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

Abstract

Aspects of the present disclosure include methods for determining location information of particles in a flowing stream of a flow cytometer. A method according to certain embodiments includes passing a composition having particles through a flow stream comprising a core flow and a sheath flow stream, irradiating the particles of the composition with a light source, detecting light from the irradiated particles, determining light emissions from the irradiated particles of the composition, and determining, based on the detected light emissions, whether or not the particles are irradiated with the light source. A location of the irradiated particles in the core stream of the flow stream is determined. In embodiments, a particle is stably associated (e.g., covalently bonded) with an irradiation power density sensitive compound that emits light whose intensity depends on the irradiation power density of the light source incident on the particle. Systems (e.g., flow cytometers) for practicing the subject methods are also described. A non-transitory computer-readable storage medium is also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the filing date of U.S. Provisional Patent Application Serial No. 63 / 394,415 filed on August 2, 2022, the disclosure of which is incorporated herein by reference. Background Art

[0003] Light detection is often used to characterize the composition of a sample (e.g., a biological sample), such as when the sample is used to diagnose a disease or medical condition. When a sample is irradiated, light can be scattered by the sample, transmitted through the sample, and emitted by the sample (e.g., by fluorescence). Changes in the composition of the sample, such as morphology, absorbance, and the presence of fluorescent markers, can result in changes in the light scattered, transmitted, or emitted by the sample. These changes can be used to characterize and identify the presence of components in the sample. To quantify these changes, light is collected and directed to the surface of a detector.

[0004] One technique that uses light detection to characterize components in a sample is flow cytometry. A flow cytometer includes a light detection system consisting of optics, photodetectors, and electronics that efficiently detect light signals and convert them 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 different types of photodetectors to detect light signals, such as those from fluorescence, side scattered light, or forward scattered light. When a light signal is incident on a photodetector, an electrical signal proportional to the incident light signal is generated at its output. Flow cytometry provides a rapid method to quantify cellular characteristics.

[0005] The intensity profile of the light used to irradiate the flow stream in a flow cytometer can vary between irradiation spots, such as the power density at the center of the laser beam being the highest, and the power density decreasing more rapidly as one moves away from the center of the beam spot. Particles that pass directly through the center of the beam spot receive a greater laser power intensity than particles that do not pass through the center of the beam spot. In some cases, fluorophores present on particles that pass through the periphery of the beam spot will be suboptimally excited, resulting in lower brightness and greater noise in the data signal from the detected light. Summary of the invention

[0006] Aspects of the present disclosure include methods for determining the position information of particles in the flow stream of a flow cytometer. The method according to certain embodiments includes: passing a composition with particles through a flow stream comprising a core flow and a sheath flow stream, irradiating the particles of the composition with a light source, detecting light from the irradiated particles, determining the light emission from the irradiated particles of the composition, and determining the position of the irradiated particles in the core stream of the flow stream based on the detected light emission. In an embodiment, particles are stably associated (e.g., covalently bonded) with an irradiation power density-sensitive compound of the emitted light, and the intensity of the light depends on the irradiation power density of the light source incident on the particles. A system (e.g., a flow cytometer) for practicing the subject method is also described. A non-transitory computer-readable storage medium is also provided.

[0007] When practicing the subject method, a composition with particles is irradiated with a light source in a flow stream having a core flow and a sheath flow stream. In an embodiment, the composition comprises particles, and the particles are stably associated with an irradiation power density sensitive compound of the emitted light, and the intensity of the light depends on the irradiation power density of the light source incident on the particle. In some cases, the irradiation power density sensitive compound is covalently bonded to the particles in the composition through a joint. In some cases, the joint is a cleavable joint. In some cases, the joint is a non-cleavable joint. In some cases, the irradiation power density sensitive compound is associated with the particles through 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 irradiation power density sensitive compound is an up-conversion nanoparticle (UCNP). In some cases, the nanoparticle is a lanthanide-doped particle. In some cases, the nanoparticle is a europium-doped or thulium-doped nanoparticle, such as NaYF4: Yb, Er nanoparticles or NaYF4: YbTm nanoparticles.

[0008] In some embodiments, the irradiated particles in the composition have a spectral emission intensity profile that changes in response to the power density of the light source. In some cases, the irradiated particles of the composition have 2 or more emission peaks in response to irradiation by the light source. In some cases, the intensity of each emission peak depends on the irradiation power intensity of the light source. In certain embodiments, one or more of the emission 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. In some cases, the irradiated particles of the composition have: a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density, and a second emission peak having the same intensity at the first irradiation power density and the second irradiation power density.

[0009] In certain embodiments, the composition is irradiated in the flow stream with a light source (e.g., a laser) having a Gaussian beam power density profile on 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, for example, wherein the major axis of the beam spot is orthogonal to the horizontal axis of the core stream. In some cases, the laser is configured to irradiate the core stream so that the irradiation power density along the horizontal axis of the flow stream has a larger power density at or near the center of the core stream, and a lower power density at or near the edge of the core stream.

[0010] In some embodiments, the method includes determining the position of the irradiated particles of the composition based on the difference between the emission intensities of the first emission peak and the second emission peak. In some cases, when the first emission peak and the second emission peak have approximately the same intensity, it is determined that the irradiated particles are passed through the core stream at approximately the center of the irradiation position of the light source. In some cases, when the first emission peak and the second emission peak have different intensities, it is determined that the irradiated particles are passed through the core stream at the edge of the irradiation position of the light source. In some cases, when the first emission peak and the second emission peak have approximately the same intensity, it is determined that the irradiated particles are passed through the core stream at approximately the center of the core stream. In some cases, when the first emission peak and the second emission peak have different intensities, it is determined that the irradiated particles are passed through the core stream at the edge of the core stream.

[0011] In some cases, the method includes determining that the irradiated particles are passed through the flow stream at or near the center of the core stream. In some cases, the method includes determining that the irradiated particles are passed through the flow stream at or near the edge of the core stream. In some cases, one or more particle population clusters are generated based on the determined positions of the irradiated particles in the core stream. In some embodiments, the particles determined to be passed through the flow stream at or near the center of the core stream are clustered together. In some embodiments, the particles determined to be passed through the flow stream at or near the edge of the core stream are clustered together. In certain embodiments, a data signal generated in response to light is discarded, the light coming from the irradiated particles of the composition determined to be passed through the flow stream at or near the edge of the core stream.

[0012] Aspects of the present disclosure include systems for determining position information of particles in a flow stream. A system according to certain embodiments includes a flow cell configured to deliver a composition having particles in a flow stream comprising a core flow and a sheath flow stream; a light source for irradiating the particles of the composition; a light detection system for detecting light from the irradiated particles; and a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to determine the position of the particles in the flow stream based on the detected light emission from the irradiated particles.

[0013] In some embodiments, the light source includes one or more lasers. In some cases, the light source is a laser configured to have a Gaussian beam power density profile on the horizontal axis of the flow stream. In some cases, the laser includes an elliptical beam spot having a major axis and a minor axis. In some cases, the laser is configured to irradiate the core flow with the elliptical beam spot so that the major axis of the beam spot is orthogonal to the horizontal axis of the flow stream. In some cases, the laser is configured to irradiate the core flow so that the irradiation power density along the horizontal axis of the flow stream has a larger power density at or near the center of the core flow, and a lower power density at or near the edge of the core flow. In some embodiments, the light source includes an optical adjustment component configured to transfer light to the flow stream so that the major axis of the beam spot is orthogonal to the horizontal axis of the flow stream. In some cases, the optical adjustment component is configured to rotate the beam spot by 90°. In some cases, the optical adjustment component includes one or more lenses. In some embodiments, the light source includes a plurality of lasers. In some cases, the light source includes at least one laser having an elliptical beam spot, the 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 orthogonal to the longitudinal axis of the flow stream; and a laser having an elliptical beam spot, the 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 orthogonal to the horizontal axis of the flow stream.

[0014] In some embodiments, the system includes a processor including a memory operably coupled to the processor, wherein the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to determine the position of the irradiated particles of the composition based on the difference between the emission intensities of the first emission peak and the second emission peak. In some cases, the memory includes instructions for determining that the irradiated particles are passed through the core flow at approximately the center of the irradiation position of the light source when the first emission peak and the second emission peak have approximately the same intensity. In some cases, the memory includes instructions for determining that the irradiated particles are passed through the core flow at the edge of the irradiation position of the light source when the first emission peak and the second emission peak have different intensities. In some cases, the memory includes instructions for determining that the irradiated particles are passed through the core flow at approximately the center of the core flow when the first emission peak and the second emission peak have approximately the same intensity. In some cases, the memory includes instructions for determining that the irradiated particles are passed through the core flow at the edge of the core flow when the first emission peak and the second emission peak have different intensities.

[0015] In some cases, the memory includes instructions for determining that the irradiated particles are passed through the flow stream at or near the center of the core stream. In some cases, the memory includes instructions for determining that the irradiated particles are passed through the flow stream at or near the edge of the core stream. In some cases, the memory includes instructions for generating one or more particle population clusters based on the determined positions of the irradiated particles in the core stream. In some embodiments, the memory includes instructions for clustering particles determined to be passed through the flow stream at or near the center of the core stream. In some embodiments, the memory includes instructions for clustering particles determined to be passed through the flow stream at or near the edge of the core stream. In certain embodiments, the memory includes instructions for discarding data signals generated in response to light, the light coming from irradiated particles of the composition determined to be passed through the flow stream at or near the edge of the core stream.

[0016] Aspects of the present disclosure also include a non-transitory computer-readable storage medium for determining position information of particles of a composition (e.g., cells of a biological sample) in a flow cytometer. In an embodiment, the non-transitory computer-readable storage medium includes: an algorithm for passing a composition having particles (e.g., cells) through a flow stream having a core flow and a sheath flow stream; an algorithm for irradiating the particles of the composition with a light source; an algorithm for detecting light from the irradiated particles; an algorithm for determining the emission intensity of the irradiated particles of the composition; and an algorithm for determining the position of the irradiated particles in the core flow of the flow stream based on the detected light emission from the irradiated particles.

[0017] In some embodiments, the irradiated particles in the composition are stably associated with an irradiation power density-sensitive compound that emits light, and the intensity of the light depends on the irradiation power density of the light source incident on the particles. In some embodiments, the irradiated particles are configured to have a spectral emission intensity profile that changes in response to the power density of the light source, for example, wherein the particles of the composition are configured to have 2 or more emission peaks in response to irradiation by the light source. In some cases, the intensity of each emission peak depends on the irradiation power intensity of the light source. In some cases, one or more of the emission peaks have a first intensity at the first irradiation power density of the light source and a second intensity at the second irradiation power density of the light source. In certain embodiments, the particles of the composition have: a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density, and a second emission peak having the same intensity at the first irradiation power density and the second irradiation power density.

[0018] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining the position of the irradiated particles of the composition based on the difference between the emission intensities of the first emission peak and the second emission peak. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining that the irradiated particles are passed through the core flow at approximately the center of the irradiation position of the light source when the first emission peak and the second emission peak have approximately the same intensity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining that the irradiated particles are passed through the core flow at the edge of the irradiation position of the light source when the first emission peak and the second emission peak have different intensities. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining that the irradiated particles are passed through the core flow at approximately the center of the core flow when the first emission peak and the second emission peak have approximately the same intensity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining that the irradiated particles are passed through the core flow at the edge of the core flow when the first emission peak and the second emission peak have different intensities.

[0019] In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating one or more particle population clusters based on the determined positions of the particles in the core flow. In some cases, the non-transitory computer-readable storage medium includes an algorithm for clustering particles that are determined to pass through the flow stream at or near the center of the core flow. In some cases, the non-transitory computer-readable storage medium includes an algorithm for clustering particles that are determined to pass through the flow stream at or near the edge of the core flow. In some cases, the non-transitory computer-readable storage medium includes an algorithm for discarding data signals generated in response to light from irradiated particles that are determined to pass through the flow stream at or near the edge of the core flow. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention can be better understood by reading the following detailed description in conjunction with the accompanying drawings, which include the following figures:

[0021] Figure 1 Determining position information for particles of a composition in a core stream according to certain embodiments is depicted.

[0022] Figure 2 Depicted is a flow chart for characterizing positional information of particles of a composition according to certain embodiments.

[0023] Figure 3 Depicted are light sources for irradiating a composition having particles in a flowing stream according to certain embodiments.

[0024] Figure 4A Depicted is a functional block diagram of a particle analysis system according to certain embodiments.

[0025] Figure 4B Depicted are flow cytometers according to certain embodiments.

[0026] Figure 5 A functional block diagram of one example of a particle analyzer control system according to certain embodiments is shown.

[0027] Fig. 6A Depicted is a schematic diagram of a particle sorting system according to certain embodiments.

[0028] Figure 6B Depicted is a schematic diagram of a particle sorting system according to certain embodiments.

[0029] Figure 7 A block diagram of a computing system is depicted in accordance with certain embodiments. DETAILED DESCRIPTION

[0030] Aspects of the present disclosure include methods for determining the position information of particles in the flow stream of a flow cytometer. The method according to certain embodiments includes: passing a composition with particles through a flow stream comprising a core flow and a sheath flow stream, irradiating the particles of the composition with a light source, detecting light from the irradiated particles, determining the light emission from the irradiated particles of the composition, and determining the position of the irradiated particles in the core stream of the flow stream based on the detected light emission. In an embodiment, particles are stably associated (e.g., covalently bonded) with an irradiation power density-sensitive compound of the emitted light, and the intensity of the light depends on the irradiation power density of the light source incident on the particles. A system (e.g., a flow cytometer) for practicing the subject method is also described. A non-transitory computer-readable storage medium is also provided.

[0031] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, and therefore may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

[0032] Where a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of the range (unless the context clearly indicates otherwise, up to one tenth of the unit of the lower limit) and any other stated or intermediate value in the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also encompassed within the present invention, but are subject to the impact of any specifically excluded limitations in the stated range. When the stated range includes one or two limits, ranges excluding one or both of the included limits are also encompassed within the present invention.

[0033] Certain ranges of values ​​given herein are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that, in the context presented, provides a substantial equivalent to the specifically recited number.

[0034] 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 the invention belongs. Representative illustrative methods and materials are now described, 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.

[0035] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with the publication cited. 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 publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0036] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0037] As will be apparent to those skilled in the art after reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any described method can be executed in the order of events described or in any other order that is logically possible.

[0038] The apparatus and method have been or will be described for grammatical fluency and functional explanation, but it should be clearly understood that unless expressly stated in accordance with 35 U.S.C. §112, the claims should not be interpreted as being necessarily limited in any way to the interpretation of "means" or "step" limitations, but should be given the full scope of the meaning and equivalents provided by the claims under the doctrine of equivalents, and the case where the claims are expressly stated in accordance with 35 U.S.C. §112 should be given all legal equivalents under 35 U.S.C. §112.

[0039] As described above, aspects of the present disclosure provide methods for determining position information of particles in a flow stream in a flow cytometer. In further describing embodiments of the present disclosure, a method for determining position information of particles having an irradiation power density sensitive compound in a core stream of a flow stream is first described, wherein the intensity of light emitted by the irradiation power density sensitive compound depends on the irradiation power density of the light source incident on the particles. Next, a system (e.g., a flow cytometer) comprising a light source and a light detection system for practicing the subject method is described. A non-transitory computer-readable storage medium is also provided.

[0040] Method for determining position information of particles in a flow stream of a flow cytometer

[0041] Aspects of the present disclosure include methods for determining the position information of particles in the flow stream of a flow cytometer. In some embodiments, the present disclosure provides a method for modifying or adjusting the data signal analysis of particles of a sample composition based on the position information of particles in the flow stream. In some embodiments, the subject method allows the flow position of particles in the core stream of a flow stream comprising a core stream and a sheath flow stream to be determined. In some cases, it can be determined that the particles flow at or near the center of the core stream. In other cases, it can be determined that the particles flow at or near the edge of the core stream. In some embodiments, the methods described herein allow the position of one or more particles relative to the position of the light source irradiating the core stream to be determined. In some cases, the particles in the composition are determined to be passed through the core stream at a position located at or near the center of the light source irradiation. In other cases, the particles in the composition are determined to be passed through the core stream at a position located at or near the edge of the light source irradiation.

[0042] The intensity profile of the light used to irradiate the flow stream in the flow cytometer can vary between irradiation spots, such as the power density at the center of the laser beam is the highest, while the power density decreases as the distance from the center of the beam spot increases. Particles that pass directly through the center of the beam spot can receive a greater laser power intensity than particles that do not pass through the center of the beam spot. In some cases, fluorophores present on particles that pass through the periphery of the beam spot will be suboptimally excited, resulting in lower brightness and greater noise in the data signal from the detected light. In some cases, the method described herein for determining position information of particles in the core stream allows for smaller variations in the irradiation of particles in the core stream, for example, where the irradiation intensity incident on the particles of the composition varies by 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less and including 0.001% or less, between the entire core stream. In certain embodiments, the subject methods allow for determination of which particles in a sample composition are suboptimally excited, and adjustment or discarding of data signals from particles that are suboptimally excited by irradiation with a light source. In certain embodiments, the subject methods improve the signal-to-noise ratio of data signals generated in response to light from irradiated particles of a sample composition by, for example, 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and including 99% or more.

[0043] In practicing the subject method, a composition having particles passing through a core stream is irradiated with a light source. In some embodiments, the light source is a continuous wave light source, e.g., where the light source provides an uninterrupted light flux and maintains irradiation of sample particles in the flow stream with little or no undesirable changes in light intensity. In some embodiments, a continuous light source emits non-pulsed or non-strobed irradiation. In certain embodiments, a continuous light source provides a substantially constant emitted light intensity. For example, the method may include irradiating a sample in a flowing stream with a continuous light source, the light source emitting light whose intensity does not vary by more than 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, such as 0.00001% or less, and including wherein the amplitude of the variation of the emitted light intensity during the irradiation time interval does not exceed 0.000001%. The intensity of the light output may be measured by any convenient scheme, including but not limited to a scanning slit profilometer, a charge coupled device (CCD, such as an intensified charge coupled device, ICCD), a positioning sensor, a power sensor (such as a thermopile power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector, and other types of photodetectors.

