] In other embodiments, the light collection system is a free-space optical relay system. The term "free-space optical relay" is used herein in its conventional sense to refer to the propagation of light using an arrangement of one or more optical components to direct light from a sample through free space to a light detection system. In certain embodiments, the free-space optical relay system includes a housing having a proximal end and a distal end, the proximal end being coupled to a light detection module. The free-space relay system may include any combination of various optical components, such as one or more lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof. For example, in some embodiments, free-space optical relay systems of interest include one or more focusing lenses. In other embodiments, free-space optical relay systems of interest include one or more mirrors. In still other embodiments, the free-space optical relay system includes a collimating lens. In certain embodiments, suitable free-space optical relay systems for transmitting light from the sample to the first set of linear variable optical filters of the optical detection system include, but are not limited to, optical relay systems such as those described in U.S. Pat. Nos. 7,643,142, 7,728,974, and 8,223,445, the disclosures of which are incorporated herein by reference.

[0113] In some embodiments, the system includes a processor operatively coupled to a memory, the memory storing instructions that, when executed by the processor, cause the processor to determine a radiation intensity of the irradiated particles of the composition and, based on the determined radiation intensity, determine a position of the irradiated particles of the composition within a core stream of the flow stream.

[0114] As described above, in some embodiments, the composition includes particles bound to an irradiance power density-sensitive compound that emits light at an intensity determined by the irradiance power density of a light source incident on the particles. In some embodiments, the irradiated particles of the composition exhibit a change in radiation intensity based on the intensity of irradiation by the light source incident on the particles. In some embodiments, the irradiated particles of the composition have a spectral profile that varies depending on the power density of the light source. In some embodiments, the particles of the composition have two or more radiation peaks in response to irradiation by the light source, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more radiation peaks. In some embodiments, one or more peaks of the spectral profile exhibit a change in intensity depending on the power density of the light source. In some embodiments, the intensity of each radiation peak is determined by the irradiance power density of the light source. In some embodiments, the memory comprises instructions for determining positional information of the particles of the composition based on the radiation intensity of one or more radiation peaks. In some cases, the memory has instructions for determining that a particle is propagating at or near a center of the core stream when the emission peak indicates a high emission intensity, and determining that a particle is propagating at or near an edge of the core stream when the emission peak indicates a low emission intensity. In some cases, the memory has instructions for determining that a particle is propagating at or near a center of the core stream when the emission peak indicates a low emission intensity, and determining that a particle is propagating at or near an edge of the core stream when the emission peak indicates a high emission intensity.

[0115] In some embodiments, the irradiated particles of the composition have a first radiation peak having a first intensity at a first radiation power density and a second intensity at a second radiation power density, and a second radiation peak having the same intensity at the first radiation power density and the second radiation power density. In some cases, the memory has instructions for determining position information of the particles of the composition based on the radiation intensities of the first radiation peak and the second radiation peak. In one example, the memory has instructions for determining that the particles are propagating at or near the center of the core stream when the first radiation peak exhibits a high radiation intensity, and determining that the particles are propagating at or near the edge of the core stream when the first radiation peak exhibits a low radiation intensity. In another example, the memory has instructions for determining that the particles are propagating at or near the center of the core stream when the first radiation peak exhibits a low radiation intensity, and determining that the particles are propagating at or near the edge of the core stream when the first radiation peak exhibits a high radiation intensity.

[0116] In other cases, the memory comprises instructions for determining the position of the particle based on a ratio of an intensity of the first radiation peak to an intensity of the second radiation peak. In some cases, the ratio of the intensity of the first radiation peak to the intensity of the second radiation peak is in the range of 1:100 to 100:1, such as 1:75 to 75:1, for example 1:50 to 50:1, such as 1:25 to 25:1, for example 1:10 to 10:1, such as 1:9 to 9:1, for example 1:8 to 8:1, for example 1:7 to 7:1, such as 1:6 to 6:1, for example 1:5 to 5:1, or for example 1:4 to 4:1. In some embodiments, the memory has instructions for determining that a particle is propagating at or near the center of the core stream when the ratio of the intensity of the first emission peak to the intensity of the second emission peak is within a range of 0.7:1 to 1:0.7, such as 0.8:1 to 1:0.8, such as 0.9:1 to 1:0.9, for example 0.95:1 to 1:0.95. In some cases, the memory has instructions for determining that a particle is propagating at or near the center of the core stream when the ratio of the intensity of the first emission peak to the intensity of the second emission peak is about 1:1 (i.e., a particle is propagating at the center of the core stream (or is at the center of illumination by the light source) when the first emission peak and the second emission peak have substantially the same emission intensity). In some embodiments, the greater the ratio of the intensity of the first emission peak to the intensity of the second emission peak, the greater the distance from the center of illumination (or the center of the core stream when the light source and core stream are aligned) that the particle is propagating.

[0117] In some embodiments, the memory has instructions for determining that particles of the composition are propagating within the flow stream at or near the center of the core stream. In some cases, the memory has instructions for determining that particles of the composition are propagating within the flow stream at or near the edge of the core stream. In some embodiments, the memory has instructions for generating one or more particle population clusters based on the determined positions of the particles within the core stream. In some cases, the memory has instructions for classifying particles as being irradiated at or near the center of the core stream based on the determined spectral radiation intensity profile. In other cases, the memory has instructions for classifying particles as being irradiated at or near the edge of the core stream based on the determined spectral radiation intensity profile. In some cases, the memory has instructions for clustering together data signals from particles classified as being irradiated at or near the center of the core stream. In some cases, the memory has instructions for clustering together data signals from particles classified as being irradiated at or near the edge of the core stream. In some embodiments, the memory has instructions for discarding data signals from particles of the composition classified as being irradiated at or near the edge of the core stream.

[0118] In certain embodiments, the system further comprises a flow cell configured to propagate the composition in a flow stream. Any convenient flow cell may be used that propagates the fluid sample to the sample interrogation region; in some embodiments, the flow cell has a proximal cylindrical portion defining a longitudinal axis and a distal frusto-conical portion terminating in a flat surface having an orifice transverse to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) may vary from 1 mm to 15 mm, e.g., 1.5 mm to 12.5 mm, e.g., 2 mm to 10 mm, e.g., 3 mm to 9 mm, e.g., 4 mm to 8 mm. The length of the distal frusto-conical portion (measured along the longitudinal axis) may also vary from 1 mm to 10 mm, e.g., 2 mm to 9 mm, e.g., 3 mm to 8 mm, e.g., 4 mm to 7 mm. The diameter of the flow cell nozzle chamber may in some embodiments vary within the range of 1 mm to 10 mm, such as 2 mm to 9 mm, such as 3 mm to 8 mm, such as 4 mm to 7 mm.

[0119] In some cases, the flow cell does not have a cylindrical portion, and the entire interior chamber of the flow cell is frustoconical. In these embodiments, the length of the frustoconical interior chamber (measured along the longitudinal axis transverse to the nozzle orifice) may be in the range of 1 mm to 15 mm, e.g., 1.5 mm to 12.5 mm, e.g., 2 mm to 10 mm, e.g., 3 mm to 9 mm, e.g., 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical interior chamber may be in the range of 1 mm to 10 mm, e.g., 2 mm to 9 mm, e.g., 3 mm to 8 mm, e.g., 4 mm to 7 mm.

[0120] In some embodiments, the sample flow stream is emitted from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow stream, the orifice of the flow cell may have any suitable shape, with cross-sectional shapes of interest including, but not limited to, rectilinear cross-sectional shapes, e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.; curved cross-sectional shapes, e.g., circular, oval, etc.; and irregular shapes, e.g., a parabolic base joined to a planar top. In certain embodiments, flow cells of interest have circular orifices. The size of the nozzle orifice may vary in some embodiments from 1 μm to 20,000 μm, such as from 2 μm to 17,500 μm, for example, from 5 μm to 15,000 μm, for example, from 10 μm to 12,500 μm, for example, from 15 μm to 10,000 μm, for example, from 25 μm to 7,500 μm, for example, from 50 μm to 5,000 μm, for example, from 75 μm to 1,000 μm, for example, from 100 μm to 750 μm, for example, from 150 μm to 500 μm. In one embodiment, the nozzle orifice is 100 μm.