[0044] In other embodiments, the composition in the flow stream is irradiated with a pulsed light source, e.g., wherein light is emitted at predetermined time intervals, each time interval having a predetermined irradiation duration (i.e., pulse width). In certain embodiments, the method comprises irradiating the sample with a pulsed light source in each interrogation region of the flow stream with a periodic flash. For example, the frequency of each light pulse can be 0.0001 kHz or greater, e.g., 0.0005 kHz or greater, e.g., 0.001 kHz or greater, e.g., 0.005 kHz or greater, e.g., 0.01 kHz or greater, e.g., 0.05 kHz or greater, e.g., 0.1 kHz or greater, e.g., 0.5 kHz or greater, e.g., 1 kHz or greater, e.g., 2.5 kHz or greater, e.g., 5 kHz or greater, e.g., 10 kHz or greater, e.g., 25 kHz or greater, e.g., 50 kHz or greater, and including 100 kHz or greater. In some cases, the pulse irradiation frequency of the light source ranges from 0.00001 kHz to 1000 kHz, such as 0.00005 kHz to 900 kHz, such as 0.0001 kHz to 800 kHz, such as 0.0005 kHz to 700 kHz, such as 0.001 kHz to 600 kHz, such as 0.005 kHz to 500 kHz, such as 0.01 kHz to 400 kHz, such as 0.05 kHz to 300 kHz, such as 0.1 kHz to 200 kHz, and including 1 kHz to 100 kHz. The light irradiation duration (i.e., pulse width) of each light pulse can vary, and can be 0.000001ms or longer, for example 0.000005ms or longer, for example 0.00001ms or longer, for example 0.00005ms or longer, for example 0.0001ms or longer, for example 0.0005ms or longer, for example 0.001ms or longer, for example 0.005ms or longer, for example 0.01ms or longer, for example 0.05ms or longer, for example 0.1ms or longer, for example 0.5ms or longer, for example 1ms or longer, for example 2ms or longer, for example 3ms or longer, for example 4ms or longer, for example 5ms or longer, for example 10ms or longer, for example 25ms or longer, for example 50ms or longer, for example 100ms or longer, and including 500ms or longer.For example, the duration of light irradiation can range from 0.000001ms to 1000ms, for example, 0.000005ms to 950ms, for example, 0.00001ms to 900ms, for example, 0.00005ms to 850ms, for example, 0.0001ms to 800ms, for example, 0.0005ms to 750ms, for example, 0.001ms to 700ms, for example, 0.005ms to 650ms, for example, 0.01ms to 600ms, for example, 0.05ms to 550ms, for example, 0.1ms to 500ms, for example, 0.5ms to 450ms, for example, 1ms to 400ms, for example, 5ms to 350ms, and including 10ms to 300ms.

[0045] The flow stream can be irradiated with any convenient light source, and can include lasers and non-laser light sources (e.g., light emitting diodes). In certain embodiments, the method comprises irradiating the particles with a laser, such as a pulsed or continuous wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near infrared diode laser. In other embodiments, the laser can 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 2 Lasers of interest include, for example, 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, and combinations thereof. In other cases, the subject system includes a solid-state laser, such as a ruby ​​laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO laser, a Nd:YV laser, a Nd:YV 4 Laser, Nd:YCa4O(BO 3 ) 3 Laser, Nd:YCOB laser, titanium sapphire laser, thulium YAG laser, ytterbium YAG laser, ytterbium 2 O 3 Laser or cerium-doped laser and a combination thereof.

[0046] In some embodiments, the light source outputs a specific wavelength, such as 200nm to 1500nm, such as 250nm to 1250nm, such as 300nm to 1000nm, such as 350nm to 900nm, and including 400nm to 800nm. In certain embodiments, the continuous wave light source emits light with a wavelength of 365nm, 385nm, 405nm, 460nm, 490nm, 525nm, 550nm, 580nm, 635nm, 660nm, 740nm, 770nm or 850nm.

[0047] The light source may irradiate the flow stream from any suitable distance, for example at 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, and including a distance of 100 mm or more. In addition, the irradiation of the flow stream may be at any suitable angle, 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°, and including an angle of 30° to 60°, for example 90°.

[0048] In some embodiments, the method includes further adjusting the light from the flow stream before detecting the light. For example, the light can pass through one or more lenses, reflectors, pinholes, slits, gratings, light refractors, and any combination thereof. In some cases, the collected light passes through one or more focusing lenses, for example to reduce the profile of the light. In other cases, the emitted light from the sample passes through one or more collimators to reduce beam divergence.

[0049] In certain embodiments, the method includes irradiating the flow stream with two or more frequency-shifted beams. A beam generator assembly with a laser and an acousto-optic device can be used to frequency-shift the laser. In these embodiments, the method includes irradiating the acousto-optic device with a laser. According to the desired wavelength of the light produced in the output laser beam (for example, for irradiating the sample in the flow stream), the laser can have a specific wavelength varying from 200nm to 1500nm (for example, 250nm to 1250nm, for example, 300nm to 1000nm, for example, 350nm to 900nm, and including 400nm to 800nm). The acousto-optic device can be irradiated with one or more lasers, for example, 2 or more lasers, for example, 3 or more lasers, for example, 4 or more lasers, for example, 5 or more lasers, and including 10 or more lasers. The laser can include any type of laser combination. For example, in some embodiments, the method includes irradiating the acousto-optic device with a laser array (eg, an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers).

[0050] When more than one laser is used, the acousto-optic device may be irradiated with the laser simultaneously or sequentially or in combination. For example, the acousto-optic device may be irradiated with each laser simultaneously. In other embodiments, the acousto-optic device is irradiated sequentially with each laser. When more than one laser is used to sequentially irradiate the acousto-optic device, the time for each laser to irradiate the acousto-optic device may be independently 0.001 microseconds or longer, such as 0.01 microseconds or longer, such as 0.1 microseconds or longer, such as 1 microsecond or longer, such as 5 microseconds or longer, such as 10 microseconds or longer, such as 30 microseconds or longer and including 60 microseconds or longer. For example, the method may include irradiating the acousto-optic device with a laser for a duration ranging from 0.001 microseconds to 100 microseconds, such as 0.01 microseconds to 75 microseconds, such as 0.1 microseconds to 50 microseconds, such as 1 microsecond to 25 microseconds and including 5 microseconds to 10 microseconds. In embodiments where the acousto-optic device is sequentially irradiated with two or more lasers, the duration that the acousto-optic device is irradiated by each laser may be the same or different.

[0051] In an embodiment, the method includes applying a radio frequency drive signal to the acousto-optic device to generate an angularly deflected laser beam. Two or more radio frequency drive signals may be applied to the acousto-optic device to generate an output laser beam having a desired number of angularly deflected laser beams, such as 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and including 100 or more radio frequency drive signals.

[0052] The angularly deflected laser beams generated by the RF drive signal each have an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal having an amplitude sufficient to generate an angularly deflected laser beam with a desired intensity. In some cases, each applied RF drive signal independently has an amplitude of about 0.001V to about 500V, such as about 0.005V to about 400V, such as about 0.01V to about 300V, such as about 0.05V to about 200V, such as about 0.1V to about 100V, such as about 0.5V to about 75V, such as about 1V to 50V, such as about 2V to 40V, such as 3V to about 30V, and including about 5V to about 25V. In some embodiments, each applied RF drive signal has a frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0053] In these embodiments, the angle deflected laser beams in the output laser beam are spatially separated. Depending on the applied RF drive signal and the desired irradiation profile of the output laser beam, the angle deflected laser beams can 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, and including 5000 μm or more. In some embodiments, the angle deflected laser beams overlap, such as overlapping with adjacent angle deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., the overlap of beam spots) can be an overlap of 0.001 μm or greater, for example, an overlap of 0.005 μm or greater, for example, an overlap of 0.01 μm or greater, for example, an overlap of 0.05 μm or greater, for example, an overlap of 0.1 μm or greater, for example, an overlap of 0.5 μm or greater, for example, an overlap of 1 μm or greater, for example, an overlap of 5 μm or greater, for example, an overlap of 10 μm or greater, and including an overlap of 100 μm or greater.

[0054] In some cases, a flow stream is irradiated with multiple frequency-shifted beams, and cells in the flow stream are imaged by fluorescence imaging using radiofrequency tagged emission (FIRE) to produce frequency-encoded images, e.g., 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, 10,40 8,758, 10,451,538, 10,620,111 and those described in U.S. 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.

[0055] In some cases, the core flow is irradiated with an elliptical beam spot having a major axis and a minor axis, for example, wherein the major axis of the beam spot is orthogonal to the horizontal axis of the core flow. In certain embodiments, the flow stream is irradiated with a light source (described in more detail below) having a Gaussian beam power density profile along the horizontal axis of the core flow. In some cases, the laser is configured to irradiate the core flow so that the irradiation power density along the horizontal axis of the flow stream has a larger power density at or near the center of the core flow and a lower power density at or near the edge of the core flow. In some cases, the intensity profile of the output laser beam includes an increase 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 intensity of the output laser beam at the edge of the output beam can be 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and including the intensity of the output laser beam at the edge of the output beam being 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 includes a Gaussian distribution along the horizontal axis of the output laser beam. In still other cases, the intensity profile of the output laser beam includes a super-Gaussian distribution along the horizontal axis of the output laser beam.

[0056] In some embodiments, the laser includes an optical adjustment component (e.g., one or more lenses) configured to rotate the output laser beam by 90° so that the major axis of the elliptical beam spot is parallel to the longitudinal axis of the flow stream (i.e., the minor axis of the elliptical beam spot is irradiated on the horizontal axis of the flow stream). In some cases, the optical adjustment component is configured to generate the output laser beam so that the irradiation power density along the horizontal axis of the flow stream has a larger power density at or near the center of the core stream and a lower power density at or near the edge of the core stream. In some cases, the light source includes an optical adjustment component for providing an intensity profile of the output laser beam, the intensity of which 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 optical adjustment assembly produces an output laser beam having an intensity at the edge that is 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and includes an output laser beam having an intensity at the edge of the output beam that is about 10% to about 50% of the intensity at the center of the output laser beam along the horizontal axis. In still other cases, the optical adjustment assembly produces an output laser beam having an intensity profile having a Gaussian distribution along the horizontal axis. In still other cases, the optical adjustment assembly produces an output laser beam having an intensity profile having a super-Gaussian distribution along the horizontal axis.

[0057] Light from the irradiated flow stream is detected using a light detection system having a photodetector. The photodetector can be any convenient light detection scheme, including but not limited to a photoelectric sensor or photodetector, such as an active pixel sensor (APS), an avalanche photodiode (APD), a quadrant photodiode, an image sensor, a charge coupled device (CCD), an intensified charge coupled device (ICCD), a light emitting diode, a photon counter, a radiation thermometer, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube, a phototransistor, a quantum dot photoconductor or a photodiode and combinations thereof, and other photodetectors. In certain embodiments, the photodetector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area per region in the range of 0.01 cm 2 Up to 10cm 2 , for example 0.05cm 2 Up to 9cm 2 , for example 0.1cm 2 Up to 8cm 2 , for example 0.5cm 2 Up to 7cm2 And including 1cm 2 Up to 5cm 2 .

[0058] The photodetector can measure light at one or more wavelengths (e.g., 2 or more wavelengths, e.g., 5 or more different wavelengths, e.g., 10 or more different wavelengths, e.g., 25 or more different wavelengths, e.g., 50 or more different wavelengths, e.g., 100 or more different wavelengths, e.g., 200 or more different wavelengths, e.g., 300 or more different wavelengths), and includes measuring light from particles in the flow stream at 400 or more different wavelengths. In some embodiments, fluorescence from the sample is detected by the light detection system within one or more wavelengths of 200nm-1200nm. In some cases, the method includes detecting fluorescence within a range of wavelengths from the sample, e.g., 200nm to 1200nm, e.g., 300nm to 1100nm, e.g., 400nm to 1000nm, e.g., 500nm to 900nm wavelengths, and includes 600nm to 800nm ​​wavelengths. In other cases, the method includes detecting fluorescence of one or more specific wavelengths. For example, fluorescence can be detected at one or more of 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, 617nm, and any combination thereof, depending on the number of different fluorescence detectors in the subject light detection system. In certain embodiments, the method includes detecting a wavelength of light corresponding to a peak fluorescence wavelength of certain fluorescent dyes present in the sample.

[0059] Light can be measured continuously or at discontinuous 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 discontinuous intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds or some other interval measurement of light. The light from the flowing stream can be measured once or more times in each discontinuous time interval, such as 2 times or more times, such as 3 times or more times, such as 5 times or more times and including 10 times or more times. In certain embodiments, the photodetector measures the light from the flowing stream 2 times or more, and in some cases the data is averaged.

[0060] Each photodetector can be positioned at any suitable distance from the flow stream, as long as a usable light signal can be detected. For example, the detector in the subject system can be positioned at a distance of 1mm or more from the flow stream, such as 5mm or more, such as 10mm or more, such as 15mm or more, such as 25mm or more, such as 50mm or more, such as 100mm or more, such as 150mm or more, such as 250mm or more, and including 500mm or more from the flow stream. The detector can also be positioned at any angle to the flow stream. For example, the detector can be at an angle of 10° to 90° with the vertical axis of the flow stream, such as 15° to 85°, such as 20° to 80°, such as 25° to 75° and including 30° to 60°. In some cases, one or more detectors are positioned at an angle of 30° to 60° with the vertical axis of the flow stream.

[0061] 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 irradiated flow stream can be detected by one or more detectors configured as forward scattering detectors. In these embodiments, the forward scattering detector is positioned on the side of the flow stream opposite to the light source, and is positioned to collect and detect forward propagation (e.g., scattered) light. In some embodiments, the light from the irradiated sample in the flow stream is detected in one or more fluorescence detector channels. In some embodiments, the light from the irradiated flow stream is detected in one or more scattered light detector channels.

[0062] In some embodiments, the method includes determining the spectral emission intensity profile of the particles of the composition transmitted by the core flow of the flow stream. In some embodiments, the flow stream includes a core flow and a sheath flow stream, and the sheath flow stream flows together with the core flow (e.g., in a laminar manner). In some embodiments, the composition includes particles, and the particles have an irradiation power density-sensitive compound that emits light, and the intensity of the light depends on the irradiation power density of the light source incident on the particles. In other words, the irradiated particles of the composition show property changes based on the irradiation intensity of the light source. In some cases, the irradiated particles show emission intensity changes based on the irradiation intensity of the light source incident on the particles. In some embodiments, in response to the increase in the irradiation power density of the light source incident on the particles, the irradiated particles show an increase in emission intensity. In some embodiments, in response to the increase in the irradiation power density of the light source incident on the particles, the irradiated particles show a reduction in emission intensity.

[0063] In some cases, the composition described herein is a biological sample, and the particles are cells. The term "biological sample" refers to a subset of a whole biological product, plant, fungus or animal tissue, cell or component in its conventional sense, and may be present in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal secretions and semen in some cases. Therefore, "biological sample" refers to both natural organisms or their tissue subsets, and also refers to homogenates, lysates or extracts prepared from organisms or their tissue subsets, including but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and urogenital tract, tears, saliva, milk, blood cells, tumors, organs. Biological samples can be any type of biological tissue, including healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or its derivatives (e.g., plasma), tears, urine, semen, etc., wherein in some cases, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger stick blood collection (wherein the blood may or may not be mixed with any reagents such as preservatives, anticoagulants, etc. prior to assay).

[0064] In certain embodiments, the source of the composition is "mammals" or "mammals", which are terms widely used to describe organisms in the class Mammalia, including 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 method can be applied to samples obtained from human subjects of both sexes at any stage of development (i.e., newborns, infants, teenagers, adolescents, adults), wherein in certain embodiments, the human subject is a teenager, adolescent, or adult. Although the present invention is applicable to samples from human subjects, it should be understood that the method can also be performed on samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0065] In an embodiment, each irradiation power density sensitive compound is stably associated with a particle. Stable association means that the irradiation power density sensitive compound is not easily dissociated from the particle and contacts with a liquid medium (e.g., an aqueous medium). In some embodiments, one or more irradiation power density sensitive compounds are covalently conjugated to the particle. In other embodiments, one or more irradiation power density sensitive compounds are physically associated with the particle (i.e., non-covalently linked). In other embodiments, one or more irradiation power density sensitive compounds are covalently conjugated to the particle, and one or more irradiation power density sensitive compounds are physically associated with the particle.

[0066] In some embodiments, the irradiation power density sensitive compound is coupled to the particles in the composition. In some cases, the irradiation power density sensitive compound is bonded to the particles in the composition by one or more of hydrogen bonding, dipole-dipole interaction or ionic bonding. In some cases, the irradiation power density sensitive compound is covalently bonded to the particles in the composition. In some cases, the irradiation power density sensitive compound is directly covalently bonded to the particles. In some cases, the irradiation power density sensitive compound is covalently bonded to the particles through a joint. The joint can be any convenient covalent connection scheme, such as a zero-length cross-linker, a homobifunctional joint, a heterobifunctional joint or a trifunctional cross-linker. In some cases, the joint is a cleavable joint. In some cases, the joint is a non-cleavable joint. The joint may include one or more functional groups as needed, such as amides, alkylamines, carbamates, carbonates, thiol ethers, alkyls, cycloalkyls or aryl moieties. In some embodiments, the joint includes a carbamate moiety. In some cases, the joint includes a chemically selective functional group, such as for conjugation with a comonomer or a biomolecule. In certain embodiments, the joint is an amine-containing joint. In some cases, the linker is an N-hydroxysuccinimide (NHS) ester with one or more maleimide groups, which allows conjugation to sulfhydryl groups. In certain embodiments, the irradiation power density sensitive compound is covalently bonded to the particles in the composition via a linker as described in U.S. Pat. No. 11,333,666, U.S. Patent Publication Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20110257374, 20120028828, 20120252986, 20130190193, 20160264737, 20160266131, 20180231530, 20180009990, 20180009989, 20180163054, and 20220089877; the disclosures of which are incorporated herein by reference.