[0121] In some embodiments, the flow cell has a sample injection port configured to deliver a sample to the flow cell. In embodiments, the sample injection system is configured to deliver a suitable flow of sample to the internal chamber of the flow cell. Depending on the desired characteristics of the flow stream, the flow rate of the sample delivered by the sample injection port to the flow cell chamber can be 1 μL / min or more, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min or more, for example, 15 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, for example, 100 μL / min or more, and in some cases, the flow rate of the sample delivered by the sample injection port to the flow cell chamber is 1 μL / sec or more, for example, 2 μL / sec or more, for example, 3 μL / sec or more, for example, 5 μL / sec or more, for example, 10 μL / sec or more, for example, 15 μL / sec or more, for example, 25 μL / sec or more, for example, 50 μL / sec or more, for example, 100 μL / sec or more.

[0122] The sample injection port may be an orifice in the wall of the internal chamber or a tube located at the proximal end of the internal chamber. When the sample injection port is an orifice in the wall of the internal chamber, the orifice may have any suitable shape. Cross-sectional shapes of interest include, but are not limited to, rectilinear shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal; curved shapes, such as circular and oval; and irregular shapes, such as a parabolic bottom joined to a flat top. In some embodiments, the sample injection port has a circular orifice. The size of the orifice of the sample injection port may vary depending on the shape, and in some cases may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., a 1.5 mm opening.

[0123] In some cases, the sample injection port is a tube located at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a tube aligned with the orifice of the flow cell. When the sample injection port is a tube aligned with the orifice of the flow cell, the cross-sectional shape of the sample injection tube may have any suitable shape. Cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes, such as circular and oval; and irregular shapes, such as a parabolic bottom joined to a flat top. The orifice of the tube may vary depending on the shape and may in some cases have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may have a beveled tip with a bevel angle within a range of 1° to 10°, for example, 2° to 9°, for example, 3° to 8°, for example, 4° to 7°, for example, a bevel angle of 5°.

[0124] In some embodiments, the flow cell further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid, e.g., along with the sample, into the internal chamber of the flow cell to generate a laminated flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the flow rate of the sheath fluid delivered to the flow cell chamber may be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, e.g., 2500 μL / sec or more.

[0125] In some embodiments, the sheath fluid injection port is an orifice in the wall of the internal chamber. The orifice of the sheath fluid injection port may have any suitable shape, and cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal, curved cross-sectional shapes, such as circular and oval, and irregular shapes, such as a parabolic bottom joined to a flat top. The size of the orifice of the sample injection port may vary depending on the shape, and in some cases may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., a 1.5 mm opening.

[0126] In some embodiments, the system further includes a pump in fluid communication with the flow cell for propagating the flow stream through the flow cell. Any convenient fluid pumping protocol may be used to control the flow of the flow stream through the flow cell. In some embodiments, the system includes a peristaltic pump, e.g., a peristaltic pump having a pulse damper. The pump in the subject systems is configured to deliver fluid through the flow cell at a rate suitable for detecting light from the sample in the flow stream. In some embodiments, the flow rate of the sample flow in the flow cell is 1 μL / min (microliters per minute) or more, e.g., 2 μL / min or more, e.g., 3 μL / min or more, e.g., 5 μL / min or more, e.g., 10 μL / min or more, e.g., 25 μL / min or more, e.g., 50 μL / min or more, e.g., 75 μL / min or more, e.g., 100 μL / min or more, e.g., 250 μL / min or more, e.g., 500 μL / min or more, e.g., 750 μL / min or more, e.g., 1000 μL / min or more. For example, the system may comprise a pump configured to flow the sample through the flow cell at a rate in the range of 1 μL / min to 500 μL / min, e.g., 1 μL / min to 250 μL / min, e.g., 1 μL / min to 100 μL / min, e.g., 2 μL / min to 90 μL / min, e.g., 3 μL / min to 80 μL / min, e.g., 4 μL / min to 70 μL / min, e.g., 5 μL / min to 60 μL / min, e.g., 10 μL / min to 50 μL / min. In an embodiment, the flow rate of the flow stream is 5 μL / min to 6 μL / min.

[0127] In certain embodiments, a light detection system having a plurality of light detectors as described above is part of or located in a particle analyzer, such as a particle sorter, hi certain embodiments, the subject system is a flow cytometry system that includes a photodiode and amplifier components as part of the light detection system for detecting light emitted by a sample in a flow stream. Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan;49(pt 1):17-28; Linden, et. al., Semin Thromb Hemost. 2004 Oct;30(5):502-11; Alison, et al. J Pathol, 2010 Dec;222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst., the disclosures of which are incorporated herein by reference. 24(3):203-255.In some cases, flow cytometry systems of interest include the BD Biosciences FACSCanto™ flow cytometer, BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Accuri™ C6 Plus flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer, and BD Biosciences FACSCalibur™ cell sorter, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter ...Verse™ flow cytometer, BD Biosciences FACSSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFor These include the BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, BD Biosciences FACSMelody™ cell sorter, and BD Biosciences FACSymphony™ S6 cell sorter.

[0128] In some embodiments, the subject system may be configured with a fusion technology similar to that disclosed in U.S. Pat. Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, 10,302,545, 10,145,793, 10,113,967, 10,006,852, 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,099,909, 9,100,100,110,120,130,140,150,160,170,172,180,190,192,194,195,196,197,198,19 ... Nos. 5494, 9092034, 8975595, 8753573, 8233146, 8140300, 7544326, 7201875, 7129505, 6821740, 6813017, 6809804, 6372506, 5700692, 5643796, 5627040, 5620842, 5602039, 4987086, and 4498766.

[0129] In some embodiments, the subject system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as described in U.S. Patent Application Publication No. 2017 / 0299493, the disclosure of which is incorporated herein by reference. In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting determination module having multiple sorting determination units, such as described in U.S. Patent Application Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference. In some embodiments, the subject system includes a particle sorting module with deflection plates, such as described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0130] In some cases, the flow cytometry system of the present invention may be implemented using the techniques described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, U.S. Patent Application Publication No. 200900222, and the like. and US Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894, the disclosures of which are incorporated herein by reference, for imaging particles in a flowstream by Fluorescence Imaging Using Radio Frequency Tag Emission (FIRE).

[0131] In some embodiments, the system is a particle analyzer, and particle analysis system 401 (FIG. 4A) can be used to analyze and characterize particles with or without physical sorting of the particles into a collection vessel. FIG. 4A is a functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, particle analysis system 401 is a flow system. The particle analysis system 401 shown in FIG. 4A can be configured to perform, for example, the methods described herein in whole or in part. Particle analysis system 401 includes a fluidic system 402. Fluidic system 402 can include or be coupled to a sample tube 405 and a moving fluid column within the sample tube through which particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0132] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. A detection station 408 generally refers to a monitoring region 407 of the common sample path. Detection, in some embodiments, may involve detecting light or one or more other properties of the particle 403 as it passes through the monitoring region 407. In FIG. 4A , one detection station 408 is shown with one monitoring region 407. In some embodiments of the particle analysis system 401, multiple detection stations may be provided. Additionally, some detection stations may monitor more than one region.

[0133] Each signal is assigned a signal value to form a data point for each particle. As described above, this data may be referred to as event data. The data points may be multidimensional data points that include values ​​for each property measured for the particle. The detection system 404 is configured to collect a series of such data points over a first time interval.

[0134] The particle analysis system 401 may further include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operatively associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the Poisson distribution and the number of data points collected by the detection system 404 during the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. The control system 406 may further compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0135] 4B shows a system 400 for flow cytometry according to an exemplary embodiment of the invention. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. Flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more longpass (LP) filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0136] Pump lasers 415a-415c emit light in the form of laser beams. The wavelengths of the laser beams emitted from pump lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively, in the exemplary system of FIG. 4B. The laser beams are first directed through one or more of beam splitters 445a and 445b. Beam splitter 445a transmits 488 nm light and reflects 633 nm light. Beam splitter 445b transmits UV light (light having a wavelength in the range of 10-400 nm) and reflects 488 nm and 633 nm light.