[0067] In some embodiments, the irradiation power density sensitive compound is a 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 irradiation power density sensitive compound described herein can be conjugated with a specific binding member. Specific binding members and irradiation power density sensitive compounds can be conjugated to each other (e.g., covalently linked) via an optional joint at any convenient position of two molecules. In some embodiments, the labeled specific binding member is anti-aggregation. As used herein, "anti-aggregation" refers to a labeled specific binding member that can form a homogeneous aqueous composition without forming an aggregated precipitate in an aqueous buffer of interest at 1 mg / ml or higher (e.g., 2 mg / ml or higher, 3 mg / ml or higher, 4 mg / ml or higher, 5 mg / ml or higher, 6 mg / ml or higher, 7 mg / ml or higher, 8 mg / ml or higher, 9 mg / ml or higher, 10 mg / mL or higher or even higher) concentration of labeled specific binding members.

[0068] As used herein, the term "specific binding member" refers to one member of a pair of molecules that have binding specificity to each other. One member of the pair of molecules may have an area or cavity on its surface that specifically binds to an area on the surface of, or a cavity therein, the other member of the pair of molecules. Thus, the members of the pair have the property of specifically binding to each other to produce a binding complex. In some embodiments, the affinity between the specific binding members in the binding complex is characterized by a K d (dissociation constant) is 10 -6 M or smaller, such as 10 -7 M or smaller, including 10 -8 M or less, for example, 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, including 10 -15 In some embodiments, the specific binding member specifically binds with high affinity. High affinity means that the binding member specifically binds with a certain apparent affinity, which is characterized by an apparent K d 10x 10 -9 M or smaller, such as 1x 10 -9 M or smaller, 3x 10 -10 M or smaller, 1x 10 -10 M or smaller, 3x 10 -11 M or smaller, 1x 10-11 M or smaller, 3x 10 -12 M or smaller, or 1x10 -12 M or smaller.

[0069] Specific binding members can be proteinaceous. As used herein, the term "proteinaceous" refers to a portion consisting of amino acid residues. The proteinaceous portion can be a polypeptide. In some cases, proteinaceous specific binding members are antibodies. In certain embodiments, proteinaceous specific binding members are antibody fragments, such as antibody binding fragments that specifically bind to polymer dyes. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides encoded by immunoglobulin genes that are substantially recognized in whole or in part. For example, in humans, recognized immunoglobulin genes include κ (k), λ (l) and heavy chain genetic loci, which together contain countless variable region genes, and constant region genes μ (u), δ (d), γ (g), σ (e) and α (a), which encode IgM, IgD, IgG, IgE and IgA isotypes, respectively. Immunoglobulin light chain or heavy chain variable region is composed of a "framework" region (FR) interrupted by three hypervariable regions, also referred to as a "complementary determining region" or "CDR". The scope of framework region and CDR has been precisely defined (see "Sequences of Proteins of Immunological Interest", E. Kabat et al., US Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light chains or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework region of the light chain and the heavy chain, is used to position and arrange the CDR. The CDR is primarily responsible for binding to the antigenic epitope. The term "antibody" is intended to include full-length antibodies and may refer to natural antibodies, engineered antibodies, or recombinantly produced antibodies from any organism for experimental, therapeutic or other purposes, as further defined below.

[0070] Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv or other antigen-binding subsequences of antibodies, which are produced by modification of intact antibodies or synthesized de novo using recombinant DNA technology. Antibodies can be monoclonal or polyclonal and can have other specific activities on cells (e.g., antagonistic antibodies, agonistic antibodies, neutralizing antibodies, inhibitory antibodies or stimulating antibodies). It is understood that antibodies can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions.

[0071] In certain embodiments, the specific binding member is a Fab fragment, F(ab') 2 In some embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or a binding fragment thereof. In some cases, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0072] In certain embodiments, the irradiation power density sensitive compound is associated with the particles of the composition through a specific binding member. For example, the irradiation power density sensitive compound can be covalently bonded to a protein binding member, such as an antibody or antibody fragment, and the antibody or antibody fragment is associated with the particles of the composition. In certain embodiments, the irradiation power density sensitive compound is covalently bonded (e.g., directly or through a linker) to a binding member having binding specificity to a receptor on the particle surface (e.g., a surface-bound protein or antigen).

[0073] The irradiation power density sensitive compound can be any compound that exhibits a property change in response to a change in the power density of the light source irradiation, such as a change in the characteristics of the emission spectrum profile or intensity. In some embodiments, the irradiation power density sensitive compound is an up-conversion nanoparticle (UCNP). In some cases, the nanoparticle is a lanthanide-doped particle. In some cases, the nanoparticle is a europium-doped or thulium-doped nanoparticle, such as NaYF4:Yb,Er nanoparticles or NaYF4:YbTm nanoparticles.

[0074] In certain embodiments, the irradiated particles in the composition have a spectral emission intensity profile that changes in response to the power density of the light source. In some embodiments, the irradiated particles of the composition have 2 or more emission peaks in response to the irradiation from the light source, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more and include 10 or more emission peaks. In some cases, one or more peaks of the spectral profile show a change in intensity in response to the power density of the light source. In some cases, the intensity of each emission peak depends on the irradiation power intensity of the light source. In an example, one or more of the emission peaks have a first emission intensity at a first irradiation power density of the light source and a second emission intensity at a second irradiation power density of the light source. In some cases, the emission peak has a higher emission intensity at a higher irradiation power density of the light source and a lower emission intensity at a lower irradiation power density. In some cases, when the particle passes through the core flow at or near the center of the light source irradiation, one or more emission peaks have a first emission intensity, and when the particle passes through the core flow at or near the edge of the light source irradiation, one or more emission peaks have a second emission intensity. In these cases, where the center of the core flow is aligned with the center of the light source irradiation (e.g., the center of the beam spot is at the center of the core flow), when the particle passes at or near the center of the core flow, one or more emission peaks have a first emission intensity, and when the particle passes at or near the edge of the core flow, one or more emission peaks have a second emission intensity.

[0075] In some embodiments, the method includes determining position information of irradiated particles of the composition based on the emission intensity of one or more emission peaks. In one example, when the emission peak exhibits a high emission intensity, the particle is determined to be passed at or near the center of the core flow, and when the emission peak exhibits a low emission intensity, the particle is determined to be passed at or near the edge of the core flow. In another example, when the emission peak exhibits a low emission intensity, the particle is determined to be passed at or near the center of the core flow, and when the emission peak exhibits a high emission intensity, the particle is determined to be passed at or near the edge of the core flow.

[0076] In certain embodiments, the irradiated particles of the composition have: a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density, and a second emission 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 emission peak depends on the irradiation power density of the light source, while the intensity of the second emission peak remains unchanged. In one example, the first emission peak has a higher emission intensity at a higher irradiation power density of the light source and a lower emission intensity at a lower irradiation power density. The second emission peak has the same emission intensity at higher and lower irradiation power densities of the light source. In some cases, when the particles are passed through the core flow at or near the irradiation center of the light source, the first emission peak has a first emission intensity, and when the particles are passed through the core flow at or near the irradiation edge of the light source, the first emission peak has a second emission intensity. When the particles are passed through the core flow at or near the irradiation center of the light source and when the particles are passed through the core flow at or near the irradiation edge of the light source, the second emission peak has the same emission intensity. In those cases, where the center of the core stream is aligned with the center of the light source irradiation (e.g., the center of the beam spot is in the center of the core stream), the first emission peak has a first emission intensity when the particle passes at or near the center of the core stream, and the first emission peak has a second emission intensity when the particle passes at or near the edge of the core stream. The second emission peak has the same emission intensity when the particle passes at or near the center of the core stream and when the particle passes at or near the edge of the core stream.

[0077] In some embodiments, the method includes determining position information of irradiated particles of the composition based on the emission intensities of the first and second emission peaks. In one example, when the first emission peak exhibits a high emission intensity, the particles are determined to be passed at or near the center of the core flow, and when the first emission peak exhibits a low emission intensity, the particles are determined to be passed at or near the edge of the core flow. In another example, when the first emission peak exhibits a low emission intensity, the particles are determined to be passed at or near the center of the core flow, and when the first emission peak exhibits a high emission intensity, the particles are determined to be passed at or near the edge of the core flow.

[0078] In other cases, the method includes determining the particle position based on the ratio of the intensity of the first emission peak to the intensity of the second emission peak. In some cases, the ratio of the intensity of the first emission peak to the intensity of the second emission peak ranges from 1:100 to 100:1, such as 1:75 to 75:1, such as 1:50 to 50:1, such as 1:25 to 25:1, such as 1:10 to 10:1, such as 1:9 to 9:1, such as 1:8 to 8:1, such as 1:7 to 7:1, such as 1:6 to 6:1, such as 1:5 to 5:1, and includes 1:4 to 4:1. In certain embodiments, when the ratio of the intensity of the first emission peak to the intensity of the second emission peak is 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 and including 0.95:1 to 1:0.95, it is determined that the particle is passed at or near the center of the core flow. In some cases, the particle is determined to be delivered at or near the center of the core stream (i.e., the particle is delivered through the center of the core stream (or when the first emission peak and the second emission peak have approximately the same emission intensity, the particle is delivered at the center of the light source irradiation) when the ratio of the intensity of the first emission peak to the intensity of the second emission peak is approximately 1:1. 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 the particle is delivered to the irradiation center (or the center of the core stream when the light source and the core stream are aligned).

[0079] Figure 1 Depicted is position information for particles of a composition in a core flow, according to certain embodiments. Particles (e.g., cells) coupled (e.g., covalently bonded) to an irradiation power density sensitive compound are irradiated in a flow stream at 100 by a light source that exhibits an elliptical beam spot, wherein the short axis of the elliptical beam spot is orthogonal to the longitudinal axis of the flow stream (i.e., the long axis of the beam spot is parallel to the longitudinal long axis of the flow stream). In embodiments, the beam spot on the horizontal axis exhibits an irradiation power density with a higher center intensity than the intensity at or near the edge of the beam spot. As Figure 1As shown, the flow stream is irradiated by an output laser beam having a Gaussian beam profile 110. The light source having a Gaussian beam profile 110 exhibits an irradiation power density, which has a greater intensity at or near the irradiation center, and the intensity decreases as it moves away from the center. The irradiation power density of the light source having a Gaussian beam profile 110 is lowest at or near the irradiation edge. The particle 100a exhibits a spectral emission profile having two emission peaks at 400nm and 800nm. The particle 100a passing through the left edge of the irradiation 101 by the light source exhibits a spectral profile 101a, wherein the emission peak at 400nm is greater than the emission peak at 800nm ​​(i.e., the ratio of the emission intensity at 400nm to the emission intensity at 800nm ​​is greater than 1). The particle 100a passing through the center of the irradiation 102 by the light source exhibits a spectral profile 102a, wherein the emission peak at 400nm is approximately the same as the emission peak at 800nm ​​(i.e., the ratio of the emission intensity at 400nm to the emission intensity at 800nm ​​is about 1). The particle 100a passing through the right edge of the irradiation 103 by the light source exhibits a spectral profile spectral profile 103a, wherein the emission peak at 400nm is greater than the emission peak at 800nm ​​(i.e., the ratio of the emission intensity at 400nm to the emission intensity at 800nm ​​is greater than 1). Based on the intensity difference between the emission peaks at 400nm and 800nm, the position of the particle can be determined. When the light source with a Gaussian beam profile 110 is aligned with the core flow of the flow stream 100, the position of the particle in the core flow can be determined. In the case where the emission peaks at 400nm and 800nm ​​have approximately the same intensity, it is determined that the particle passes at or near the center of the core flow. In the case where the emission peaks at 400nm and 800nm ​​have different intensities, it is determined that the particle passes at or near the edge of the core flow. In some cases, the distance from the center of the core flow is determined based on the intensity ratio between the emission peaks at 400nm and 800nm.

[0080] In certain embodiments, one or more particle population clusters are generated based on the determined positions of the irradiated particles in the core stream. In some cases, particles are classified as being irradiated at or near the center of the core stream based on the determined spectral emission intensity profile. In other cases, particles are classified as being irradiated at or near the edge of the core stream based on the determined spectral emission 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 certain embodiments, data signals from particles of a sample composition classified as being irradiated at or near the edge of the core stream are discarded.

[0081] Figure 2A flow chart for characterizing position information of particles of a composition according to certain embodiments is depicted. In step 201, a light source is irradiated in a core stream with an irradiation power density sensitive compound (e.g., Figure 1 In step 205a, particles transmitted at or near the center of the core flow are clustered together, while in step 205b, particles transmitted at or near the edge of the core flow are clustered together. In certain embodiments, data signals from particles of the composition determined to be transmitted at or near the edge of the core flow are discarded (step 210) and no further analysis is performed.

[0082] In certain embodiments, the particles of the composition also include one or more stably associated (e.g., by covalent bonds or by specific binding members as described above) fluorophores. In some embodiments, each particle includes 2 or more different types of fluorophores. If any two fluorophores differ from each other in one or more aspects of molecular formula, excitation maximum, and emission maximum, they are considered to be different or unique. Therefore, different or unique fluorophores may differ from each other in chemical composition or in one or more characteristics of fluorophores. For example, different fluorophores may differ from each other in at least one aspect of excitation maximum and emission maximum. In some cases, different fluorophores differ from each other in their excitation maximum. In some cases, different fluorophores differ from each other in their emission maximum. In some cases, different fluorophores differ from each other in both excitation maximum and emission maximum. Therefore, in embodiments including the first and second fluorophores, the first and second fluorophores may differ from each other in at least one aspect of excitation maximum and emission maximum. For example, the first and second fluorophores may differ from each other in excitation maximum, emission maximum, or excitation maximum and emission maximum. A given set of fluorophores can be considered different if they differ from each other in excitation maximum or emission maximum, where the magnitude of such difference in some cases is 5 nm or more, such as 10 nm or more, including 15 nm or more, where the magnitude of the difference in some cases ranges from 5 to 400 nm, such as 10 to 200 nm, including 15 to 100 nm, such as 25 to 50 nm.

[0083] According to some embodiments, the fluorophore of interest has an excitation maximum of 100nm to 800nm, such as 150nm to 750nm, such as 200nm to 700nm, such as 250nm to 650nm, such as 300nm to 600nm, and including 400nm to 500nm. According to some embodiments, the fluorophore of interest has an emission maximum of 400nm to 1000nm, such as 450nm to 950nm, such as 500nm to 900nm, such as 550nm to 850nm, and including 600nm to 800nm. In some cases, the fluorophore is a luminescent dye, such as a fluorescent dye having a peak emission wavelength of 200 nm or longer, such as 250 nm or longer, such as 300 nm or longer, such as 350 nm or longer, such as 400 nm or longer, such as 450 nm or longer, such as 500 nm or longer, such as 550 nm or longer, such as 600 nm or longer, such as 650 nm or longer, such as 700 nm or longer, such as 750 nm or longer, such as 800 nm or longer, such as 850 nm or longer, such as 900 nm or longer, such as 950 nm or longer, such as 1000 nm or longer, and including 1050 nm or longer. For example, the fluorophore can be a fluorescent dye having a peak emission wavelength range of 200 nm to 1200 nm, such as 300 nm to 1100 nm, such as 400 nm to 1000 nm, such as 500 nm to 900 nm, and includes a fluorescent dye having a peak emission wavelength of 600 nm to 800 nm. In certain embodiments, the subject multispectral particles allow stable excitation by lasers at or near wavelengths of 349 nm (UV laser), 488 nm (blue laser), 532 nm (Nd: YAG solid-state laser), 640 nm (red laser), and 405 nm (violet laser). In certain cases, the subject multispectral particles allow stable excitation by light sources with a full spectrum detection band (e.g., 350 nm to 850 nm).

[0084] In some cases, each particle comprises a fluorophore that emits fluorescence in response to irradiation with a light source. In some embodiments, the fluorophore of interest may include, but is not limited to, dyes suitable for analytical applications (e.g., flow cytometry, imaging, etc.), such as acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenylmethane dyes), chlorophyll-containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazo dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone imine dyes, azine dyes, eurhodin dyes, safranin dyes, indoleamines, indophenol dyes, fluorine dyes, oxazine dyes, oxone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronine dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, and dyes combining (e.g., in series) two or more of the above dyes, polymer dyes having one or more monomer dye units, and mixtures of two or more of the above dyes. A large number of dyes are commercially available from various 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 may 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 protein, 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; o-aminobenzamide; brilliant yellow; coumarin and its derivatives, such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151); cyanine and its derivatives, such as cyanine, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 4',6-dimethoxybenzylamine; Amino-2-phenylindole (DAPI); 5',5"-dibromopyrogallosulfonphthalein (bromopyrogallo red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonic acid Acyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); Eosin and derivatives, such as eosin and eosin isothiocyanate; Erythrosine and derivatives, such as erythrosine B and erythrosine isothiocyanate; Ethidium; Fluorescein and 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), chlorotriazinylfluorescein, naphthofluorescein and QFITC ​​(XRITC); Fluoresceinamine; IR144; IR1446; Green fluorescent protein (GFP); Reef coral fluorescent protein (RCFP); Lissamine. TM ; Lissamine, rhodamine, fluorescent yellow; malachite green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; pararosaniline; Nile red; Oregon green; phenol red; B-phycoerythrin; o-phthalaldehyde; pyrene and its derivatives, such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron TMBrilliant Red 3B-A); Rhodamine and its derivatives, such as 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), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rose hip acid and terbium chelate derivatives; xanthene; dye-conjugated polymers (i.e., polymer-attached dyes) such as fluorescein isothiocyanate-dextran, as well as dyes combining two or more dyes (e.g., in tandem), polymer dyes having one or more monomeric dye units, and mixtures of two or more of the above dyes, or combinations thereof.

[0085] In some cases, fluorophore is polymer dye. In some cases of method, polymer dye comprises conjugated polymer. Conjugated polymer (CP) is characterized in that has delocalized electronic structure, and this structure comprises the skeleton of unsaturated bond (such as double bond and / or triple bond) and saturated bond (such as single bond) alternating, and wherein π electron can move from one bond to another bond. Therefore, conjugated skeleton can give polymer dye extended linear structure, wherein the bond angle between polymer repeating unit is limited. For example, although protein and nucleic acid are also polymer, in some cases, can not form extended rod-like structure, but fold into higher order three-dimensional shape. In addition, CP can form " rigid rod " polymer backbone, and along polymer backbone, experience the limited distortion (such as torsion) angle between monomer repeating unit. In some cases, polymer dye comprises CP with rigid rod-like structure. The structural feature of polymer dye can affect the fluorescence characteristics of molecule.

[0086] Polymeric dyes of interest include, but are not limited to, those described in U.S. Patent Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20110257374, 20120028828, 20120252986, 20130190193, 20160264737, 20160266131, 20180231530, 20180009990, 20180009989, and 20180163054 to Gaylord et al., the disclosures of which are incorporated herein by reference in their entireties; and Gaylord 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), pp12600-12607, the disclosures of which are incorporated herein by reference in their entirety.

[0087] The polymer dye may have one or more desired spectral properties, such as a specific absorption maximum wavelength, a specific emission maximum wavelength, an extinction coefficient, a quantum yield, etc. (see, e.g., Chattopadhyay et al., Brilliant violetfluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments, Cytometry Part A, 81A (6), 456-466, 2012). In some embodiments, the absorption curve of the polymer dye is between 280 nm and 475 nm. In certain embodiments, the absorption maximum (excitation maximum) of the polymer dye is within the range of 280 nm and 475 nm. In some embodiments, the polymer dye absorbs incident light with a wavelength between 280 nm and 475 nm. In some embodiments, the maximum emission wavelength of the polymer dye ranges from 400 nm to 850 nm, such as 415 nm to 800 nm, wherein specific examples of the maximum emission wavelength 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 cases, the maximum emission wavelength range of the polymer dye is selected from 410nm to 430nm, 500nm to 520nm, 560nm to 580nm, 590nm to 610nm, 640nm to 660nm, 700nm to 720nm and 775nm to 795nm. In certain embodiments, the maximum emission wavelength of the polymer dye is 421nm. In some cases, the emission maximum wavelength of the polymer dye is 510nm. In some cases, the emission maximum wavelength of the polymer dye is 570nm. In certain embodiments, the emission maximum wavelength of the polymer dye is 602nm. In some cases, the emission maximum wavelength of the polymer dye is 650nm. In some cases, the emission maximum wavelength of the polymer dye is 711nm. In some embodiments, the emission maximum wavelength of the polymer dye is 786nm. In some cases, the emission maximum wavelength of the polymer dye is 421nm ± 5nm. In some embodiments, the emission maximum wavelength of the polymer dye is 510nm ± 5nm. In some cases, the emission maximum wavelength of the polymer dye is 570nm ± 5nm. In some cases, the polymer dye has an emission maximum wavelength of 602 nm ± 5 nm. In some embodiments, the polymer dye has an emission maximum wavelength of 650 nm ± 5 nm. In some cases, the polymer dye has an emission maximum wavelength of 711 nm ± 5 nm. In some cases, the polymer dye has an emission maximum wavelength of 786 nm ± 5 nm.In certain embodiments, the polymeric dye has a maximum emission wavelength selected from 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm.

[0088] Specific polymer dyes that may be used include, but are not limited to, BD Horizon Brilliant TM Dyes, such as BD Horizon Brilliant TM Violet dyes (e.g., BV421, BV510, BV605, BV650, BV711, BV786); BD Horizon Brilliant TM UV dyes (e.g., BUV395, BUV496, BUV737, BUV805); and BD HorizonBrilliant TM Blue dye (eg, BB515) (BD Biosciences, San Jose, CA).

[0089] System configured to determine position information of particles in a flow stream

[0090] Aspects of the present disclosure include systems for determining position information of particles in a flow stream. The system according to certain embodiments includes a flow cell configured to pass a composition having particles in a flow stream comprising a core flow and a sheath flow stream; a light source for irradiating particles of the composition; a light detection system for detecting light from the irradiated particles; and a processor having a memory operably coupled to the processor, wherein the memory contains instructions stored thereon, which when executed by the processor causes the processor to determine the position of the particles in the flow stream based on the detected light emission from the irradiated particles. In some embodiments, the irradiated particles in the composition are stably associated with an irradiation power density-sensitive compound that emits light, the intensity of which depends on the irradiation power density of the light source incident on the particles.

[0091] In embodiments, system includes the light source for irradiating flow stream.In embodiments, light source can be any suitable broadband or narrowband light source.According to the component (for example, cell, bead, non-cellular particle etc.) in sample, light source can be configured to emit the light of multiple wavelengths, and the variation range of this wavelength is 200nm to 1500nm, for example 250nm to 1250nm, for example 300nm to 1000nm, for example 350nm to 900nm, and includes 400nm to 800nm.For example, light source can include the broadband light source that emission wavelength is the light of 200nm to 900nm.In other cases, light source includes the narrowband light source that emission wavelength range is 200nm to 900nm.For example, light source can be the narrowband LED (1nm-25nm) that emission wavelength range is the light of 200nm to 900nm.

[0092] 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 2 lasers, such as argon fluorine (ArF) excimer lasers, krypton fluorine (KrF) excimer lasers, xenon chlorine (XeCl) excimer lasers or xenon fluorine (XeF) excimer lasers or combinations thereof; dye lasers, such as stilbene, coumarin or rhodamine lasers; metal vapor lasers, such as helium cadmium (HeCd) lasers, helium mercury (HeHg) lasers, helium selenium (HeSe) lasers, helium silver (HeAg) lasers, strontium lasers, neon copper (NeCu) lasers, copper lasers or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO 4 Laser, Nd:YCa 4 O(BO 3 ) 3 Laser, Nd:YCOB laser, Ti:Sapphire laser, Thallium YAG laser, Ytterbium YAG laser, Ytterbium 2 O 3 Laser or cerium-doped laser and combinations thereof; semiconductor diode laser, optically pumped semiconductor laser (OPSL) or frequency doubled or tripled implementations of any of the above lasers.

[0093] In other embodiments, the light source is a non-laser light source, such as a lamp, including 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 super-irradiance light emitting diode, a semiconductor light emitting diode, a wide spectrum LED white light source, and a multi-LED integration. In some cases, the non-laser light source is a stable fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0094] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted light beams. In some cases, the beam generator includes a laser, a radio frequency generator configured to apply a radio frequency drive signal to the acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous wave laser. For example, the laser in the beam generator of interest can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO 2 lasers, such as argon fluorine (ArF) excimer lasers, krypton fluorine (KrF) excimer lasers, xenon chlorine (XeCl) excimer lasers or xenon fluorine (XeF) excimer lasers or combinations thereof; dye lasers, such as stilbene, coumarin or rhodamine lasers; metal vapor lasers, such as helium cadmium (HeCd) lasers, helium mercury (HeHg) lasers, helium selenium (HeSe) lasers, helium silver (HeAg) lasers, strontium lasers, neon copper (NeCu) lasers, copper lasers or gold lasers and combinations thereof; solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO 4 Laser, Nd:YCa 4 O(BO 3 ) 3 Laser, Nd:YCOB laser, Ti:Sapphire laser, Thallium YAG laser, Ytterbium YAG laser, Ytterbium 2 O 3 Laser or cerium-doped laser and a combination thereof.

[0095] The acousto-optic device can be any convenient acousto-optic scheme that is configured to frequency shift the laser using an applied acoustic wave. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied RF drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source (e.g., a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator).

[0096] In an embodiment, the controller is configured to apply an RF drive signal to the acousto-optic device to produce a desired number of angularly deflected laser beams in the output laser beam, for example, configured to apply 3 or more RF drive signals, for example, 4 or more RF drive signals, for example, 5 or more RF drive signals, for example, 6 or more RF drive signals, for example, 7 or more RF drive signals, for example, 8 or more RF drive signals, for example, 9 or more RF drive signals, for example, 10 or more RF drive signals, for example, 15 or more RF drive signals, for example, 25 or more RF drive signals, for example, 50 or more RF drive signals, and including being configured to apply 100 or more RF drive signals.

[0097] In some cases, to produce an intensity profile of an angularly deflected laser beam in the output laser beam, the controller is configured to apply a RF drive signal having an amplitude varying from 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 50 V, e.g., about 2 V to 40 V, e.g., 3 V to about 30 V, and including about 5 V to about 25 V). In some embodiments, each applied RF drive signal has a frequency of about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, such as about 0.01 MHz to about 300 MHz, such as about 0.05 MHz to about 200 MHz, such as about 0.1 MHz to about 100 MHz, such as about 0.5 MHz to about 90 MHz, such as about 1 MHz to about 75 MHz, such as about 2 MHz to about 70 MHz, such as about 3 MHz to about 65 MHz, such as about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0098] In some embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam with a desired intensity profile. For example, the memory may include instructions to generate two or more (e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 10 or more, e.g., 25 or more, e.g., 50 or more) angularly deflected laser beams having the same intensity, and the memory may include instructions to generate 100 or more angularly deflected laser beams having the same intensity. In other embodiments, instructions may be included to generate two or more (e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 10 or more, e.g., 25 or more, e.g., 50 or more) angularly deflected laser beams having different intensities, and the memory may include instructions to generate 100 or more angularly deflected laser beams having different intensities.

[0099] In certain embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity increases from its edge to its center along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam can be 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from its edge to its center along the horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam can be 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and includes 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 other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile with a Gaussian distribution along a horizontal axis. In yet other embodiments, the controller has a processor having a memory operably coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile with a top-hat distribution along a horizontal axis.

[0100] In an embodiment, the beam generator of interest can be configured to generate an angle-deflected laser beam in a spatially separated output laser beam. Depending on the applied RF drive signal and the desired irradiation profile of the output laser beam, the angle-deflected laser beams can 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, and including 5000 μm or more. In some embodiments, the system is configured to generate an angle-deflected laser beam in the output laser beam, which angle-deflected laser beam overlaps, for example, with an adjacent angle-deflected laser beam along the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., the overlap of beam spots) can be an overlap of 0.001 μm or greater, for example, an overlap of 0.005 μm or greater, for example, an overlap of 0.01 μm or greater, for example, an overlap of 0.05 μm or greater, for example, an overlap of 0.1 μm or greater, for example, an overlap of 0.5 μm or greater, for example, an overlap of 1 μm or greater, for example, an overlap of 5 μm or greater, for example, an overlap of 10 μm or greater, and including an overlap of 100 μm or greater.

[0101] In some cases, a beam generator configured to generate two or more frequency-shifted beams includes a laser excitation module as described in U.S. Patent Nos. 9,423,353, 9,784,661, and 10,006,852 and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0102] The light source can be positioned at any suitable distance from the flow stream, such as 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, and including a distance of 100 mm or more. In addition, the light source irradiates the flow stream at any suitable angle (e.g., relative to the vertical axis of the flow stream), such as an angle in the range of 10 ° to 90 °, such as 15 ° to 85 °, such as 20 ° to 80 °, such as 25 ° to 75 °, and including an angle of 30 ° to 60 °, such as 90 °.

[0103] The light source can be configured to irradiate the composition in the flowing stream continuously or at discontinuous intervals. In some cases, the system includes a light source configured to continuously irradiate the sample, such as with a continuous wave laser, which continuously irradiates the flowing stream at the probe point in the flow cytometer. In other cases, the system of interest includes a light source configured to irradiate the sample at discontinuous intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds, or some other interval. When the light source is configured to irradiate the sample at discontinuous intervals, the system may include one or more additional components to intermittently irradiate the sample with the light source. For example, the subject system in these embodiments may include one or more laser beam choppers, manually or computer-controlled beam stops, for blocking the sample and exposing the sample to the light source.

[0104] In some cases, the light source is configured to irradiate the core flow with an elliptical beam spot having a major axis and a minor axis, for example, wherein the major axis of the beam spot is orthogonal to the horizontal axis of the core flow. 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 along the horizontal axis of the core flow. In some cases, the laser is configured to irradiate the core flow so that the irradiation power density along the horizontal axis of the flow stream has a larger power density at or near the center of the core flow and a lower power density at or near the edge of the core flow. In some cases, the intensity profile of the output laser beam includes an increase 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 intensity of the output laser beam at the edge of the output beam can be 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and including the intensity of the output laser beam at the edge of the output beam being 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 includes a Gaussian distribution along the horizontal axis of the output laser beam. In still other cases, the intensity profile of the output laser beam includes a super-Gaussian distribution along the horizontal axis of the output laser beam.

[0105] In some embodiments, the laser includes an optical adjustment component (e.g., one or more lenses) configured to rotate the output laser beam by 90° so that the major axis of the elliptical beam spot is parallel to the longitudinal axis of the flow stream (i.e., the minor axis of the elliptical beam spot is irradiated on the horizontal axis of the flow stream). In some cases, the optical adjustment component is configured to generate the output laser beam so that the irradiation power density along the horizontal axis of the flow stream has a larger power density at or near the center of the core stream and a lower power density at or near the edge of the core stream. In some cases, the laser includes an optical adjustment component for providing an intensity profile of the output laser beam, the intensity of which 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 optical adjustment assembly produces an output laser beam having an intensity at the edge that is 0.1% to about 99%, such as 0.5% to about 95%, such as 1% to about 90%, such as about 2% to about 85%, such as about 3% to about 80%, such as about 4% to about 75%, such as about 5% to about 70%, such as about 6% to about 65%, such as about 7% to about 60%, such as about 8% to about 55%, and includes an output laser beam having an intensity at the edge of the output beam that is about 10% to about 50% of the intensity at the center of the output laser beam along the horizontal axis. In still other cases, the optical adjustment assembly produces an output laser beam having an intensity profile having a Gaussian distribution along the horizontal axis. In still other cases, the optical adjustment assembly produces an output laser beam having an intensity profile having a super-Gaussian distribution along the horizontal axis.

[0106] In some embodiments, the light source includes a plurality of lasers. In some embodiments, the light source includes a plurality of lasers, wherein the first laser is positioned to irradiate the flow stream at a first position, the second laser is positioned to irradiate the flow stream at a second position, the third laser is positioned to irradiate the flow stream at a third position, and the fourth laser is positioned to irradiate the flow stream at a fourth position, wherein the second position is downstream of the first position, the third position is downstream of the second position, and the fourth position is downstream of the third position. In some embodiments, the first laser is a triggering laser, for example, wherein the system is configured to trigger after the light emitted by the second laser after irradiating the particles is detected by the light detection system. In some cases, the light source includes at least one laser having an elliptical beam spot, the elliptical beam spot having a major axis and a minor axis, the laser being configured to irradiate the flow stream, so that the major axis of the beam spot is orthogonal to the longitudinal axis of the flow stream; and a laser having an elliptical beam spot, the elliptical beam spot having a major axis and a minor axis, the laser being configured to irradiate the flow stream, so that the major axis of the beam spot is orthogonal to the horizontal axis of the flow stream.

[0107] The distance between irradiations of the flow stream by each laser can vary, with the inter-irradiation spacing independently being 0.0001 μm or greater, such as 0.0005 μm or greater, such as 0.001 μm or greater, such as 0.005 μm or greater, such as 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 2 μm or greater, such as 3 μm or greater, such as 4 μm or greater, such as 5 μm or greater, such as 6 μm or greater, such as 7 μm or greater, such as 8 μm or greater, such as 9 μm or greater and including 10 μm or greater. In some cases, the lasers in the subject light sources are configured to irradiate locations on the flow stream that are immediately adjacent to each other (i.e., with no inter-irradiation space).

[0108] Depending on the desired wavelength of light generated in the output laser beam (e.g., for irradiating a sample in a flow stream), each laser can have a specific wavelength varying from 200nm to 1500nm (e.g., 250nm to 1250nm, e.g., 300nm to 1000nm, e.g., 350nm to 900nm, and including 400nm to 800nm). In certain embodiments, the laser of interest may include one or more of a 405nm laser, a 488nm laser, a 561nm laser, and a 628nm laser. In some embodiments, each laser outputs light of different wavelengths. In some cases, the lasers of the system are positioned so that each downstream laser outputs light of a longer wavelength. For example, when the system includes four lasers, the wavelength of the light from the second laser is longer than the wavelength of the light from the first laser, the wavelength of the light from the third laser is longer than the wavelength of the light from the second laser, and the wavelength of the light from the fourth laser is longer than the wavelength of the light from the third laser. In some cases, the system includes four lasers, wherein the wavelength of the light from the first laser is 488nm, the wavelength of the light from the second laser is 405nm, the wavelength of the light from the third laser is 561nm, and the wavelength of the light from the fourth laser is 628nm. In some embodiments, the light from the second laser is configured to have a Gaussian beam power density profile on the horizontal axis of the flow stream. In some cases, the second laser includes an elliptical beam spot having a major axis and a minor axis. In some embodiments, the second laser is coupled to an optical adjustment component that is configured to transmit light to the flow stream so that the major axis of the beam spot is orthogonal to the horizontal axis of the flow stream. In some cases, the optical adjustment component is configured to rotate the beam spot 90°. In some cases, the optical adjustment component includes one or more lenses. In some cases, the first, third, and fourth lasers are configured to have a major axis and a minor axis and are coupled to an optical adjustment component that propagates the light from the first, third, and fourth lasers to the flow stream so that the major axes of the beam spots are orthogonal to the horizontal axis of the flow stream.

[0109] The light source may also include one or more optical adjustment components. The term "optical adjustment" is used here in its conventional sense to refer to any device capable of changing the spatial width of the irradiation or some other characteristics of the irradiation from one or more lasers (e.g., irradiation direction, wavelength, beam width, beam intensity, focus, and pulse width). The optical adjustment scheme may be any convenient device for adjusting one or more characteristics of the laser, including but not limited to lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation schemes, 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 reduction 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 an optical fiber. In some embodiments, the beams from each laser are combined by a beam combiner (e.g., a dichroic beam combiner). In these embodiments, the beam combiner combines the beams from each laser and propagates the light to a beam shaping assembly.