[0137] The laser beam is then directed to a focusing lens 420, which focuses the laser beam onto the portion of the fluid stream where the sample particles reside in a flow chamber 425. The flow chamber is the part of the fluid system that directs particles in the stream, typically one at a time, into the focused laser beam for investigation. The flow chamber can comprise a flow cell in a benchtop cytometer or a nozzle tip in a stream-in air cytometer.

[0138] Light from one or more laser beams interacts with particles in the sample by diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at a variety of different wavelengths depending on the particle's characteristics, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. The fluorescent radiation, as well as the diffracted, refracted, reflected, and scattered light, may be sent through one or more of beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collection lens 440 to one or more of forward scatter detector 430, side scatter detector 435, and one or more fluorescence detectors 460a-460f.

[0139] The fluorescence collection lens 440 collects light emitted from particle-laser beam interactions and directs it toward one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the bandpass filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number represents the extent of the spectral band. Thus, a 510 / 20 filter extends 10 nm on either side of the center of the spectral band, from 500 nm to 520 nm. Shortpass filters transmit light with wavelengths below a specified wavelength. Longpass filters, such as longpass filters 455a-455b, transmit light with wavelengths above a specified wavelength. For example, longpass filter 455a, a 670 nm longpass filter, transmits light above 670 nm. Filters are often selected to optimize the specificity of the detector for a particular fluorochrome. The filter may be configured so that the spectral band of light transmitted to the detector approximates the emission peak of the fluorescent dye.

[0140] Beam splitters direct light of different wavelengths in different directions. Beam splitters can be characterized by filter properties such as short-pass and long-pass. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 445g transmits light with wavelengths of 620 nm or less and reflects light with wavelengths longer than 620 nm in different directions. In one embodiment, beam splitters 445a-445g can include optical mirrors such as dichroic mirrors.

[0141] The forward scatter detector 430 is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light, or excitation light traveling mostly forward through or around the particle. The intensity of light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector may include a photodiode. The side scatter detector 435 is configured to detect refracted and reflected light from the particle's surface and internal structure, which tends to increase as the particle's structure becomes more complex. Fluorescent emission from fluorescent molecules bound to the particle may be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected by the forward scatter detector 430, side scatter detector 435, and fluorescence detector may be converted to electronic signals (voltage) by the detectors. This data may provide information about the sample.

[0142] Those skilled in the art will recognize that flow cytometers according to embodiments of the present invention are not limited to the flow cytometer shown in Figure 4B, but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in a variety of different configurations.

[0143] During operation, the operation of the flow cytometer is controlled by the controller / processor 490, and measurement data from the detectors may be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detectors to receive output signals from the detectors, and may further be coupled to the electrical and electromechanical components of the flow cytometer 410 to control lasers, fluid flow parameters, etc. An input / output (I / O) functionality 497 may also be provided in the system. The memory 495, controller / processor 490, and I / O functionality 497 may be provided entirely as an integral part of the flow cytometer 410. In such an embodiment, a display may also form part of the I / O functionality 497 to present experimental data to a user of the flow cytometer 410. Alternatively, some or all of the memory 495 and the controller / processor 490 and I / O functionality may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of memory 495 and controller / processor 490 may be in wireless or wired communication with flow cytometer 410. Controller / processor 490 in conjunction with memory 495 and I / O functionality 497 may be configured to perform a variety of functions related to the preparation and analysis of flow cytometer experiments.

[0144] The system illustrated in FIG. 4B includes six different detectors that detect fluorescence within six different wavelength bands (which may be referred to herein as "filter windows" for a given detector) as determined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in a flow cytometer experiment emit light in their own unique wavelength bands. The particular fluorescent labels used in the experiment and their associated fluorescence emission bands may be selected to roughly match the filter windows of the detectors. However, as more detectors are provided and more labels are utilized, perfect correspondence between filter windows and fluorescence emission spectra is not possible. While the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of one particular detector, it is generally true that a portion of that label's emission spectrum also overlaps with the filter window of one or more other detectors. This may be referred to as spillover. I / O functionality 497 may be configured to receive data for a flow cytometer experiment with a panel of fluorescent labels and multiple cell populations with multiple markers (each cell population having a subset of multiple markers). I / O functionality 497 may be further configured to receive biological data assigning one or more markers to one or more cell populations, marker concentration data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experimental data, such as label spectral characteristics and flow cytometer configuration data, may be further stored in memory 495. Controller / processor 490 may be configured to evaluate one or more assignments of labels to markers.

[0145] 5 is a functional block diagram of an example particle analysis control system for analyzing and displaying biological events, such as an analysis controller 500. The analysis controller 500 can be configured to perform various processes for controlling the graphical display of biological events.

[0146] A particle analyzer or particle sorting system 502 may be configured to acquire the biological event data. For example, a flow cytometer may generate flow cytometry event data. The particle analyzer 502 may be configured to provide the biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer or particle sorting system 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel.

[0147] The analysis controller 500 may be configured to receive biological event data from the particle analyzer or particle sorting system 502. The biological event data received from the particle analyzer or particle sorting system 502 may include flow cytometry event data. The analysis controller 500 may be configured to provide a graphical display including a first plot of the biological event data on the display device 506. The analysis controller 500 may be further configured to render a region of interest as a gate around a population of the biological event data displayed by the display device 506, e.g., overlaid on the first plot. In some embodiments, the gate may be a logical combination of one or more illustrated regions of interest plotted on a histogram or bivariate plot of a parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0148] Analysis controller 500 may further be configured to display the in-gate biological event data on display device 506 differently from other events in the outside-gate biological event data. For example, analysis controller 500 may be configured to render the color of the biological event data included within the gate differently from the color of the outside-gate biological event data. Display device 506 may be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0149] The analysis controller 500 may be configured to receive a gate selection signal identifying a gate from a first input device. For example, the first input device may be implemented as a mouse 510. The mouse 510 may initiate a gate selection signal to the analysis controller 500 identifying a gate to be displayed or manipulated via the display device 506 (e.g., by clicking on or within the desired gate when the cursor is over the desired gate). In some embodiments, the first device may be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, a pen, a photodetector, or a voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function may be considered an input device. For example, as shown in FIG. 5, the mouse 510 may include a right mouse button and a left mouse button, and the right mouse button and the left mouse button may each generate a trigger event.

[0150] A trigger event can cause the analysis controller 500 to change how the data is displayed, which portions of the data are actually displayed on the display device 506, and / or provide input to further processing, such as selecting a population of interest for particle sorting.

[0151] In some embodiments, the analysis controller 500 may be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 may further be configured to automatically modify the visualization of the plot to facilitate gating. This modification may be made based on a particular distribution of the biological event data received by the analysis controller 500.

[0152] The analysis controller 500 may be connected to a storage device 504. The storage device 504 may be configured to receive and store biological event data from the analysis controller 500. The storage device 504 may be further configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 may be further configured to enable retrieval of biological event data, such as flow cytometry event data, by the analysis controller 500.

[0153] A display device 506 may be configured to receive display data from the analysis controller 500. The display data may include plots of the biological event data and gates outlining sections of the plots. The display device 506 may be further configured to modify the information displayed in response to input received from the analysis controller 500 in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0154] In some embodiments, the analysis controller 500 can generate a user interface for receiving example events for filtering. For example, the user interface can include controls for receiving example events or example images. The example events or images, or example gates, can be provided prior to collection of event data for a sample or based on an initial set of events for a portion of the sample.

[0155] FIG. 6A is a schematic diagram illustrating a particle sorting system 600 (e.g., particle analyzer or particle sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in FIG. 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, include, or be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are aligned in a single file and traverse a monitoring region 611 (e.g., intersecting a laser stream) that is illuminated by an illumination source 612 (e.g., a laser). Vibration of droplet-forming transducer 602 causes moving fluid column 608 to break up into multiple droplets 610 , some of which contain particles 609 .