[0110] In some embodiments, the light from each laser is propagated to the flow stream through a reflector assembly. In some cases, the reflector assembly may include a first reflector and a second reflector, the second reflector being positioned to propagate light from the first reflector to the flow stream. In an embodiment, the second reflector is positioned to propagate light from the first reflector at an angle varying relative to the first reflector, the range of the variation being, for example, 1° to 90°, for example, 5° to 85°, for example, 10° to 80°, for example, 15° to 75°, for example, 20° to 70°, for example, 25° to 65°, and including 30° to 60°. In some cases, the second reflector is positioned to propagate light from the first reflector orthogonally. In other embodiments, the second reflector is positioned to propagate light from the first reflector at an angle that varies relative to the laser, such as 1° to 90°, such as 5° to 85°, such as 10° to 80°, such as 15° to 75°, such as 20° to 70°, such as 25° to 65°, and including 30° to 60°. In some cases, the second reflector is positioned to propagate light in an orthogonal manner relative to the laser. In some embodiments, the second reflector is also a beam combiner that is configured to combine beams from two or more lasers. In these embodiments, the second reflector can be a dichroic mirror that selectively passes wavelengths of light as needed.

[0111] Figure 3A light source for irradiating a composition having particles in a flow stream according to certain embodiments is depicted. Light source 300 includes laser modules 301 and 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 flow stream with an elliptical beam spot (shown in 304), wherein the major axis of the elliptical beam spot is orthogonal to the horizontal axis of the flow stream. In some cases, this beam spot configuration provides a Gaussian beam power density profile on the horizontal axis of the flow stream. Laser module 301 is in optical communication with a first optical adjustment component 303a, which transmits light from lasers 305, 306, 307, and 308 to the flow stream. In some embodiments, the output laser beams from lasers 305, 306, 307, and 308 are configured such that the major axis of the elliptical beam spot is orthogonal to the longitudinal axis of the flow stream (i.e., orthogonal to the beam spot from laser 302a). Laser module 302 is in optical communication with a second optical adjustment component 303b that is configured to rotate the beam spot from laser 302a by 90° to produce the beam profile shown in 304 and to provide a Gaussian beam power density profile on the horizontal axis of the flow stream. Figure 3 An example of an optical adjustment assembly 303b is shown.

[0112] The system of the present disclosure also includes a light detection system having a photodetector for detecting light from the irradiated core stream. The photodetector can be any convenient light detection scheme, including but not limited to a photoelectric sensor or photodetector, such as an active pixel sensor (APS), an avalanche photodiode (APD), a four-quadrant photodiode, an image sensor, a charge-coupled device (CCD), an intensified charge-coupled device (ICCD), a light-emitting diode, a photon counter, a radiation thermometer, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube, a phototransistor, a quantum dot photoconductor or a photodiode and combinations thereof, and other photodetectors. In certain embodiments, the photodetector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area per area in the range of 0.01 cm 2 Up to 10cm 2 , for example 0.05cm 2 Up to 9cm 2 , for example 0.1cm 2 Up to 8cm 2 , for example 0.5cm 2 Up to 7cm 2 And including 1cm 2 Up to 5cm2 .

[0113] In some embodiments, the light detection system of interest includes a plurality of photodetectors. In some cases, the light detection system includes a plurality of solid-state detectors, such as photodiodes. In certain embodiments, the light detection system includes a photodetector array, such as a photodiode array. In these embodiments, the photodetector array may include 4 or more photodetectors, such as 10 or more photodetectors, such as 25 or more photodetectors, such as 50 or more photodetectors, such as 100 or more photodetectors, such as 250 or more photodetectors, such as 500 or more photodetectors, such as 750 or more photodetectors, and include 1000 or more photodetectors. For example, the detector may be a photodiode array having 4 or more photodiodes, such as 10 or more photodiodes, such as 25 or more photodiodes, such as 50 or more photodiodes, such as 100 or more photodiodes, such as 250 or more photodiodes, such as 500 or more photodiodes, such as 750 or more photodiodes, and including 1000 or more photodiodes.

[0114] The photodetectors can be arranged in any geometric configuration as desired, where arrangements of interest include, but are not limited to, square configurations, rectangular configurations, trapezoidal configurations, triangular configurations, hexagonal configurations, heptagonal configurations, octagonal configurations, nonagonal configurations, decagonal configurations, dodecagonal configurations, circular configurations, elliptical configurations, and irregular pattern configurations. The photodetectors in the photodetector array can be oriented relative to each other (with reference to the XZ plane) at an angle 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° and including 45° to 90°). The photodetector array can be of any suitable shape, and can be a rectilinear shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved shape, such as a circle, an ellipse; and an irregular shape, such as a parabolic bottom coupled to a plane top. In some embodiments, the photodetector array has a rectangular effective surface.

[0115] Each photodetector (e.g., photodiode) in the array may have an active surface having a width in the range of 5 μm to 250 μm, 10 μm to 225 μm, 15 μm to 200 μm, 20 μm to 175 μm, 25 μm to 150 μm, 30 μm to 125 μm, and including 50 μm to 100 μm and a length in the range of 5 μm to 250 μm, 10 μm to 225 μm, 15 μm to 200 μm, 20 μm to 175 μm, 25 μm to 150 μm, 30 μm to 125 μm, and including 50 μm to 100 μm, wherein the surface area of ​​each photodetector (e.g., photodiode) in the array is in the range of 25 μm to 100 μm. 2 Up to 10000μm 2 , 50 to μm 2 Up to 9000μm 2 , 75 to μm 2 Up to 8000μm 2 , 100 to μm 2 Up to 7000μm 2 , 150 to μm 2 Up to 6000μm 2 and includes 200 to μm 2 Up to 5000μm 2 .

[0116] 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 range in length from 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm, and including 5 mm to 25 mm. The width of the photodetector array may also vary, ranging from 0.01 mm to 100 mm, such as 0.05 mm to 90 mm, such as 0.1 mm to 80 mm, such as 0.5 mm to 70 mm, such as 1 mm to 60 mm, such as 2 mm to 50 mm, such as 3 mm to 40 mm, such as 4 mm to 30 mm, and including 5 mm to 25 mm. Thus, the effective surface of the photodetector may range from 0.1 mm to 100 mm. 2 Up to 10000mm 2 , for example 0.5mm 2 Up to 5000mm 2 , for example 1mm 2 Up to 1000mm 2 , for example 5mm 2 Up to 500mm 2 And including 10mm 2 Up to 100mm 2 .

[0117] The photodetector can measure light at one or more wavelengths (e.g., 2 or more wavelengths, e.g., 5 or more different wavelengths, e.g., 10 or more different wavelengths, e.g., 25 or more different wavelengths, e.g., 50 or more different wavelengths, e.g., 100 or more different wavelengths, e.g., 200 or more different wavelengths, e.g., 300 or more different wavelengths), and includes measuring light from particles in the flow stream at 400 or more different wavelengths. In some embodiments, fluorescence from the sample is detected by the light detection system within one or more wavelengths of 200nm-1200nm. In some cases, the method includes detecting fluorescence within a range of wavelengths from the sample, e.g., 200nm to 1200nm, e.g., 300nm to 1100nm, e.g., 400nm to 1000nm, e.g., 500nm to 900nm, and including 600nm to 800nm. In other cases, the method includes detecting fluorescence of one or more specific wavelengths. For example, fluorescence can 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, and any combination thereof, depending on the number of different fluorescence detectors in the subject light detection system.

[0118] Light can be measured continuously or at discontinuous 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 discontinuous intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds and including every 1000 milliseconds or some other interval measurement of light. The light from the flowing stream can be measured once or more times in each discontinuous time interval, such as 2 times or more times, such as 3 times or more times, such as 5 times or more times and including 10 times or more times. In certain embodiments, the photodetector measures the light from the flowing stream 2 times or more, and in some cases the data is averaged.

[0119] Each photodetector can be positioned at any suitable distance from the flow stream, as long as a usable light signal can be detected. For example, the detector in the subject system can be positioned at a distance of 1mm or more from the flow stream, such as 5mm or more, such as 10mm or more, such as 15mm or more, such as 25mm or more, such as 50mm or more, such as 100mm or more, such as 150mm or more, such as 250mm or more, and including 500mm or more from the flow stream. The detector can also be positioned at any angle to the flow stream. For example, the detector can be at an angle of 10° to 90° with the vertical axis of the flow stream, such as 15° to 85°, such as 20° to 80°, such as 25° to 75° and including 30° to 60°. In some cases, one or more detectors are positioned at an angle of 30° to 60° with the vertical axis of the flow stream.

[0120] 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 irradiated flow stream can be detected by one or more detectors configured as forward scattering detectors. In these embodiments, the forward scattering detector is positioned on the side of the flow stream opposite to the light source, and is positioned to collect and detect forward propagation (e.g., scattered) light. In some embodiments, the light from the irradiated sample in the flow stream is detected in one or more fluorescence detector channels. In some embodiments, the light from the irradiated sample in the flow stream is detected in one or more scattered light detector channels.

[0121] In some embodiments, the system includes an optical collection system for collecting and directing light from the flow stream to the light detection system. The optical collection system can be physically coupled to the light detection system, such as by an adhesive, co-molding, or integration into the light detection system. In certain embodiments, the optical collection system and the light detection system are integrated into a single unit. In other embodiments, the optical collection system is coupled to the light detection system through a connector, such as by hook and loop fasteners, magnets, latches, notches, countersunk holes, counterbores, grooves, pins, tethers, hinges, Velcro, non-permanent adhesives, or combinations thereof.

[0122] In other embodiments, the light detection system and the optical collection system are in optical communication but not in physical contact. For example, the optical collection system can be positioned 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 10 mm or more, such as 25 mm or more, such as 50 mm or more, and including 100 mm or more from the light detection system.

[0123] In some embodiments, the optical collection system includes an optical fiber. For example, the optical collection system can be a fiber optic optical relay bundle, and the light from the flow stream is transmitted to the first set of linear variable optical filters of the optical detection system through the fiber optic optical relay bundle. Any fiber optic optical relay system can be used to transmit light, wherein in some embodiments, suitable fiber optic optical relay systems include, but are not limited to, fiber optic optical relay systems, such as those described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0124] In other embodiments, the optical 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 light propagation using a configuration of one or more optical components to guide light from a sample to a light detection system through free space. 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 different optical components, such as one or more of a lens, a reflector, a slit, a pinhole, a wavelength separator, or a combination thereof. For example, in some embodiments, the free space optical relay system of interest includes one or more focusing lenses. In other embodiments, the subject free space optical relay system includes one or more reflectors. In yet 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, those described in, for example, U.S. Patent Nos. 7,643,142; 7,728,974; and 8,223,445, the disclosures of which are incorporated herein by reference.

[0125] In some embodiments, a system includes a processor having a memory operably coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine an emission intensity of irradiated particles of a composition and determine a position of the irradiated particles of the composition in a core stream of a flow stream based on the determined emission intensity.

[0126] As described above, in some embodiments, the composition includes particles, which are coupled to a sensitive compound for the irradiation power density of the emitted light, and the intensity of the light depends on the irradiation power density of the light source incident on the particles. In some cases, the irradiated particles of the composition show emission intensity changes based on the irradiation intensity of the light source incident on the particles. In certain embodiments, the irradiated particles of the composition have a spectral profile that changes in response to the power density of the light source. In some embodiments, the particles of the composition have 2 or more emission peaks in response to the irradiation by the light source, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more and include 10 or more emission peaks. In some cases, one or more peaks of the spectral profile show changes in intensity in response to the power density of the light source. In some cases, the intensity of each emission peak depends on the irradiation power intensity of the light source. In some embodiments, the memory includes instructions for determining the position information of the particles of the composition based on the emission intensity of one or more emission peaks. In some cases, the memory includes instructions for determining that the particle passes at or near the center of the core flow when the emission peak exhibits a high emission intensity, and instructions for determining that the particle passes at or near the edge of the core flow when the emission peak exhibits a low emission intensity. In some cases, the memory includes instructions for determining that the particle passes at or near the center of the core flow when the emission peak exhibits a low emission intensity, and instructions for determining that the particle passes at or near the edge of the core flow when the emission peak exhibits a high emission intensity.

[0127] In certain embodiments, the irradiated particles of the composition have: a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density, and a second emission peak having the same intensity at the first irradiation power density and the second irradiation power density. In some cases, the memory contains instructions for determining the position information of the particles of the composition based on the emission intensities of the first and second emission peaks. In one example, the memory contains instructions for determining that the particles are passed at or near the center of the core flow when the first emission peak exhibits a high emission intensity, and instructions for determining that the particles are passed at or near the edge of the core flow when the first emission peak exhibits a low emission intensity. In another example, the memory contains instructions for determining that the particles are passed at or near the center of the core flow when the first emission peak exhibits a low emission intensity, and instructions for determining that the particles are passed at or near the edge of the core flow when the first emission peak exhibits a high emission intensity.

[0128] In other cases, the memory includes instructions for determining the position of the particle based on the ratio of the intensity of the first emission peak to the intensity of the second emission peak. In some cases, the ratio of the intensity of the first emission peak to the intensity of the second emission peak ranges from 1:100 to 100:1, such as 1:75 to 75:1, such as 1:50 to 50:1, such as 1:25 to 25:1, such as 1:10 to 10:1, such as 1:9 to 9:1, such as 1:8 to 8:1, such as 1:7 to 7:1, such as 1:6 to 6:1, such as 1:5 to 5:1, and includes 1:4 to 4:1. In certain embodiments, the memory includes instructions for determining that the particle is passing at or near the center of the core flow when the ratio of the intensity of the first emission peak to the intensity of the second emission peak is 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 and including 0.95:1 to 1:0.95. In some cases, the memory includes instructions for determining that the particle is passed at or near the center of the core flow (i.e., the particle is passed through the center of the core flow (or when the first emission peak and the second emission peak have approximately the same emission intensity, the particle is passed in the center of the light source irradiation) when the ratio of the intensity of the first emission peak to the intensity of the second emission peak is approximately 1:1. 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 the particle is passed to the irradiation center (or the center of the core flow when the light source and the core flow are aligned).

[0129] In some embodiments, the memory includes instructions for determining that particles of the composition are passed through the flow stream at or near the center of the core flow. In some cases, the memory includes instructions for determining that particles of the composition are passed through the flow stream at or near the edge of the core flow. In certain embodiments, the memory includes instructions for generating one or more particle population clusters based on the determined position of the particles in the core flow. In some cases, the memory includes instructions for classifying particles irradiated at or near the center of the core flow based on a determined spectral emission intensity profile. In other cases, the memory includes instructions for classifying particles irradiated at or near the edge of the core flow based on a determined spectral emission intensity profile. In some cases, the memory includes instructions for clustering data signals of particles irradiated at or near the center of the core flow. In some cases, the memory includes instructions for clustering data signals of particles irradiated at or near the edge of the core flow. In certain embodiments, the memory includes instructions for discarding data signals of particles from the composition, and the particles are classified as irradiated at or near the edge of the core flow.

[0130] In certain embodiments, the system also includes a flow cell, which is configured to transfer compositions in a flowing stream. Any convenient flow cell for transferring a fluid sample to a sample exploration region can be used, wherein in some embodiments, the flow cell includes a proximal cylindrical portion defining a longitudinal axis and a distal truncated cone portion terminating in a flat surface, the flat surface having an orifice transverse to the longitudinal axis. The length of the proximal cylindrical portion (as measured along the longitudinal axis) can vary in the range of 1mm to 15mm, for example, from 1.5mm to 12.5mm, for example, from 2mm to 10mm, for example, from 3mm to 9mm, and including from 4mm to 8mm. The length of the distal truncated cone portion (as measured along the longitudinal axis) can also vary in the range of 1mm to 10mm, for example, from 2mm to 9mm, for example, from 3mm to 8mm, and including from 4mm to 7mm. The diameter of the flow cell nozzle chamber can vary, in some embodiments, ranging from 1 mm to 10 mm, such as from 2 mm to 9 mm, such as from 3 mm to 8 mm, and including from 4 mm to 7 mm.

[0131] In some cases, the flow cell does not include a cylindrical portion, and the entire flow cell chamber is frustoconical. In these embodiments, the length of the frustoconical chamber (as measured along a longitudinal axis transverse to the nozzle orifice) can range from 1 mm to 15 mm, such as from 1.5 mm to 12.5 mm, such as from 2 mm to 10 mm, such as from 3 mm to 9 mm, and including from 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical chamber can range from 1 mm to 10 mm, such as from 2 mm to 9 mm, such as from 3 mm to 8 mm, and including from 4 mm to 7 mm.

[0132] In some embodiments, the sample flow stream flows out of an orifice at the far end of the flow cell. Depending on the desired characteristics of the flow stream, the flow cell orifice can be any suitable shape, where the cross-sectional shapes of interest include, but are not limited to: straight cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, hexagonal, etc.; curved cross-sectional shapes, such as circular, elliptical; and irregular shapes, such as a parabolic bottom coupled to a flat top. In certain embodiments, the flow cell of interest has a circular orifice. The size of the nozzle orifice can vary, in some embodiments, ranging from 1 μm to 20000 μm, such as from 2 μm to 17500 μm, such as from 5 μm to 15000 μm, such as from 10 μm to 12500 μm, such as from 15 μm to 10000 μm, such as from 25 μm to 7500 μm, such as from 50 μm to 5000 μm, such as from 75 μm to 1000 μm, such as from 100 μm to 750 μm, and including from 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0133] In some embodiments, the flow cell includes a sample injection port configured to provide a sample to the flow cell. In embodiments, the sample injection system is configured to provide an appropriate sample flow to the flow cell chamber. Depending on the desired characteristics of the flow stream, the rate at which the sample is delivered to the flow cell chamber by the sample injection port can be 1 μL / min or faster, such as 2 μL / min or faster, such as 3 μL / min or faster, such as 5 μL / min or faster, such as 10 μL / min or faster, such as 15 μL / min or faster, such as 25 μL / min or faster, such as 50 μL / min or faster and including 100 μL / min or faster, wherein in some cases, the rate at which the sample is delivered to the flow cell chamber by the sample injection port is 1 μL / sec or faster, such as 2 μL / sec or faster, such as 3 μL / sec or faster, such as 5 μL / sec or faster, such as 10 μL / sec or faster, such as 15 μL / sec or faster, such as 25 μL / sec or faster, such as 50 μL / sec or faster and including 100 μL / sec or faster.

[0134] The sample injection port can be an orifice located on the inner chamber wall, or can be a conduit located at the proximal end of the inner chamber. When the sample injection port is an orifice located on the inner chamber wall, the sample injection port orifice can be any suitable shape, wherein the cross-sectional shape of interest includes but is not limited to: a straight cross-sectional shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved cross-sectional shape, such as a circle, an ellipse, etc.; and an irregular shape, such as a parabola bottom coupled to a plane top. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port may vary depending on the shape, and in certain cases, the opening ranges from 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and includes 1.25 mm to 1.75 mm, such as 1.5 mm.

[0135] In some cases, the sample injection port is a conduit located at the proximal end of the flow cell chamber. For example, the sample injection port can be a conduit positioned so that the orifice of the sample injection port is aligned with the orifice of the flow cell. When the sample injection port is a conduit aligned with the orifice of the flow cell, the cross-sectional shape of the sample injection tube can be any suitable shape, wherein the cross-sectional shape of interest includes but is not limited to: a straight cross-sectional shape, such as a square, rectangle, trapezoid, triangle, hexagon, etc.; a curved cross-sectional shape, such as a circle, an ellipse; and an irregular shape, such as a parabola bottom coupled to a plane top. The orifice of the conduit may vary in shape, and in some cases, the opening range is 0.1mm to 5.0mm, such as 0.2 to 3.0mm, such as 0.5mm to 2.5mm, such as 0.75mm to 2.25mm, such as 1mm to 2mm and includes 1.25mm to 1.75mm, such as 1.5mm. The shape of the tip of the sample injection port may be the same or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip having a bevel angle ranging from 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7°, and including a bevel angle of 5°.

[0136] In some embodiments, the flow cell further comprises a sheath liquid injection port configured to provide sheath liquid to the flow cell. In an embodiment, the sheath liquid injection system is configured to provide a sheath liquid flow to the flow cell chamber, for example, together with the sample, to generate a sheath liquid laminar flow around the sample flow. Depending on the desired characteristics of the flow stream, the rate of the sheath liquid delivered to the flow cell chamber can be 25 μL / sec or faster, such as 50 μL / sec or faster, such as 75 μL / sec or faster, such as 100 μL / sec or faster, such as 250 μL / sec or faster, such as 500 μL / sec or faster, such as 750 μL / sec or faster, such as 1000 μL / sec or faster, and includes 2500 μL / sec or faster.

[0137] In some embodiments, the sheath injection port is an orifice located in the inner chamber wall. The sheath injection port orifice can be any suitable shape, where the cross-sectional shapes of interest include, but are not limited to: straight cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, hexagonal, etc.; curved cross-sectional shapes, such as circular, elliptical; and irregular shapes, such as a parabolic bottom coupled to a flat top. The size of the sample injection port can vary depending on the shape, and in some cases, the opening ranges from 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm and includes 1.25 mm to 1.75 mm, such as 1.5 mm.

[0138] In some embodiments, the system also includes a pump connected to the flow cell fluid so that the flow stream is transmitted through the flow cell. Any convenient fluid pump scheme can be used to control the flow of the flow stream through the flow cell. In some cases, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in the subject system is configured to be suitable for detecting the speed of light from the sample in the flow stream by the fluid transport through the flow cell. In some cases, the rate of the sample flow in the flow cell is 1 μL / min (microliter / minute) or faster, such as 2 μL / min or faster, such as 3 μL / min or faster, such as 5 μL / min or faster, such as 10 μL / min or faster, such as 25 μL / min or faster, such as 50 μL / min or faster, such as 75 μL / min or faster, such as 100 μL / min or faster, such as 250 μL / min or faster, such as 500 μL / min or faster, such as 750 μL / min or faster and including 1000 μL / min or faster. For example, the system may include a pump configured to flow the sample through the flow cell at a rate ranging from 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 and including 10 μL / min to 50 μL / min). In certain embodiments, the flow rate of the flow stream is from 5 μL / min to 6 μL / min.

[0139] In some embodiments, a light detection system having a plurality of photodetectors as described above is part of or located in a particle analyzer (e.g., a particle sorter). In some embodiments, the subject system is a flow cytometry system that includes a photodiode and an amplifier assembly as part of a light detection system for detecting light emitted by a sample in a flowing stream. Suitable flow cytometry systems may include, but are not limited to, 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 edition, Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt 1): 17-28; Linden, et al., Semin Throm 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. 24(3):203-255; the disclosure of which is incorporated herein by reference. In some cases, flow cytometry systems of interest include the BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortessaTM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD BiosciencesAria TM Cell sorter, BD Biosciences FACSAria TM II cell sorter, BD Biosciences FACSAria TM III cell sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorter, BD Biosciences FACSymphony TM S6 cell sorter, etc.

[0140] In some embodiments, the subject system is a flow cytometry system, such as 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, 9,947,834, and 9,113,967. those described in, for example, 4,084,975,595, 4,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766, the disclosures of which are incorporated herein by reference in their entireties.

[0141] In some embodiments, the subject system is a particle sorting system configured to sort particles using a closed particle sorting module, such as those described in U.S. Patent Publication No. 2017 / 0299493, the disclosure of which is incorporated herein by reference. In certain embodiments, a sorting decision module having a plurality of sorting decision units is used to sort particles (e.g., cells) of a sample, such as those described in U.S. Patent 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 a deflection plate, such as those described in U.S. Patent Publication No. 2017 / 0299493 filed on March 28, 2017, the disclosure of which is incorporated herein by reference.

[0142] In some cases, the flow cytometry systems of the present invention are configured to image particles in a flow stream by fluorescence imaging using radio frequency labeled emission (FIRE), such as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and in U.S. Pat. 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, 10,40 8,758, 10,451,538, 10,620,111 and those described in U.S. 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.

[0143] In some embodiments, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4A ) can be used to analyze and characterize particles, whether or not the particles are physically sorted into collection containers. Figure 4A A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, particle analysis system 401 is a flow system. Figure 4A The particle analysis system 401 shown in the figure can be configured to perform the methods described herein in whole or in part, for example. The particle analysis system 401 includes a fluid system 402. The fluid system 402 may include or be coupled with a sample tube 405 and a moving fluid column within the sample tube, wherein particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0144] The particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as it passes through one or more detection stations along a common sample path. The detection stations 408 are generally referred to as monitored regions 407 of the common sample path. In some embodiments, the detection can include detecting light or one or more other characteristics of the particles 403 as they pass through the monitored regions 407. Figure 4A , a detection station 408 is shown having a monitored area 407. Some embodiments of particle analysis system 401 may include multiple detection stations. In addition, some detection stations may monitor multiple areas.

[0145] Each signal is assigned a signal value to form a data point for each particle. As described above, this data can be referred to as event data. The data point can be a multi-dimensional data point, including values ​​of various characteristics measured for the particle. The detection system 404 is configured to collect a series of such data points over a first time interval.

[0146] The particle analysis system 401 may also include a control system 306. The control system 406 may include one or more processors, an amplitude control circuit, and / or a frequency control circuit. The control system shown 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 a 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 a portion of the first time interval. In addition, the control system 406 may compare the experimental signal frequency with the calculated signal frequency or the predetermined signal frequency.

[0147] Figure 4B A system 400 for flow cytometry according to an illustrative embodiment of the invention is shown. The system 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The 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 long pass ("LP") filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0148] The excitation lasers 115a-c emit light in the form of laser beams. Figure 4B In the example system of , the wavelengths of the laser beams emitted by the excitation lasers 415a-415c are 488nm, 633nm and 325nm, respectively. The laser beams are first directed through one or more beam splitters 445a and 445b. Beam splitter 445a transmits 488nm light and reflects 633nm light. Beam splitter 445b transmits ultraviolet light (light with a wavelength range of 10 to 400nm) and reflects 488nm and 633nm light.

[0149] The laser beam is then directed to a focusing lens 420, which focuses the beam onto the portion of the fluid stream where the sample particles are located within a flow cell 425. The flow cell is the portion of the fluid system that directs the particles in the stream (usually one at a time) to the focused laser beam for interrogation. The flow cell may include a flow cell in a benchtop cytometer or a nozzle head in an air flow cytometer.

[0150] Light from the laser beam interacts with particles in the sample by diffraction, refraction, reflection, scattering, and absorption, where it is re-emitted at various wavelengths depending on the characteristics of the particle (e.g., its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or in the particle). The fluorescent emission and the diffracted, refracted, reflected, and scattered light can be routed to one or more forward scatter detectors 430, side scatter detectors 435, and one or more fluorescence detectors 460a-460f via one or more beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collection lenses 440.

[0151] Fluorescence collection lens 440 collects light emitted from the particle laser beam interaction and routes the light to 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 filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10nm on each side of the center of the spectral band, or from 500nm to 520nm. Shortpass filters transmit light with a wavelength equal to or shorter than a specified wavelength. Longpass filters, such as longpass filters 455a-455b, transmit light with a wavelength equal to or longer than a specified wavelength. For example, longpass filter 455a, which is a 670nm longpass filter, transmits light with a wavelength equal to or longer than 670nm. Filters are typically selected to optimize the specificity of the detector for a particular fluorescent dye. The filter can be configured so that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0152] Beam splitters direct light of different wavelengths to 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, which means that beam splitter 445g transmits light with a wavelength of 620nm or shorter and reflects light with a wavelength greater than 620nm to different directions. In one embodiment, beam splitters 445a-445g can include optical mirrors, such as dichroic mirrors.

[0153] The forward scattering detector 430 is located at a position slightly deviated from the axis by the direct beam of light passing through the flow cell, and is configured to detect diffracted light, i.e., excitation light passing through or around the particles mainly in the forward direction. The intensity of the light detected by the forward scattering detector depends on the overall size of the particles. The forward scattering detector may include a photodiode. The side scattering detector 435 is configured to detect refracted and reflected light from the surface and internal structure of the particles, and tends to increase with the increase of the complexity of the particle structure. The fluorescence emission of the fluorescent molecules associated with the particles can be detected by one or more fluorescence detectors 460a-460f. The side scattering detector 435 and the fluorescence detector may include a photomultiplier tube. The signals detected at the forward scattering detector 430, the side scattering detector 435 and the fluorescence detector may be converted into electronic signals (voltage) by the detector. The data may provide information about the sample.

[0154] Those skilled in the art will recognize that flow cytometers according to embodiments of the present invention are not limited to Figure 4B The flow cytometer shown in , but can include any flow cytometer known in the art. For example, the flow cytometer can have any number of lasers, beam splitters, filters and detectors, and they have various wavelengths and various different configurations.

[0155] In operation, the cytometer operation is controlled by the controller / processor 490, and the measurement data from the detector can be stored in the memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 190 is coupled to the detector to receive the output signal from the detector, and can also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control the laser, fluid flow parameters, etc. Input / output (I / O) functions 497 can also be provided in the system. The memory 495, the controller / processor 490 and the I / O 497 can be provided entirely as an integral part of the flow cytometer 410. In such an embodiment, the display can also form part of the I / O function 497 for presenting experimental data to the user of the cytometer 400. Alternatively, some or all of the memory 495 and the controller / processor 490 and the I / O function can be part of one or more external devices (e.g., a general purpose computer). In some embodiments, some or all of the memory 495 and the controller / processor 490 can communicate wirelessly or wired with the cytometer 410. Controller / processor 490 in conjunction with memory 495 and I / O 497 may be configured to perform various functions associated with the preparation and analysis of flow cytometric experiments.

[0156] Figure 4BThe system shown in includes six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as the "filter windows" of a given detector), which are defined by the configuration of filters and / or spectrometers in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in flow cytometer experiments will emit light with their own characteristic wavelength bands. Specific fluorescent markers and their associated fluorescence emission bands for the experiment can be selected to generally coincide with the filter windows of the detectors. However, as more detectors are provided and more markers are used, a perfect correspondence between the filter windows and the fluorescence emission spectra is impossible. Typically, although the peak of the emission spectrum of a specific fluorescent molecule may be located within the filter window of a specific detector, some of the emission spectra of the marker will also overlap with the filter windows of one or more other detectors. This can be called overflow. I / O 497 can be configured to receive data about a flow cytometer experiment having a set of fluorescent markers and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. The I / O 497 may also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data (e.g., marker spectral characteristics and flow cytometer configuration data) may also be stored in the memory 495. The controller / processor 490 may be configured to evaluate the assignment of one or more labels to a marker.

[0157] Figure 5 A functional block diagram of one example of a particle analyzer control system, such as analysis controller 500, for analyzing and displaying biological events is shown. Analysis controller 500 may be configured to implement various processes for controlling the graphical display of biological events.

[0158] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0159] The analysis controller 500 can be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 can include flow cytometry event data. The analysis controller 500 can be configured to provide a graphical display of a first graph including the biological event data to a display device 506. The analysis controller 500 can be further configured to render a region of interest as a gate around a group of biological event data shown by the display device 506, for example, overlaid on the first graph. In some embodiments, the gate can be a logical combination of one or more graphical regions of interest plotted on a single parameter histogram or a bivariate graph. In some embodiments, the display can be used to display particle parameters or saturation detector data.

[0160] Analysis controller 500 may be further configured to display biological event data within the gate differently from other events in the biological event data outside the gate on display device 506. For example, analysis controller 500 may be configured to render the color of the biological event data contained within the gate different from the color of the biological event data outside the gate. Display device 506 may be implemented as a monitor, tablet computer, smart phone, or other electronic device configured to present a graphical interface.

[0161] The analysis controller 500 can be configured to receive a gate selection signal for identifying a gate from a first input device. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a gate selection signal to the analysis controller 500, thereby identifying the gate to be displayed on the display device 506 or manipulated by the display device 506 (for example, providing a click on or in a desired door when the cursor is located on the desired door). In some embodiments, the first device can be implemented as a keyboard 508 or other device for providing input signals to the analysis controller 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function can be considered an input device. For example, if Figure 5 As shown, the mouse 510 may include a right mouse button and a left mouse button, each of which may generate a trigger event.

[0162] A triggering event may cause the analysis controller 500 to change the way the data is displayed, which portions of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selecting a population of interest for particle sorting.

[0163] In some embodiments, the analysis controller 500 can be configured to detect when the mouse 510 initiates a gating selection. The analysis controller 500 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a particular distribution of the bio-event data received by the analysis controller 500.

[0164] Analysis controller 500 may be connected to storage device 504. Storage device 504 may be configured to receive and store biological event data from analysis controller 500. Storage device 504 may also be configured to receive and store flow cytometry event data from analysis controller 500. Storage device 504 may further be configured to allow retrieval of biological event data, such as flow cytometry event data, by analysis controller 500.

[0165] Display device 506 can be configured to receive display data from analysis controller 500. Display data can include graphs of biological event data and gates outlining portions of the graph. Display device 506 can be further configured to change the information presented based on input received from analysis controller 500 in conjunction with input from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0166] In some embodiments, the analysis controller 500 can generate a user interface to receive sample events for sorting. For example, the user interface can include a controller for receiving sample events or sample images. Sample events or images or sample gating can be provided before collecting event data for a sample or based on an initial event set of a portion of the sample.

[0167] Fig. 6A is a schematic diagram of a particle sorter system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorter system 600 is a cell sorter system. Fig. 6A As shown, a droplet formation sensor 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which can be coupled to, can include, or can 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 arranged in a single file to pass through a monitoring area 611 (e.g., where the laser streams intersect) irradiated by an irradiation source 612 (e.g., a laser). The vibration of the droplet formation sensor 602 causes the moving fluid column 608 to break up into a plurality of droplets 610, some of which contain particles 609.

[0168] In operation, a detection station 614 (e.g., an event detector) identifies when a particle of interest (or a cell of interest) passes through the monitoring area 611. The detection station 614 feeds a timing circuit 628, which in turn feeds a flash charge circuit 630. At the droplet breakoff point, informed by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 608 such that the droplet of interest is charged. The droplets of interest may include 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 droplets into a container (e.g., a collection tube or a multi-porous or micro-well sample plate), where a well or micro-well can be associated with a particular droplet of interest. As Fig. 6A As shown, the droplets can be collected in a drain container 638.

[0169] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal when a particle of interest passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference. The detection system 616 allows the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 616 can feed an amplitude signal 620 and / or a phase 618 signal, which in turn feeds (via amplifier 622) an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 in turn control the droplet formation sensor 602. The amplitude control circuit 626 and / or the frequency control circuit 624 can be included in the control system.

[0170] 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 thereon. The sorting decision can be included in the event data of the particle. In some embodiments, the detection system 616 and the detection station 614 can be implemented or communicatively coupled as a single detection unit so that event measurements can be collected by one of the detection system 616 or the detection station 614 and provided to a non-collecting element.

[0171] Figure 6B is a schematic diagram of a particle sorter system according to one embodiment presented herein. Figure 6B The particle sorter system 600 shown in FIG. 6 includes deflection plates 652 and 654. A charge can be applied via a stream of charging wires in the barbs. This produces a droplet stream 610 containing particles 610 for analysis. The particles can be irradiated with one or more light sources (e.g., lasers) to produce light scattering and fluorescence information. The information of the particles is obtained, for example, by sorting electronics or other detection systems ( Figure 6BDeflector plates 652 and 654 can be independently controlled to attract or repel charged droplets to direct the droplets toward a target collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, 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., exhibits no scattering or illumination information within a specified sorting range), the deflector plates can allow the particle to continue along flow path 664. Such uncharged droplets can be directed, for example, by an aspirator 670 into a waste container.

[0172] Sorting electronics may be included to initiate the collection of measurements, receive the fluorescence signal of the particles, and determine how to adjust the deflection plates to sort the particles. Figure 6B An exemplary implementation of the embodiment shown in FIG. 1 includes a BD FACSAria commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ). TM Serial flow cytometer.