[0156] During operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitoring region 611. The detection station 614 feeds a timing circuit 628, which in turn feeds a flash charge circuit 630. At a droplet break-off point, signaled by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 608 so that the droplet of interest carries a charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect the charged droplets into a receptacle, such as a collection tube or a multi-well or microwell sample plate, where a well or microwell can be specifically associated with the droplet of interest. As shown in FIG. 6A, the droplets can be collected in a drain receptacle 638.

[0157] A detection system 616 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal as a particle of interest passes through the monitoring region 611. An exemplary droplet boundary detector is described in U.S. Pat. No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 616 enables the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may provide an amplitude signal 620 and / or a phase signal 618, which are then provided (via an amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be provided within the control system.

[0158] In some embodiments, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled to a memory configured to store detected events and sorting decisions based on the detected events. The sorting decisions can be included in the event data for the particles. In some embodiments, detection system 616 and detection station 614 can be implemented as a single detection unit or can be communicatively coupled such that event measurements can be collected by either detection system 616 or detection station 614 and provided to a non-collection element.

[0159] FIG. 6B is a schematic diagram illustrating a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in FIG. 6B includes deflection plates 652 and 654. An electric charge can be applied via a stream of charging wires within the barbs, generating a stream of droplets 610 containing particles 609 for analysis. The particles can be illuminated using one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The information about the particles is analyzed by sorting electronics or other detection systems (not shown in FIG. 6B). Deflection plates 652 and 654 can be independently controlled to attract or repel the charged droplets and direct them toward a desired collection vessel (e.g., one of 672, 674, 676, or 678). 6B, deflector plates 652 and 654 can be controlled to direct particles along a first path 662 toward a container 674 or along a second path 668 toward a container 678. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflector plates may allow the particle to continue along path 664. Such uncharged droplets may be directed into a waste container, such as via an aspirator 670.

[0160] Sorting electronics can be included to initiate measurement collection, receive fluorescent signals for the particles, and determine how to adjust the deflection plates to sort the particles. Exemplary implementations of the embodiment shown in Figure 6B include the BD FACSAria™ system of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0161] Computer Control System Aspects of the present disclosure further include a computer control system, the system further comprising one or more computers for fully or partially automating the methods described herein. In some embodiments, the system comprises a computer having a computer-readable storage medium having a computer program stored thereon, the computer program having instructions, when loaded into the computer, for determining positional information of particles within a flow stream of a flow cytometer according to the subject methods described herein. In some embodiments, the computer program has instructions for propagating a composition having particles (e.g., cells) through a flow stream, including a core stream and a sheath flow stream; illuminating the particles of the composition with a light source; detecting light from the illuminated particles; determining the emission intensity of the illuminated particles of the composition; and determining the position of the illuminated particles within the core stream of the flow stream based on the detected emitted light from the illuminated particles. In some embodiments, the composition is a biological composition, and the particles of interest in the composition are cells.

[0162] In some embodiments, the computer program has instructions for determining a position of the irradiated particle of the composition based on a difference between the radiation intensity of the first radiation peak and the radiation intensity of the second radiation peak. In some embodiments, the computer program has instructions for determining that the irradiated particle is propagating in the core stream at approximately the center of the light source irradiation position when the first radiation peak and the second radiation peak have approximately the same intensity. In some embodiments, the computer program has instructions for determining that the irradiated particle is propagating in the core stream at an edge of the light source irradiation position when the first radiation peak and the second radiation peak have different intensities. In some embodiments, the computer program has instructions for determining that the irradiated particle is propagating in the core stream at approximately the center of the core stream when the first radiation peak and the second radiation peak have approximately the same intensity. In some embodiments, the computer program has instructions for determining that the irradiated particle is propagating in the core stream at an edge of the core stream when the first radiation peak and the second radiation peak have different intensities.

[0163] In some embodiments, the computer program has instructions for generating one or more particle population clusters based on the determined positions of the particles within the core stream. In some cases, the computer program has instructions for clustering particles determined to be propagating within the flow stream at or near the center of the core stream. In some cases, the computer program has instructions for clustering particles determined to be propagating within the flow stream at or near the edge of the core stream. In some cases, the computer program has instructions for discarding data signals generated in response to light from illuminated particles determined to be propagating within the flow stream at or near the edge of the core stream.

[0164] In an embodiment, the system comprises an input module, a processing module, and an output module. The subject systems may comprise both hardware and software components, and the hardware components may take the form of one or more platforms, for example in the form of servers, such that the functional elements of the system, i.e., elements of the system that perform particular tasks (e.g., managing the input and output of information, processing information, etc.), may be performed by the execution of software applications on and across one or more computer platforms represented by the system.

[0165] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module has a processor that can access a memory in which instructions for executing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, an input / output controller, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are or become available. The processor executes an operating system that interfaces with firmware and hardware in a well-known manner to facilitate the processor's coordination and execution of the functions of various computer programs, which may be written in a variety of programming languages, e.g., Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other elements of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor has analog electronics that allow a user to manually align the light source with the flow stream based on the first optical signal and the second optical signal, hi some embodiments, the processor has analog electronics that provide feedback control, e.g., negative feedback control.

[0166] The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-write compact disk, flash memory device, or other memory storage device. The memory storage device may be any of a variety of known or future devices, including a compact disk drive, tape drive, removable hard disk drive, or diskette drive. These types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk, magnetic tape, removable hard disk, or magnetic disk. Any of these program storage media, or other program storage media now in use or that may be developed in the future, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or on program storage devices used in conjunction with the memory storage devices.

[0167] In some embodiments, a computer program product is described that includes a computer-usable medium having stored thereon control logic (a computer software program including program code). The control logic, when executed by a processor, a computer, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementation of hardware state machines to perform the functions described herein will be apparent to one skilled in the relevant art.

[0168] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic or optical disk, tape, RAM, or any other suitable device, fixed or portable). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium storing the necessary program code. The program may be provided to the processor remotely via a communications channel or pre-recorded on a computer program product, such as a memory, or on other portable or fixed computer-readable storage media using any of these devices connected to the memory. For example, a magnetic or optical disk may store the program and be read by a disk writer / reader. The system of the present invention further comprises a program, e.g., in the form of a computer program product, an algorithm for use in implementing the method as described above. The program of the present invention may be recorded on a computer-readable medium, e.g., any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media, such as magnetic disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; portable flash drives; and hybrids of these categories, such as magnetic / optical storage media.

[0169] The processor may also access a communication channel to communicate with a user at a remote location, meaning that the user does not have direct contact with the system but instead relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0170] In some embodiments, a system according to the present disclosure may be configured to include a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communications interface may be configured for wired or wireless communications, including, but not limited to, radio frequency (RF) communications such as radio frequency identification (RFID), ZigBee communications protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocol, and cellular communications such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0171] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces, such as a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment) configured for similar complementary data communication.

[0172] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, allowing the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that a user may use in conjunction with the device.

[0173] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing the Internet Protocol (IP) via a cellular network, short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0174] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communications interface using common standards such as 802.11 or Bluetooth® RF protocols or the IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer; or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touchscreen.

[0175] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored in the subject system, e.g., any data storage unit, with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0176] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input / output interface between the system and the user and for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved using a network or other type of remote communication in alternative embodiments. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network, according to known techniques. Presentation of data by the output manager may be performed according to various known techniques. In some examples, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The platform or platforms present in the subject system are typically a class of computers commonly referred to as servers, but may be any type of computer platform now known or later developed. However, the platforms may also be mainframe computers, workstations, or other computer types. The platforms may be networked or not, and may be connected via any type of cabling now known or later, or other communication systems, including wireless systems. The platforms may be co-located or physically separated.Various operating systems may be used on any of the computer platforms, depending in some cases on the type and / or configuration of the computer platform selected. Suitable operating systems include Windows, iOS, Oracle Solaris, Linux, IBM i, Unix, etc.