[0173] Computer control system

[0174] Aspects of the present disclosure also include a computer control system, wherein the system also includes one or more computers for fully automating or partially automating the methods described herein. In some embodiments, the system includes a computer with a computer-readable storage medium, on which a computer program is stored, wherein the computer program includes instructions for determining the position information of particles in the flow stream of a flow cytometer according to the subject method described herein when loaded on the computer. In some embodiments, the computer program includes: instructions for passing a composition with particles (e.g., cells) through a flow stream comprising a core flow and a sheath flow stream; instructions for irradiating particles of the composition with a light source; instructions for detecting light from irradiated particles; instructions for determining the emission intensity of irradiated particles of the composition; and instructions for determining the position of irradiated particles in the core flow of the flow stream based on the detected light emission from the irradiated particles. In some embodiments, the composition is a biological composition, and the particles of interest in the composition are cells.

[0175] In some embodiments, the computer program includes instructions for determining the position of the irradiated particles of the composition based on the difference between the emission intensities of the first emission peak and the second emission peak. In some cases, the computer program includes instructions for determining that the irradiated particles are passed through the core flow at approximately the center of the irradiation position of the light source when the first emission peak and the second emission peak have approximately the same intensity. In some cases, the computer program includes instructions for determining that the irradiated particles are passed through the core flow at the edge of the irradiation position of the light source when the first emission peak and the second emission peak have different intensities. In some cases, the computer program includes instructions for determining that the irradiated particles are passed through the core flow at approximately the center of the core flow when the first emission peak and the second emission peak have approximately the same intensity. In some cases, the computer program includes instructions for determining that the irradiated particles are passed through the core flow at the edge of the core flow when the first emission peak and the second emission peak have different intensities.

[0176] In certain embodiments, the computer program includes instructions for generating one or more particle population clusters based on the determined position of the particles in the core flow. In some cases, the computer program includes instructions for clustering particles that are determined to be passed through the flow stream at or near the center of the core flow. In some cases, the computer program includes instructions for clustering particles that are determined to be passed through the flow stream at or near the edge of the core flow. In some cases, the computer program includes instructions for abandoning data signals generated in response to light, and the light comes from irradiated particles that are determined to be passed through the flow stream at or near the edge of the core flow.

[0177] In an embodiment, the system includes an input module, a processing module, and an output module. The subject system may include both hardware and software components, wherein the hardware components may take the form of one or more platforms, for example, in the form of a server, so that functional elements, i.e., those elements that perform specific tasks of the system (e.g., manage the input and output of information, process information, etc.) can be performed by executing software applications on and across one or more computer platforms represented by the system.

[0178] The system may include a display and an operator input device. The operator input device may be a keyboard, a mouse, etc. The processing module includes a processor that can access a memory on which instructions for performing the subject method steps are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input-output controller, a cache, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other existing or upcoming processors. The processor executes an operating system, and the operating system interfaces with firmware and hardware in a well-known manner and helps the processor coordinate and execute the functions of various computer programs, which can be written in various 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 coordinates and executes the functions of other components of the computer in collaboration with the processor. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all of which are consistent with known technologies. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow a user to manually align a light source with a flow stream based on the first and second light signals. In some embodiments, the processor includes analog electronics that provide feedback control (eg, negative feedback control).

[0179] The system memory may be any known or future memory storage device. Examples include any commonly used random access memory (RAM), magnetic media (e.g., resident hard disk or tape), optical media (e.g., read-write optical disk), flash memory device, or other memory storage device. The memory storage device may be any known or future device, including an optical disk drive, a tape drive, a removable hard disk drive, or a floppy disk drive. This type of memory storage device typically reads and / or writes a program storage medium (not shown) such as an optical disk, a tape, a removable hard disk, or a floppy disk. Any of these program storage media, or other program storage media currently 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 system memory and / or program storage devices used in conjunction with the memory storage device.

[0180] In some embodiments, a computer program product is described that includes a computer usable medium having control logic (computer software program, including program code) stored therein. When the control logic is executed by a computer processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are primarily implemented in hardware, such as using a hardware state machine. It is apparent to those skilled in the relevant art that a hardware state machine is implemented to perform the functions described herein.

[0181] The memory may be any suitable device in which the processor can store and retrieve data, such as a magnetic, optical or solid-state storage device (including a magnetic or optical disk or tape or RAM, or any other suitable device, whether fixed or portable). The processor may include a general-purpose digital microprocessor, which is appropriately programmed from a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely through a communication channel, or pre-stored in a computer program product (such as a memory or some other portable or fixed computer-readable storage medium) using any of these devices associated with the memory. For example, a magnetic or optical disk may carry the program and may be read by a disk writer / reader. The system of the present invention also includes a program, such as in the form of a computer program product, for practicing the algorithm of the above-mentioned method. The program according to the present invention may be recorded on a computer-readable medium, such as 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 floppy disks, hard disk storage media and tapes; 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.

[0182] The processor may also access a communication channel to communicate with a user at a remote location. A remote location refers to a user who is not directly engaging with the system but instead passes input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), a telephone network, a satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smart phone).

[0183] In some embodiments, a system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication scheme, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Communication schemes and cellular communications, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0184] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as a USB port, an RS-232 port, or any other suitable electrical connection port, to allow data communications between the subject system and other external devices, such as computer terminals (e.g., in a doctor's office or hospital environment), which are configured for similar complementary data communications.

[0185] In one embodiment, the communication interface is configured for infrared communication, communications or any other suitable wireless communication scheme to enable 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 subject system.

[0186] In one embodiment, the communication interface is configured to provide a data transmission connection using an Internet protocol (IP) through a cellular telephone network, a 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.

[0187] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, for example, using a common standard such as 802.11 or RF scheme, or IrDA infrared scheme. The server device can be another portable device, such as a smart phone, a personal digital assistant (PDA), or a notebook computer; or a larger device, such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as a button, a keyboard, a mouse, or a touch screen.

[0188] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the subject system (e.g., the optional data storage unit) to a network or server device using one or more of the communication schemes and / or mechanisms described above.

[0189] The output controller may include a controller for any of various known display devices for presenting information to a user (whether a person or a machine, whether local or remote). If a display device provides visual information, the information can generally be logically and / or physically organized into an array of image elements. A graphical user interface (GUI) controller may include various known or future software programs for providing a graphical input and 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. In alternative embodiments, some of these communications may be implemented using a network or other types of remote communications. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, a telephone, or a satellite network, according to known techniques. The presentation of data by the output manager may be implemented according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, emails, or other files or other forms of data. The data may include an Internet URL address so that a user can retrieve other SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or a type to be developed in the future, although they generally belong to a class of computers commonly referred to as servers. However, they may also be mainframe computers, workstations, or other computer types. They may be connected by any known or future type of cable or other communication system (including wireless systems, whether networked or otherwise). They may be co-located or physically separated. A variety of operating systems may be used on any computer platform, which may depend on the type and / or brand of computer platform selected. Suitable operating systems include Windows, iOS, Oracle Solaris, Linux, IBM i, Unix, etc.

[0190] Figure 7 Depicted is the general architecture of an example computing device 700 in accordance with certain embodiments. Figure 7 The general architecture of the computing device 700 shown in FIG. 7 includes an arrangement of computer hardware and software components. The computing device 700 may include Figure 7700. The more (or less) elements shown in. However, it is not necessary to display all these conventional elements to provide feasible disclosure. As shown, computing device 700 includes processing unit 710, network interface 720, computer readable medium driver 730, input / output device interface 740, display 750 and input device 760, all of which can communicate with each other through communication bus. Network interface 720 can provide connectivity with one or more networks or computing systems. Therefore, processing unit 710 can receive information and instructions from other computing systems or services through the network. Processing unit 710 can also communicate back and forth with memory 770, and further provide output information for optional display 750 through input / output device interface 740. Input / output device interface 740 can also accept input from optional input device 760 (such as keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope or other input device).

[0191] The memory 770 may contain computer program instructions (grouped into modules or components in some embodiments) that the processing unit 710 executes to implement one or more embodiments. The memory 770 typically includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. The memory 770 may store an operating system 772 that provides computer program instructions that the processing unit 710 uses in the general management and operation of the computing device 700. The memory 770 may also include computer program instructions and other information for implementing aspects of the present disclosure.

[0192] Non-transitory computer readable storage medium

[0193] Aspects of the present disclosure also include non-transitory computer-readable storage media having instructions for determining the position information of particles in the flow stream of a flow cytometer according to the subject method described herein. Computer-readable storage media can be used on one or more computers to fully automate or partially automate the system for practicing the methods described herein. In certain embodiments, instructions according to the methods described herein can be encoded in the form of "programming" on a computer-readable medium, wherein the term "computer-readable medium" as used herein 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 floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state disks, and network attached storage (NAS), whether these devices are inside or outside a computer. Files containing information can be "stored" on a computer-readable medium, wherein "storage" means recording information so that a computer can access and retrieve the information later. The computer-implemented method described herein can be executed using a program, which can be written in one or more computer programming languages ​​of any number. Such languages ​​include, for example, Python, Java, JavaScript, C, C#, C++, Go, R, Swift, PHP, and many others.

[0194] In some embodiments, the irradiated particles in the composition are stably associated with the irradiation power density sensitive compound of the emitted light, and the intensity of the light depends on the irradiation power density of the light source incident on the particles. In some embodiments, the irradiated particles are configured to have a spectral emission intensity profile that changes in response to the power density of the light source, for example, wherein the particles of the composition are configured to have 2 or more emission peaks in response to irradiation by the light source. In some cases, the intensity of each emission peak depends on the irradiation power intensity of the light source. In some cases, one or more of the emission peaks have a first intensity at the first irradiation power density of the light source and a second intensity at the second irradiation power density of the light source. In certain embodiments, the particles of the composition have: a first emission peak having a first intensity at the first irradiation power density and a second intensity at the second irradiation power density, and a second emission peak having the same intensity at the first irradiation power density and the second irradiation power density.

[0195] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining the position of the irradiated particles of the composition based on the difference between the emission intensities of the first emission peak and the second emission peak. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining that the irradiated particles are passed through the core flow at approximately the center of the irradiation position of the light source when the first emission peak and the second emission peak have approximately the same intensity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining that the irradiated particles are passed through the core flow at the edge of the irradiation position of the light source when the first emission peak and the second emission peak have different intensities. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining that the irradiated particles are passed through the core flow at approximately the center of the core flow when the first emission peak and the second emission peak have approximately the same intensity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining that the irradiated particles are passed through the core flow at the edge of the core flow when the first emission peak and the second emission peak have different intensities.

[0196] In some cases, the non-transitory computer-readable storage medium includes an algorithm for generating one or more particle population clusters based on the determined positions of the particles in the core flow. In some cases, the non-transitory computer-readable storage medium includes an algorithm for clustering particles that are determined to pass through the flow stream at or near the center of the core flow. In some cases, the non-transitory computer-readable storage medium includes an algorithm for clustering particles that are determined to pass through the flow stream at or near the edge of the core flow. In some cases, the non-transitory computer-readable storage medium includes an algorithm for discarding data signals generated in response to light from irradiated particles that are determined to pass through the flow stream at or near the edge of the core flow.

[0197] The non-transitory computer-readable storage medium can be used in one or more computer systems with a display and an operator input device. The operator input device can be a keyboard, a mouse, etc. The processing module includes a processor that can access a memory on which instructions for performing the subject method steps are stored. The processing module can include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input-output controller, a cache, a data backup unit, and many other devices. The processor can be a commercially available processor or one of other existing or upcoming processors. The processor executes the operating system, and the operating system interfaces with firmware and hardware in a well-known manner and helps the processor coordinate and execute the functions of various computer programs, which can be written in various programming languages ​​(such as those mentioned above, other high-level or low-level languages, and combinations thereof), as known in the art. The operating system usually coordinates and executes the functions of other components of the computer in cooperation with the processor. The operating system also provides scheduling, input-output control, file and data management, storage management, and communication control and related services, all of which are in accordance with known technologies.

[0198] Reagent test kit

[0199] Aspects of the present disclosure also include kits, wherein in some embodiments, the kit comprises one or more compositions for determining the position of particles in the core flow of a flow as described herein. In some embodiments, the kit comprises a composition having particles stably associated with an irradiation power density sensitive compound that emits light, the intensity of which depends on the irradiation power density of the light incident on the particles. As described above, in some cases, the irradiation power density sensitive compound is covalently bonded to the particles in the composition, for example, via a cleavable or non-cleavable linker. In other cases, the irradiation power density sensitive compound is associated with the particles via a specific binding member such as an antibody.

[0200] In certain embodiments, the kit comprises one or more compositions for stably associating particles of a sample composition (e.g., a biological composition in which the particles of interest are cells) with an irradiation power density sensitive compound. In some cases, the irradiation power density sensitive compound is conjugated to a specific binding member. For example, in a composition of interest, the irradiation power density sensitive compound can be covalently bonded to a protein specific binding member, such as an antibody. In other embodiments, the composition can be formulated to covalently bond the irradiation power density sensitive compound to the particles in the composition, for example, by a cleavable or non-cleavable linker.

[0201] In some cases, the particles stably associated with the irradiation power density sensitive compound have 2 or more emission peaks in response to the irradiation of the light source. In certain embodiments, one or more of the emission peaks have a first intensity at the first irradiation power density of the light source and a second intensity at the second irradiation power density of the light source. In some cases, the particles of the composition have: a first emission peak having a first intensity at the first irradiation power density and a second intensity at the second irradiation power density, and a second emission peak having the same intensity at the first irradiation power density and the second irradiation power density. In some embodiments, the irradiation power density sensitive compound is an upper conversion nanoparticle (UCNP). In some cases, the upper conversion nanoparticle is a lanthanide doped particle. In some cases, the upper conversion nanoparticle is a europium doped or thulium doped nanoparticle, such as NaYF4: Yb, Er particles or NaYF4: YbTm nanoparticles.

[0202] In some embodiments, the kit further comprises an optical adjustment component configured to orthogonally rotate the Gaussian beam profile. In certain embodiments, the kit comprises a plurality of photodetectors and programming for the subject system, for example in the form of a computer readable medium (e.g., a flash drive, USB memory, optical disc, DVD, Blu-ray disc, etc.), or instructions for downloading the programming from an Internet network solution or cloud server. The kit may also comprise an optical adjustment component, such as a lens, a reflector, a filter, an optical fiber, a wavelength separator, a pinhole, a slit, a collimation scheme, and combinations thereof.

[0203] Test kit can also include the explanation for practicing the subject method.These explanations can be present in the subject test kit in various forms, one or more of which can be present in the test kit.A form in which these explanations can exist is as the packaging, package insert etc. of printed information, test kit on suitable medium or substrate (for example, one or more sheets of paper printed with information).Another form of these explanations is the computer-readable medium having recorded information thereon, for example, floppy disk, CD (Cd), portable flash drive etc.Another form of these explanations is the website address, the information on the remote site can be accessed using this address by the Internet.

[0204] use

[0205] The subject methods, systems, and computer systems can be used in various applications where it is necessary to optimize the processors and light detection systems for flow cytometry. The subject methods and systems can also be used in light detection systems with multiple photodetectors, which are used to analyze and sort particle components in samples (e.g., biological samples) in fluid media. The present disclosure can also be used in flow cytometry, where it is necessary to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection. In an embodiment, the present disclosure reduces the need for user input or manual adjustment during sample analysis using a flow cytometer. In certain embodiments, the subject methods and systems provide a fully automated solution so that little manual input is required for adjustment of the flow cytometer during use.

[0206] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses:

[0207] 1. A method for determining the position of a particle in a core stream of a flow stream passing through a flow cell, the method comprising:

[0208] irradiating a composition comprising particles passing through a flow stream comprising a core flow stream and a sheath flow stream with a light source, wherein the particles are stably associated with an irradiation power density-sensitive compound that emits light whose intensity depends on the irradiation power density of the light source incident on the particles;

[0209] detecting light emission from the irradiated particles; and

[0210] Based on the detected light emissions from the irradiated particles, the positions of the particles in the core flow are determined.

[0211] 2. The method according to item 1, wherein the composition is a biological composition and the particles are cells.

[0212] 3. The method according to any one of items 1-2, wherein the irradiation power density sensitive compound is covalently bonded to the particles in the composition.

[0213] 4. The method according to any one of items 1 to 3, wherein the irradiation power density-sensitive compound is covalently bonded to the particles in the composition via a linker.

[0214] 5. The method according to item 4, wherein the linker is a cleavable linker.

[0215] 6. The method according to item 4, wherein the linker is a non-cleavable linker.

[0216] 7. The method according to any one of items 1 to 2, wherein the irradiation power density-sensitive compound is associated with the particle via a specific binding member.

[0217] 8. The method according to item 7, wherein the specific binding member is an antibody.

[0218] 9. The method according to any one of items 1 to 8, wherein the irradiation power density sensitive compound comprises upconversion nanoparticles (UCNPs).

[0219] 10. The method according to item 9, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0220] 11. The method according to item 9, wherein the upconversion nanoparticles comprise europium-doped or thulium-doped nanoparticles.

[0221] 12. The method according to item 11, wherein the upconversion nanoparticles comprise NaYF4:Yb,Er nanoparticles or NaYF4:YbTm nanoparticles.

[0222] 13. The method according to any one of items 1 to 12, wherein the irradiated particles in the composition are configured to have a spectral emission intensity profile that varies in response to the power density of the light source.

[0223] 14. The method according to any one of items 1 to 13, wherein the irradiated particles in the composition are configured to have 2 or more emission peaks in response to irradiation by the light source.

[0224] 15. A method according to item 14, wherein the intensity of each emission peak depends on the irradiation power intensity of the light source.

[0225] 16. The method according to any of items 14-15, wherein one or more of the emission peaks has 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.

[0226] 17. The method according to item 16, wherein the irradiated particles have:

[0227] a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density; and

[0228] A second emission peak has the same intensity at the first irradiation power density and the second irradiation power density.