[0177] FIG. 7 illustrates a general configuration of an exemplary computing device 700 according to one embodiment. The general configuration of computing device 700 illustrated in FIG. 7 includes the arrangement of computer hardware and software components. Computing device 700 may include more (or fewer) elements than those illustrated in FIG. 7 . However, not all of these typical conventional elements need be shown to provide a useful disclosure. As illustrated, computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750, and input devices 760, all of which may communicate with each other via a communications bus. Network interface 720 may provide connectivity to one or more networks or computing systems. Thus, processing unit 710 may receive information and instructions from other computing systems or services via a network. Processing unit 710 may further communicate with memory 770 and may further provide output information for an optional display 750 via input / output device interface 740. The input / output device interface 740 may further accept input from any input device 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0178] Memory 770 may include computer program instructions (grouped in some embodiments as modules or components) that processing unit 710 executes to implement one or more embodiments. Memory 770 typically includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by processing unit 710 in the general management and operation of computing device 700. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0179] Non-transitory computer-readable storage medium Aspects of the present disclosure further include a non-transitory computer-readable storage medium having instructions for determining particle position information within a flow stream of a flow cytometer according to the subject methods described herein. The computer-readable storage medium may be used by one or more computers for fully or partially automating a system for implementing the methods described herein. In certain embodiments, instructions for the methods described herein may be encoded on a computer-readable medium in the form of a "program," and as used herein, the term "computer-readable medium" refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include magnetic disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray® disks, solid-state disks, and network-attached storage devices (NAS), regardless of whether such devices are internal or external to the computer. A file containing information may be "stored" on a computer-readable medium, where "storing" means recording information so that the information can be subsequently accessed and retrieved by a computer. The computer-implemented methods described herein may be performed using programs that may be written in one or more of any number of computer programming languages, including, for example, Python, Java, Java Script, C, C#, C++, Go, R, SWIFT®, PHP, and many others.

[0180] In some embodiments, the irradiated particles in the composition are stably bound to an irradiance power density-sensitive compound that emits light at an intensity determined by the irradiance power density of a light source incident on the particles. In some embodiments, the irradiated particles are configured to have a spectral radiation intensity profile that varies depending on the power density of the light source, for example, the particles of the composition are configured to have two or more radiation peaks in response to irradiation by the light source. In some cases, the intensity of each radiation peak depends on the irradiance power density of the light source. In some cases, one or more of the radiation peaks have a first intensity at a first irradiance power density of the light source and a second intensity at a second irradiance power density of the light source. In some embodiments, the particles of the composition have a first radiation peak that has a first intensity at a first irradiance power density and a second intensity at a second irradiance power density, and a second radiation peak that has the same intensity at both the first and second irradiance power densities.

[0181] In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for determining a position of an irradiated particle of the composition based on a difference between the radiation intensity of the first radiation peak and the radiation intensity of the second radiation peak. In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for determining that an irradiated particle is propagating in the core stream at approximately the center of the light source irradiation position when the first radiation peak and the second radiation peak have approximately the same intensity. In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for determining that an irradiated particle is propagating in the core stream at approximately the center of the light source irradiation position when the first radiation peak and the second radiation peak have different intensities. In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for determining that an irradiated particle is propagating in the core stream at approximately the center of the core stream when the first radiation peak and the second radiation peak have approximately the same intensity. In some cases, the non-transitory computer-readable storage medium has an algorithm for determining that the irradiated particle is propagating within the core stream at an edge of the core stream when the first emission peak and the second emission peak have different intensities.

[0182] In some embodiments, the non-transitory computer-readable storage medium comprises an algorithm for generating one or more particle population clusters based on the determined positions of the particles within the core stream. In some cases, the non-transitory computer-readable storage medium comprises an algorithm for clustering particles determined to be propagating within the flow stream at or near the center of the core stream. In some cases, the non-transitory computer-readable storage medium comprises an algorithm for clustering particles determined to be propagating within the flow stream at or near the edge of the core stream. In some cases, the non-transitory computer-readable storage medium comprises an algorithm for discarding data signals generated in response to light from illuminated particles determined to be propagating within the flow stream at or near the edge of the core stream.

[0183] The non-transitory computer-readable storage medium may be used in one or more computer systems having a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module has a processor that can access a memory in which instructions for performing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, an input / output controller, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are or become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor's coordination and execution of the functions of various computer programs, which may be written in various programming languages ​​such as those described above, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other elements of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

[0184] kit Aspects of the present disclosure further include kits, which in some embodiments comprise one or more compositions for determining the position of particles within a core stream of a stream, as described herein. In some embodiments, the kit comprises a composition including particles stably bound to an irradiance power density-sensitive compound that emits light at an intensity that depends on the irradiance power density of the light incident on the particles. As described above, in some cases, the irradiance power density-sensitive compound is covalently bound to the particles in the composition, for example, via a cleavable or non-cleavable linker. In other cases, the irradiance power density-sensitive compound is bound to the particles via a specific binding member, such as an antibody.

[0185] In some embodiments, the kits include one or more compositions for stably binding particles of a sample composition (e.g., a biological composition in which the particles of interest are cells) to an irradiance power density-sensitive compound. In some cases, the irradiance power density-sensitive compound is conjugated to a specific binding member. For example, in the composition of interest, the irradiance power density-sensitive compound may be covalently bound to a proteinaceous specific binding member, such as an antibody. In other embodiments, the composition may be formulated to covalently bind the irradiance power density-sensitive compound to particles in the composition, for example, via a cleavable or non-cleavable linker.

[0186] In some embodiments, the particles stably associated with the irradiance power density-sensitive compound have two or more emission peaks in response to irradiation by a light source. In some embodiments, one or more of the emission peaks have a first intensity at a first irradiance power density of the light source and a second intensity at a second irradiance power density of the light source. In some embodiments, the particles of the composition have a first emission peak having a first intensity at a first irradiance power density and a second intensity at a second irradiance power density, and a second emission peak having the same intensity at the first and second irradiance power densities. In some embodiments, the irradiance power density-sensitive compound is an upconversion nanoparticle (UCNP). In some embodiments, the upconversion nanoparticle is a lanthanide-doped particle. In some embodiments, the upconversion nanoparticle is a europium-doped nanoparticle or a thulium-doped nanoparticle, such as NaYF4:Yb, Er, or NaYF4:YbTm nanoparticle.

[0187] In some embodiments, the kit further comprises optical adjustment components configured to orthogonally rotate the Gaussian beam profile. In certain embodiments, the kit comprises a plurality of photodetectors and programming for the subject systems, e.g., in the form of instructions for downloading the programming from a computer-readable medium (e.g., a flash drive, USB storage, compact disc, DVD, Blu-ray disc, etc.) or an Internet web protocol or cloud server. The kit may further comprise optical adjustment components, e.g., lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof.

[0188] The kits may further comprise instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, and one or more of these instructions may be present in the kit. One form in which these instructions may be provided is as information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, kit packaging, a package insert, etc. Another form in which these instructions may be present is as a computer-readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Another form in which these instructions may be present is a website address that may be used via the internet to access the information at a remote location.

[0189] usefulness The subject methods, systems, and computer systems find use in a variety of applications where it is desirable to optimize the processor and optical detection system of a flow cytometer. The subject methods and systems are further used in optical detection systems having multiple optical detectors used to analyze and sort particle components in samples in fluid media, such as biological samples. The present disclosure finds further use in flow cytometry, where it is desirable to provide a flow cytometer with increased cell sorting accuracy, improved particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and improved particle deflection during cell sorting. In embodiments, the present disclosure reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In certain embodiments, the subject methods and systems provide fully automated protocols, such that adjustments to the flow cytometer during use require little, if any, human input.

[0190] Regardless of the scope of the appended claims, the present disclosure is further defined by the following notes.

[0191] Clause 1. A method for determining the position of a particle within a core stream of a flow stream propagating within a flow cell, comprising: illuminating a composition comprising particles propagating within the flow streams, including the core stream and the sheath flow stream, with a light source; Detecting radiation from the irradiated particles; determining the position of the particle within the core stream based on the detected radiation from the illuminated particle; The method, wherein the particles are stably associated with an irradiance power density sensitive compound that emits light with an intensity determined by the irradiance power density of a light source incident on the particles.

[0192] Appendix 2. The method of Appendix 1, wherein the composition is a biological composition and the particles are cells.

[0193] Appendix 3. The method of Appendix 1 or 2, wherein the irradiation power density sensitive compound is covalently bonded to particles in the composition.

[0194] Appendix 4. The method of any one of Appendixes 1 to 3, wherein the irradiation power density sensitive compound is covalently attached to the particle in the composition via a linker.

[0195] Appendix 5. The method of Appendix 4, wherein the linker is a cleavable linker.

[0196] Appendix 6. The method of Appendix 4, wherein the linker is a non-cleavable linker.

[0197] Clause 7. The method of clause 1 or clause 2, wherein the radiation power density sensitive compound is bound to the particle via a specific binding member.

[0198] Clause 8. The method of Clause 7, wherein the specific binding member is an antibody.

[0199] Appendix 9. The method of any one of appendices 1 to 8, wherein the irradiation power density sensitive compound comprises an upconversion nanoparticle (UCNP).

[0200] Clause 10. The method of Clause 9, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0201] Clause 11. The method of clause 9, wherein the upconversion nanoparticles comprise europium-doped nanoparticles or thulium-doped nanoparticles.

[0202] Clause 12. The method of clause 11, wherein the upconversion nanoparticles comprise NaYF4:Yb, Er nanoparticles, or NaYF4:YbTm nanoparticles.

[0203] Clause 13. The method of any one of clauses 1-12, wherein the irradiated particles in the composition are configured to have a spectral radiant intensity profile that varies depending on the power density of the light source.

[0204] Clause 14. The method of any one of clauses 1-13, wherein the irradiated particles in the composition are configured to have two or more emission peaks upon irradiation by the light source.

[0205] Appendix 15. The method of Appendix 14, wherein the intensity of each radiation peak is determined by the irradiating power density of the light source.

[0206] Clause 16. The method of clause 14 or clause 15, wherein one or more of the emission peaks have a first intensity at a first illumination power density of the light source and a second intensity at a second illumination power density of the light source.

[0207] Appendix 17. The particles to be irradiated are: a first radiation peak having a first intensity at a first irradiance power density and a second intensity at a second irradiance power density; a second radiation peak having the same intensity at the first irradiation power density and the second irradiation power density; 17. The method of claim 16, comprising:

[0208] Clause 18. The method of any one of clauses 1 to 17, wherein the light source has a Gaussian beam power density profile across the horizontal axis of the flow stream.

[0209] Clause 19. The method of clause 18, wherein the light source comprises a laser.

[0210] Item 20. The method of items 18 or 19, wherein the flow stream is irradiated with an elliptical beam spot having a major axis and a minor axis.

[0211] 21. The method of claim 20, wherein the flow stream is irradiated with the beam spot such that the long axis of the beam spot is perpendicular to the horizontal axis of the flow stream.

[0212] Clause 22. The method of any one of clauses 13-21, wherein the position of the irradiated particle within the core stream is determined based on a spectral radiation intensity profile.

[0213] Appendix 23. The method of any one of Appendixes 13-22, wherein the position of the particles of the composition is determined based on a difference between the radiation intensity of the first radiation peak and the radiation intensity of the second radiation peak.

[0214] Clause 24. The method of clause 23, wherein the particle of the composition is determined to be propagating within the flow stream at approximately the center of the core stream when the first emission peak and the second emission peak have the same intensity.

[0215] Clause 25. The method of clause 23, wherein determining that a particle of the composition is propagating within the flow stream at or near the edge of the core stream when the first emission peak and the second emission peak have different intensities.

[0216] Clause 26. The method of any one of clauses 22-25, generating one or more particle population clusters based on the determined positions of the particles within the core stream.

[0217] Clause 27. The method of clause 26, clustering particles determined to be propagating in the flow stream at or near the center of the core stream.

[0218] Clause 28. The method of clause 26 or 27, clustering particles determined to be propagating in the flow stream at or near the edge of the core stream.

[0219] Clause 29. The method of clause 28, discarding data signals generated in response to light from illuminated particles determined to be propagating within the flow stream at or near the edge of the core stream.

[0220] Clause 30. A flow cell configured to propagate a composition comprising particles within a flow stream comprising a core stream and a sheath flow stream. a light source for irradiating particles of the composition; a light detection system for detecting light from the illuminated particles; and a processor to which the memory is operatively coupled It is equipped with the particles are stably bound to an irradiation power density-sensitive compound that emits light of an intensity determined by the irradiation power density of a light source incident on the particles; The memory stores instructions that, when executed by the processor, cause the processor to determine a position of the particle within the flow stream based on detected radiation from the illuminated particle.

[0221] Item 31. The system of item 30, wherein the composition is a biological composition and the particles are cells.

[0222] Item 32. The system of item 30 or 31, wherein the irradiance power density sensitive compound is covalently bonded to particles in the composition.

[0223] Addendum 33. A system described in any one of Addendums 30 to 32, wherein the irradiation power density sensitive compound is covalently attached to the particle in the composition via a linker.

[0224] Clause 34. The system of clause 33, wherein the linker is a cleavable linker.

[0225] Clause 35. The system of clause 33, wherein the linker is a non-cleavable linker.

[0226] Addendum 36. The system of Addendum 30 or 31, wherein the irradiation power density sensitive compound is bound to the particle via a specific binding member.

[0227] Clause 37. The system of clause 36, wherein the specific binding member is an antibody.

[0228] Addendum 38. The system of any one of Addendums 30-37, wherein the irradiation power density sensitive compound comprises an upconversion nanoparticle (UCNP).

[0229] Clause 39. The system of clause 38, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0230] Clause 40. The system of clause 38, wherein the upconversion nanoparticles comprise europium-doped nanoparticles or thulium-doped nanoparticles.

[0231] Clause 41. The system of clause 40, wherein the upconversion nanoparticles comprise NaYF4:Yb, Er nanoparticles, or NaYF4:YbTm nanoparticles.

[0232] Addendum 42. The system of any one of Addendums 30 to 41, wherein the light source comprises a laser.

[0233] Clause 43. The system of clause 42, wherein the laser has a Gaussian beam power density profile across the horizontal axis of the flow stream.

[0234] Clause 44. The system of clause 42 or 43, wherein the laser has an elliptical beam spot having a major axis and a minor axis.

[0235] Clause 45. The system of clause 44, wherein the laser is configured to project an elliptical beam spot onto the core stream such that the major axis of the beam spot is perpendicular to the horizontal axis of the flow stream.

[0236] Clause 46. The system of clause 45, wherein the light source has optical adjustment components configured to propagate light from the laser so that the long axis of the beam spot is perpendicular to the horizontal axis of the flow stream.

[0237] Addendum 47. The system of Addendum 46, wherein the optical adjustment component is configured to rotate the beam spot by 90 degrees.

[0238] Clause 48. The system of clause 47, wherein the optical adjustment component comprises one or more lenses.

[0239] Addendum 49. The system of any one of Addendums 42-48, wherein the light source comprises multiple lasers.

[0240] Appendix 50. The light source is at least one laser having an elliptical beam spot having a major axis and a minor axis, the laser configured to irradiate the flowstream such that the major axis of the beam spot is perpendicular to the longitudinal axis of the flowstream; a laser having an elliptical beam spot having a major axis and a minor axis, the laser being configured to irradiate the flow stream such that the major axis of the beam spot is perpendicular to the horizontal axis of the flow stream; 49. The system of claim 49, comprising:

[0241] Addendum 51. The system of any one of Addendums 30-50, wherein the irradiated particles in the composition are configured to have a spectral radiant intensity profile that varies depending on the power density of the light source.

[0242] Clause 52. The system of clause 51, wherein the irradiated particles in the composition are configured to have two or more emission peaks upon irradiation by the light source.

[0243] Addendum 53. The system of Addendum 52, wherein the intensity of each radiation peak is determined by the irradiance power density of the light source.

[0244] Addendum 54. The system of any one of Addendums 51 to 53, wherein one or more of the radiation peaks have a first intensity at a first illumination power density of the light source and a second intensity at a second illumination power density of the light source.

[0245] Appendix 55. The particles to be irradiated are: a first radiation peak having a first intensity at a first irradiance power density and a second intensity at a second irradiance power density; a second radiation peak having the same intensity at the first irradiation power density and the second irradiation power density; 55. The system of claim 54, comprising:

[0246] Addendum 56. The system of any one of Addendums 51-55, wherein the memory stores instructions that, when executed by the processor, cause the processor to determine a position of the irradiated particle within the core stream based on the spectral radiation intensity profile.

[0247] Addendum 57. The system of Addendum 56, wherein the memory stores instructions that, when executed by the processor, cause the processor to determine a position of a particle of the composition based on a difference between the radiation intensity of the first radiation peak and the radiation intensity of the second radiation peak.

[0248] Addendum 58. The system of Addendum 57, wherein the memory stores instructions that, when executed by the processor, cause the processor to determine that a particle of the composition is propagating within the flow stream at approximately the center of the core stream when the first emission peak and the second emission peak have the same intensity.

[0249] Addendum 59. The system of Addendum 57, wherein the memory stores instructions that, when executed by the processor, cause the processor to determine that a particle of the composition is propagating within the flow stream at or near the edge of the core stream when the first emission peak and the second emission peak have different intensities.

[0250] Addendum 60. The system of any one of Addendums 57-59, wherein the memory stores instructions that, when executed by the processor, cause the processor to generate one or more particle population clusters based on the determined positions of the particles within the core stream.

[0251] Addendum 61. The system of Addendum 60, wherein the memory stores instructions that, when executed by the processor, cause the processor to cluster particles determined to be propagating in the flow stream at or near the center of the core stream.

[0252] Addendum 62. The system of Addendum 60, wherein the memory stores instructions that, when executed by the processor, cause the processor to cluster particles determined to be propagating in the flow stream at or near the edge of the core stream.

[0253] Addendum 63. The system of Addendum 62, wherein the memory stores instructions that, when executed by the processor, cause the processor to discard data signals generated in response to light from illuminated particles determined to be propagating in the flow stream at or near the edge of the core stream.

[0254] Clause 64. Store instructions for determining a position of a particle within a core stream of a flow stream propagating within a flow cell, the instructions comprising: an algorithm for illuminating with a light source a composition comprising particles propagating within a flow stream, the composition comprising particles propagating within the flow stream, the core stream and the sheath flow stream; an algorithm for detecting light from the illuminated particles; Algorithms for determining particle positions within the core stream based on detected radiation from the irradiated particles. and A non-transitory computer-readable storage medium, wherein the particles are stably associated with an irradiance power density sensitive compound that emits light with an intensity determined by the irradiance power density of a light source incident on the particles.

[0255] Clause 65. The non-transitory computer-readable storage medium of Clause 64, wherein the irradiated particles in the composition are configured to have a spectral radiant intensity profile that varies depending on the power density of the light source.

[0256] Clause 66. The non-transitory computer-readable storage medium of any one of clauses 64 to 65, wherein the irradiated particles in the composition are configured to have two or more emission peaks upon irradiation by the light source.

[0257] Clause 67. The non-transitory computer-readable storage medium of Clause 65, wherein the intensity of each radiation peak is determined by the illumination power density of the light source.

[0258] Addendum 68. The non-transitory computer-readable storage medium of Addendum 66 or 67, wherein one or more of the radiation peaks have a first intensity at a first illumination power density of the light source and a second intensity at a second illumination power density of the light source.

[0259] Appendix 69. The particles to be irradiated are: a first radiation peak having a first intensity at a first irradiance power density and a second intensity at a second irradiance power density; a second radiation peak having the same intensity at the first irradiation power density and the second irradiation power density; 69. The non-transitory computer-readable storage medium of claim 68, having:

[0260] Clause 70. The non-transitory computer-readable storage medium of any one of Clauses 65-69, having an algorithm for determining a position of an irradiated particle within a core stream based on a spectral radiation intensity profile.

[0261] Addendum 71. The non-transitory computer-readable storage medium of any one of Addendums 65-70, having an algorithm for determining a position of a particle of the composition based on a difference between the radiation intensity of the first radiation peak and the radiation intensity of the second radiation peak.

[0262] Clause 72. The non-transitory computer-readable storage medium of Clause 71, having an algorithm for determining that a particle of the composition is propagating within the flow stream at approximately the center of the core stream when the first emission peak and the second emission peak have the same intensity.

[0263] Clause 73. The non-transitory computer-readable storage medium of Clause 71, having an algorithm for determining that a particle of the composition is propagating within the flow stream at or near an edge of the core stream when the first emission peak and the second emission peak have different intensities.

[0264] Clause 74. The non-transitory computer-readable storage medium of any one of Clauses 71-73, having an algorithm for generating one or more particle population clusters based on determined positions of particles within a core stream.

[0265] Clause 75. The non-transitory computer-readable storage medium of Clause 74 having an algorithm for clustering particles determined to be propagating in the flow stream at or near the center of the core stream.

[0266] Clause 76. The non-transitory computer-readable storage medium of any one of Clauses 74 to 75, having an algorithm for clustering particles determined to be propagating in the flow stream at or near the edge of the core stream.

[0267] Clause 77. The non-transitory computer-readable storage medium of Clause 76 having an algorithm for discarding data signals generated in response to light from illuminated particles determined to be propagating within the flow stream at or near the edge of the core stream.

[0268] Addendum 78. The non-transitory computer-readable storage medium of any one of Addendums 64-77, wherein the composition is a biological composition and the particles are cells.

[0269] Clause 79. The non-transitory computer-readable storage medium of any one of clauses 64-78, wherein the irradiance power density sensitive compound is covalently bonded to particles in the composition.

[0270] Clause 80. The non-transitory computer-readable storage medium of any one of clauses 64-79, wherein the irradiation power density sensitive compound is covalently attached to the particle in the composition via a linker.

[0271] Clause 81. The non-transitory computer-readable storage medium of Clause 80, wherein the linker is a cleavable linker.

[0272] Clause 82. The non-transitory computer-readable storage medium of Clause 80, wherein the linker is a non-cleavable linker.

[0273] Clause 83. The non-transitory computer-readable storage medium of any one of clauses 64-78, wherein the irradiation power density sensitive compound is bound to the particle via a specific binding member.

[0274] Clause 84. The non-transitory computer-readable storage medium of Clause 83, wherein the specific binding member is an antibody.

[0275] Addendum 85. The non-transitory computer-readable storage medium of any one of Addendums 64 to 84, wherein the irradiation power density sensitive compound comprises an upconversion nanoparticle (UCNP).

[0276] Clause 86. The non-transitory computer-readable storage medium of Clause 85, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0277] Clause 87. The non-transitory computer-readable storage medium of Clause 85, wherein the upconversion nanoparticles comprise europium-doped nanoparticles or thulium-doped nanoparticles.

[0278] Clause 88. The non-transitory computer-readable storage medium of Clause 87, wherein the upconversion nanoparticles comprise NaYF4:Yb, Er nanoparticles, or NaYF4:YbTm nanoparticles.

[0279] Item 89. A kit for determining the location of a particle within a core stream of a flow stream propagating within a flow cell of a flow cytometer, comprising: A kit comprising a composition comprising particles stably bound to an irradiance power density sensitive compound that emits light of an intensity determined by the irradiance power density of the light incident on the particles.

[0280] Item 90. The kit of item 89, wherein the irradiation power density sensitive compound is covalently bound to particles in the composition.

[0281] Addendum 91. The kit of Addendum 89 or 90, wherein the irradiation power density-sensitive compound is covalently bound to the particles in the composition via a linker.

[0282] Item 92. The kit of item 91, wherein the linker is a cleavable linker.

[0283] Item 93. The kit of item 91, wherein the linker is a non-cleavable linker.

[0284] Item 94. The kit of item 89, wherein the irradiation power density sensitive compound is bound to the particle via a specific binding member.

[0285] Item 95. The kit of item 94, wherein the specific binding member is an antibody.

[0286] Addendum 96. A kit according to any one of Addendums 89 to 95, wherein the irradiation power density sensitive compound comprises an upconversion nanoparticle (UCNP).

[0287] 97. The kit of claim 96, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0288] Item 98. The kit of item 97, wherein the upconversion nanoparticles comprise europium-doped nanoparticles or thulium-doped nanoparticles.

[0289] Item 99. The kit of item 97, wherein the upconversion nanoparticles comprise NaYF4:Yb, Er nanoparticles, or NaYF4:YbTm nanoparticles.

[0290] Appendix 100. The kit of any one of appendices 89 to 99, wherein the irradiance power density sensitive compound exhibits two or more emission peaks when irradiated by the light source.

[0291] Addendum 101. The kit of Addendum 100, wherein the irradiance power density sensitive compound exhibits a spectral radiant intensity profile that varies depending on the power density of the light source.

[0292] Addendum 102. The kit of any one of Addendums 100 or 101, wherein one or more of the radiation peaks have a first intensity at a first irradiation power density of the light source and a second intensity at a second irradiation power density of the light source.

[0293] Note 103. Particles are a first radiation peak having a first intensity at a first irradiance power density and a second intensity at a second irradiance power density; a second radiation peak having the same intensity at the first irradiation power density and the second irradiation power density; 103. The kit of claim 102, comprising:

[0294] Addendum 104. The kit of any one of Addendums 89 to 103, further comprising an optical adjustment component configured to orthogonally rotate the light beam.

[0295] Addendum 105. The kit of Addendum 104, wherein the optical adjustment component comprises one or more lenses.

[0296] Clause 106. A kit for determining the location of a particle within a core stream of a flow stream propagating within a flow cell of a flow cytometer, comprising: a composition comprising a compound for stably associating with particles in a sample composition; The compound is an irradiance power density sensitive compound that emits light with an intensity determined by the irradiance power density of light incident on the particle.

[0297] Item 107. The kit of Item 106, wherein the sample composition is a biological composition and the particles are cells.

[0298] Clause 108. The kit of clause 106 or 107, wherein the radiation power density sensitive compound is conjugated to a specific binding member.

[0299] Clause 109. The kit of clause 108, wherein the irradiance power density sensitive compound is covalently bound to the proteinaceous binding member.

[0300] Item 110. The kit of item 109, wherein the irradiation power density sensitive compound is covalently bound to the antibody.

[0301] Item 111. The kit of item 89, wherein the composition is formulated to covalently bond the irradiation power density sensitive compound to particles in the sample composition.

[0302] Clause 112. The kit of clause 111, wherein the composition is formulated to covalently bond the irradiation power density sensitive compound to particles in the sample composition via a linker.

[0303] Item 113. The kit of item 112, wherein the linker is a cleavable linker.

[0304] Item 114. The kit of item 112, wherein the linker is a non-cleavable linker.

[0305] Addendum 115. A kit according to any one of Addendums 106 to 114, wherein the irradiation power density sensitive compound comprises an upconversion nanoparticle (UCNP).

[0306] Clause 116. The kit of clause 115, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0307] Clause 117. The kit of clause 115, wherein the upconversion nanoparticles comprise europium-doped nanoparticles or thulium-doped nanoparticles.

[0308] Addendum 118. The kit of Addendum 109, wherein the upconversion nanoparticles comprise NaYF4:Yb, Er nanoparticles, or NaYF4:YbTm nanoparticles.

[0309] Addendum 119. A kit according to any one of Addendums 106 to 118, wherein the irradiance power density sensitive compound exhibits two or more emission peaks when irradiated by the light source.

[0310] Addendum 120. The kit of Addendum 119, wherein the irradiance power density sensitive compound exhibits a spectral radiant intensity profile that varies depending on the power density of the light source.

[0311] Addendum 121. The kit of Addendum 119 or 120, wherein one or more of the radiation peaks have a first intensity at a first irradiation power density of the light source and a second intensity at a second irradiation power density of the light source.

[0312] Note 122. Particles are a first radiation peak having a first intensity at a first irradiance power density and a second intensity at a second irradiance power density; a second radiation peak having the same intensity at the first irradiation power density and the second irradiation power density; 122. The kit of claim 121, comprising:

[0313] Addendum 123. The kit of any one of Addendums 106-122, further comprising an optical adjustment component configured to orthogonally rotate the light beam.

[0314] Addendum 124. The kit of any one of Addendums 106-122, further comprising one or more fluorophores for labeling particles of the sample composition.

[0315] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of the invention, certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

[0316] Accordingly, the foregoing merely illustrates the essence of the present invention. It is clear that those skilled in the art will be able to devise various configurations that embody the essence of the present invention and are within the spirit and scope of the present invention, although not explicitly described or shown herein. Furthermore, all examples and conditional language set forth herein are intended essentially to aid the reader in understanding the essence of the present invention and the concepts provided by the inventors to advance the art, and should not be construed as limiting the scope of the present invention to the specifically set forth examples and conditions. Furthermore, all statements herein that describe the essence, aspects, and embodiments of the present invention, as well as specific examples of the present invention, are intended to encompass both structural and functional equivalents of the present invention. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., all elements developed that perform the same function, regardless of structure. Furthermore, the descriptions disclosed herein are not intended to be publicly disclosed, regardless of whether such disclosure is explicitly recited in the claims.

[0317] Accordingly, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. With respect to claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) are expressly provided to be invoked with respect to a limitation in a claim only when the precise phrase "means for" or "step for" appears at the beginning of such limitation in the claim; if such precise phrase is not used in a claim limitation, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0318] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 394,415, filed August 2, 2022, the disclosure of which is incorporated herein by reference.

Claims

1. 1. A method for determining the location of a particle within a core stream of a flow stream propagating within a flow cell, comprising: illuminating a composition comprising particles propagating within the flow streams, including the core stream and the sheath flow stream, with a light source; Detecting radiation from the irradiated particles; determining a position of the particle within the core stream based on the detected radiation from the illuminated particle; The method, wherein the particles are stably associated with an irradiance power density sensitive compound that emits light with an intensity determined by the irradiance power density of the light source incident on the particles.

2. The method of claim 1 , wherein the composition is a biological composition and the particles are cells.

3. The method of claim 1 or 2, wherein the radiation power density sensitive compound is covalently bonded to particles in the composition.

4. The method according to any one of claims 1 to 3, wherein the radiation power density sensitive compound is covalently bound to particles in the composition via a linker.

5. The method of claim 4 , wherein the linker is a cleavable linker.

6. The method of claim 4 , wherein the linker is a non-cleavable linker.

7. 3. The method of claim 1, wherein the radiation power density sensitive compound is bound to the particle via a specific binding member.

8. The method of claim 7, wherein the specific binding member is an antibody.

9. The method of any one of claims 1 to 8, wherein the radiation power density sensitive compound comprises an upconversion nanoparticle (UCNP).

10. The method of claim 9 , wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

11. The method of claim 9 , wherein the upconversion nanoparticles comprise europium-doped nanoparticles or thulium-doped nanoparticles.

12. The upconversion nanoparticles are NaYF 4 : Yb, Er nanoparticles or NaYF 4 12. The method of claim 11, comprising: YbTm nanoparticles.

13. The method of any one of claims 1 to 12, wherein the irradiated particles in the composition are configured to have a spectral radiant intensity profile that varies depending on the power density of the light source.

14. a flow cell configured to propagate a composition comprising particles within a flow stream comprising a core stream and a sheath flow stream; a light source for irradiating particles of said composition; a light detection system for detecting light from the illuminated particles; and a processor to which the memory is operatively coupled It is equipped with the particles are stably bound to an irradiation power density sensitive compound that emits light of an intensity determined by the irradiation power density of the light source incident on the particles; The memory stores instructions that, when executed by the processor, cause the processor to determine a position of a particle in the flow stream based on detected radiation from an illuminated particle.

15. 1. A kit for determining the location of a particle within a core stream of a flow stream propagating within a flow cell of a flow cytometer, comprising: A kit comprising a composition comprising particles stably bound to an irradiance power density sensitive compound that emits light of an intensity determined by the irradiance power density of the light incident on the particles.

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