[0229] 18. A method according to any one of items 1 to 17, wherein the light source has a Gaussian beam power density profile in a horizontal axis of the flow stream.

[0230] 19. A method according to clause 18, wherein the light source comprises a laser.

[0231] 20. The method according to any of items 18-19, wherein the flow stream is irradiated with an elliptical beam spot having a major axis and a minor axis.

[0232] 21. A method according to item 20, wherein the flow stream is irradiated such that the major axis of the beam spot is orthogonal to the horizontal axis of the flow stream.

[0233] 22. Method according to any of items 13-21, wherein the positions of irradiated particles in the core flow are determined based on the spectral emission intensity profile.

[0234] 23. The method according to any one of items 13 to 22, wherein the method comprises determining the position of the particles of the composition based on the difference between the emission intensities of the first emission peak and the second emission peak.

[0235] 24. The method according to item 23, wherein the method comprises determining that when the first emission peak and the second emission peak have the same intensity, the particles of the composition pass through the flow stream at approximately the center of the core stream.

[0236] 25. The method according to item 23, wherein the method comprises determining that when the first emission peak and the second emission peak have different intensities, the particles of the composition pass through the flow stream at or near an edge of the core stream.

[0237] 26. Method according to any of clauses 22-25, wherein the method comprises generating one or more particle population clusters based on the determined positions of the particles in the core flow.

[0238] 27. A method according to clause 26, wherein the method comprises clustering particles determined to pass through the flow stream at or near the centre of the core stream.

[0239] 28. A method according to any of clauses 26-27, wherein the method comprises clustering particles determined to pass through the flow stream at or near an edge of the core stream.

[0240] 29. A method according to clause 28, wherein the method comprises discarding data signals generated in response to light from irradiated particles determined to pass through the flow stream at or near an edge of the core stream.

[0241] 30. A system comprising:

[0242] a flow cell configured to deliver a composition comprising particles in a flow stream comprising a core flow and a sheath flow stream;

[0243] a light source for irradiating said particles of said composition;

[0244] a light detection system for detecting light from the irradiated particles, wherein the particles are stably associated with an irradiation power density-sensitive compound that emits light whose intensity depends on the irradiation power density of the light source incident on the particles; and

[0245] A processor comprising a memory operably coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to determine a position of the particle in the flow stream based on the detected light emissions from the irradiated particle.

[0246] 31. The system according to item 30, wherein the composition is a biological composition and the particles are cells.

[0247] 32. The system according to any of items 30-31, wherein the irradiation power density sensitive compound is covalently bonded to the particles in the composition.

[0248] 33. The system according to any of items 30-32, wherein the irradiation power density sensitive compound is covalently bonded to the particles in the composition via a linker.

[0249] 34. The system according to item 33, wherein the linker is a cleavable linker.

[0250] 35. The system according to item 33, wherein the linker is a non-cleavable linker.

[0251] 36. The system according to any of items 30-31, wherein the irradiation power density sensitive compound is associated with the particle via a specific binding member.

[0252] 37. The method according to item 36, wherein the specific binding member is an antibody.

[0253] 38. The system according to any of items 30-37, wherein the irradiation power density sensitive compound comprises upconversion nanoparticles (UCNPs).

[0254] 39. The system of item 38, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0255] 40. The system according to item 38, wherein the upconversion nanoparticles comprise europium-doped or thulium-doped nanoparticles.

[0256] 41. The system according to item 40, wherein the upconversion nanoparticles comprise NaYF4:Yb,Er nanoparticles or NaYF4:YbTm nanoparticles.

[0257] 42. A system according to any of items 30-41, wherein the light source comprises a laser.

[0258] 43. A system according to item 42, wherein the laser has a Gaussian beam power density profile on a horizontal axis of the flow stream.

[0259] 44. A system according to any of items 42-43, wherein the laser comprises an elliptical beam spot having a major axis and a minor axis.

[0260] 45. The system of item 44, wherein the laser is configured to irradiate the core flow with the elliptical beam spot such that the major axis of the beam spot is orthogonal to the horizontal axis of the flow stream.

[0261] 46. ​​The system of item 45, wherein the light source comprises an optical adjustment component configured to transmit light from the laser such that the major axis of the beam spot is orthogonal to the horizontal axis of the flow stream.

[0262] 47. A system according to item 46, wherein the optical adjustment component is configured to rotate the light beam spot by 90°.

[0263] 48. A system according to item 47, wherein the optical adjustment component comprises one or more lenses.

[0264] 49. A system according to any of items 42-48, wherein the light source comprises a plurality of lasers.

[0265] 50. The system according to item 49, wherein the light source comprises:

[0266] at least one 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 orthogonal to a longitudinal axis of the flow stream; and

[0267] A laser having an elliptical beam spot having a major axis and a minor axis is configured to irradiate the flow stream such that the major axis of the beam spot is orthogonal to the horizontal axis of the flow stream.

[0268] 51. The system according to any of items 30-50, wherein the irradiated particles in the composition are configured to have a spectral emission intensity profile that varies in response to the power density of the light source.

[0269] 52. The system according to item 51, wherein the irradiated particles in the composition are configured to have 2 or more emission peaks in response to irradiation by the light source.

[0270] 53. A system according to item 52, wherein the intensity of each emission peak depends on the irradiation power intensity of the light source.

[0271] 54. A system according to any of items 51-53, wherein one or more of the emission peaks has 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.

[0272] 55. The system according to item 54, wherein the irradiated particles have:

[0273] a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density; and

[0274] A second emission peak has the same intensity at the first irradiation power density and the second irradiation power density.

[0275] 56. A system according to any of items 51-55, wherein the memory includes instructions stored thereon, which when executed by the processor cause the processor to determine the position of the irradiated particles in the flow stream based on the spectral emission intensity profile.

[0276] 57. A system according to item 56, wherein the memory includes instructions stored thereon, which when executed by the processor cause the processor to determine the position of the particles of the composition based on the difference between the emission intensities of the first emission peak and the second emission peak.

[0277] 58. A system according to item 57, wherein the memory includes instructions stored thereon, which when executed by the processor cause the processor to determine that when the first emission peak and the second emission peak have the same intensity, the particles of the composition are passed through the flow stream at approximately the center of the core stream.

[0278] 59. A system according to item 57, wherein the memory includes instructions stored thereon, which when executed by the processor cause the processor to determine that when the first emission peak and the second emission peak have different intensities, the particles of the composition pass through the flow stream at or near the edge of the core stream.

[0279] 60. A system according to any of items 57-59, wherein the memory includes instructions stored thereon, which when executed by the processor cause the processor to generate one or more particle population clusters based on the determined positions of the particles in the core flow.

[0280] 61. A system according to item 60, wherein the memory includes instructions stored thereon, which when executed by the processor cause the processor to determine a cluster of particles passing through the flow stream at or near the center of the core flow.

[0281] 62. A system according to item 60, wherein the memory includes instructions stored thereon, which when executed by the processor cause the processor to determine a cluster of particles that pass through the flow stream at or near an edge of the core stream.

[0282] 63. A system according to item 62, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to discard data signals generated in response to light from irradiated particles determined to be passing through the flow stream at or near an edge of the core stream.

[0283] 64. A non-transitory computer readable storage medium comprising instructions stored thereon for determining a location of a particle in a core stream of a flow stream passing through a flow cell, the instructions comprising:

[0284] an algorithm for irradiating a composition comprising particles passing through a flow stream comprising a core flow and a sheath flow stream with a light source, wherein the particles are stably associated with an irradiation power density-sensitive compound that emits light, the intensity of which depends on the irradiation power density of the light source incident on the particles;

[0285] an algorithm for detecting light from the irradiated particles; and

[0286] An algorithm for determining a position of an irradiated particle in the core flow based on detected light emissions from the particle.

[0287] 65. The non-transitory computer-readable storage medium of item 64, wherein the irradiated particles in the composition are configured to have a spectral emission intensity profile that varies in response to the power density of the light source.

[0288] 66. The non-transitory computer-readable storage medium according to any one of items 64-65, wherein the irradiated particles in the composition are configured to have 2 or more emission peaks in response to irradiation by the light source.

[0289] 67. The non-transitory computer-readable storage medium of item 65, wherein the intensity of each emission peak depends on the irradiation power intensity of the light source.

[0290] 68. A non-transitory computer-readable storage medium according to any of items 66-67, wherein one or more of the emission peaks has 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.

[0291] 69. The non-transitory computer-readable storage medium according to item 68, wherein the irradiated particles have:

[0292] a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density; and

[0293] A second emission peak has the same intensity at the first irradiation power density and the second irradiation power density.

[0294] 70. The non-transitory computer-readable storage medium of any one of items 65 to 69, wherein the non-transitory computer-readable storage medium comprises an algorithm for determining a position of an irradiated particle in the core flow based on the spectral emission intensity profile.

[0295] 71. The non-transitory computer-readable storage medium according to any one of items 65-70, wherein the non-transitory computer-readable storage medium comprises an algorithm for determining the position of the particles of the composition based on the difference between the emission intensities of the first emission peak and the second emission peak.

[0296] 72. A non-transitory computer-readable storage medium according to item 71, wherein the non-transitory computer-readable storage medium comprises an algorithm for determining that the particles of the composition pass through 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.

[0297] 73. A non-transitory computer-readable storage medium according to item 71, wherein the non-transitory computer-readable storage medium comprises an algorithm for determining that the particles of the composition pass through 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.

[0298] 74. A non-transitory computer-readable storage medium according to any one of items 71-73, wherein 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 in the core flow.

[0299] 75. The non-transitory computer-readable storage medium of item 74, wherein the non-transitory computer-readable storage medium comprises an algorithm for clustering particles that are determined to pass through the flow stream at or near the center of the core flow.

[0300] 76. A non-transitory computer-readable storage medium according to any of items 74-75, wherein the non-transitory computer-readable storage medium comprises an algorithm for clustering particles that are determined to pass through the flow stream at or near an edge of the core flow.

[0301] 77. A non-transitory computer-readable storage medium according to item 76, wherein the non-transitory computer-readable storage medium includes an algorithm for discarding data signals generated in response to light, wherein the light comes from irradiated particles determined to pass through the flow stream at or near the edge of the core stream.

[0302] 78. The non-transitory computer-readable storage medium according to any one of items 64 to 77, wherein the composition is a biological composition and the particles are cells.

[0303] 79. The non-transitory computer-readable storage medium according to any one of items 64-78, wherein the irradiation power density sensitive compound is covalently bonded to the particles in the composition.

[0304] 80. The non-transitory computer readable storage medium according to any one of items 64-79, wherein the radiation power density sensitive compound is covalently bonded to the particles in the composition via a linker.

[0305] 81. The non-transitory computer-readable storage medium according to item 80, wherein the linker is a cleavable linker.

[0306] 82. The non-transitory computer-readable storage medium according to item 80, wherein the linker is a non-cleavable linker.

[0307] 83. The non-transitory computer readable storage medium according to any one of items 64-78, wherein the irradiation power density sensitive compound is associated with the particle via a specific binding member.

[0308] 84. The non-transitory computer readable storage medium according to item 83, wherein the specific binding member is an antibody.

[0309] 85. The non-transitory computer readable storage medium according to any one of items 64-84, wherein the irradiation power density sensitive compound comprises upconversion nanoparticles (UCNPs).

[0310] 86. The non-transitory computer-readable storage medium of item 85, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0311] 87. The non-transitory computer-readable storage medium according to item 85, wherein the upconversion nanoparticles comprise europium-doped or thulium-doped nanoparticles.

[0312] 88. The non-transitory computer-readable storage medium of item 87, wherein the upconversion nanoparticles comprise NaYF4:Yb,Er nanoparticles or NaYF4:YbTm nanoparticles.

[0313] 89. A kit for determining the position of particles in a core stream of a flow stream passing through a flow cell of a flow cytometer, the kit comprising a composition comprising particles stably associated with an irradiance power density sensitive compound that emits light, the intensity of which depends on the irradiance power density of light incident on the particles.

[0314] 90. The kit according to item 89, wherein the radiation power density sensitive compound is covalently bonded to the particles in the composition.

[0315] 91. The kit according to any of items 89-90, wherein the radiation power density sensitive compound is covalently bonded to the particles in the composition via a linker.

[0316] 92. The kit according to item 91, wherein the linker is a cleavable linker.

[0317] 93. The kit according to item 91, wherein the linker is a non-cleavable linker.

[0318] 94. The kit according to item 89, wherein the irradiation power density sensitive compound is associated with the particle via a specific binding member.

[0319] 95. The kit according to item 94, wherein said specific binding member is an antibody.

[0320] 96. The kit according to any of items 89-95, wherein the irradiation power density sensitive compound comprises upconversion nanoparticles (UCNPs).

[0321] 97. The kit according to item 96, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles.

[0322] 98. The kit according to item 97, wherein the upconversion nanoparticles comprise europium-doped or thulium-doped nanoparticles.

[0323] 99. The kit according to item 97, wherein the upconversion nanoparticles comprise NaYF4:Yb,Er nanoparticles or NaYF4:YbTm nanoparticles.

[0324] 100. The kit according to any of items 89 to 99, wherein the irradiation power density sensitive compound exhibits 2 or more emission peaks when irradiated by a light source.

[0325] 101. The kit according to item 100, wherein said irradiation power density sensitive compound exhibits a spectral emission intensity profile that varies in response to said power density of said light source.

[0326] 102. The kit according to any of items 100-101, wherein one or more of said emission peaks has a first intensity at a first irradiation power density of said light source; and a second intensity at a second irradiation power density of said light source.

[0327] 103. The kit according to item 102, wherein the particles have:

[0328] a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density; and

[0329] A second emission peak has the same intensity at the first irradiation power density and the second irradiation power density.

[0330] 104. The kit according to any of items 89 to 103, wherein the kit further comprises an optical adjustment assembly configured to orthogonally rotate the light beam.

[0331] 105. The kit according to item 104, wherein the optical adjustment component comprises one or more lenses.

[0332] 106. A kit for determining the position of particles in a core stream of a flow stream passing through a flow cell of a flow cytometer, the kit comprising a composition comprising a compound for stably associating with particles in a sample composition, wherein the compound is an irradiance power density sensitive compound that emits light, the intensity of which depends on the irradiance power density of light incident on the particles.

[0333] 107. The kit according to item 106, wherein the sample composition is a biological composition and the particles are cells.

[0334] 108. The kit according to any of items 106-107, wherein the irradiation power density sensitive compound is conjugated to a specific binding member.

[0335] 109. The kit according to item 108, wherein the irradiation power density sensitive compound is covalently bonded to the protein binding member.

[0336] 110. The kit according to item 109, wherein the irradiation power density sensitive compound is covalently bonded to an antibody. 111. The kit according to item 89, wherein the composition is formulated to covalently bond the irradiation power density sensitive compound to the particles in the sample composition.

[0337] 112. The kit according to item 111, wherein the composition is formulated such that the irradiation power density sensitive compound is covalently bonded to the particles in the sample composition via a linker.

[0338] 113. The kit according to item 112, wherein the linker is a cleavable linker.

[0339] 114. The kit according to item 112, wherein the linker is a non-cleavable linker.

[0340] 115. The kit according to any of items 106-114, wherein the irradiation power density sensitive compound comprises upconversion nanoparticles (UCNPs).

[0341] 116. The kit according to item 115, wherein the upconversion nanoparticles comprise lanthanide-doped nanoparticles. 117. The kit according to item 115, wherein the upconversion nanoparticles comprise europium-doped or thulium-doped nanoparticles.

[0342] 118. The kit according to item 109, wherein the upconversion nanoparticles comprise NaYF4:Yb,Er nanoparticles or NaYF4:YbTm nanoparticles.

[0343] 119. The kit according to any one of items 106 to 118, wherein the irradiation power density-sensitive compound exhibits 2 or more emission peaks when irradiated by a light source.

[0344] 120. The kit according to item 119, wherein said irradiation power density sensitive compound exhibits a spectral emission intensity profile that varies in response to said power density of said light source.

[0345] 121. The kit according to any of items 119-120, wherein one or more of said emission peaks has a first intensity at a first irradiation power density of said light source; and a second intensity at a second irradiation power density of said light source.

[0346] 122. The kit according to item 121, wherein the particles have:

[0347] a first emission peak having a first intensity at a first irradiation power density and a second intensity at a second irradiation power density; and

[0348] A second emission peak has the same intensity at the first irradiation power density and the second irradiation power density.

[0349] 123. The kit according to any of items 106-122, wherein the kit further comprises an optical adjustment assembly configured to orthogonally rotate the light beam.

[0350] 124. The kit according to any of items 106 to 122, wherein said kit further comprises one or more fluorophores for labelling said particles of said sample composition.

[0351] 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 apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0352] Therefore, the foregoing only illustrates the principles of the present invention.It should be understood that those skilled in the art will be able to think of various arrangements, although not explicitly described or shown herein, these arrangements embody the principles of the present invention and are included in its spirit and scope.In addition, all examples and conditional language recorded herein are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventors to promote this area, and should be interpreted as not being limited to such specific examples and conditions of recording.In addition, all statements of the principles, aspects and embodiments of the present invention and their specific examples recorded herein are intended to cover their structural and functional equivalents.In addition, such equivalents are intended to include currently known equivalents and equivalents developed in the future, that is, no matter how the structure, any element of the developed performance of the same function.In addition, any content disclosed herein is not intended to be dedicated to the public, regardless of whether such disclosures are clearly recorded in the claims.

[0353] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112 (f) or 35 U.S.C. § 112 (6) is expressly defined as being cited for such a limitation in a claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of a certain limitation in a claim; if such exact phrase is not used in the limitation in a claim, 35 U.S.C. § 112 (f) or 35 U.S.C. § 112 (6) is not cited.

Claims

1. A method for determining the position of a particle in a core stream of a flow stream passing through a flow cell, the method comprising: irradiating a composition comprising particles passing through a flow stream comprising a core flow stream and a sheath flow stream with a light source, wherein the particles are stably associated with an irradiation power density-sensitive compound that emits light whose intensity depends on the irradiation power density of the light source incident on the particles; detecting light emission from the irradiated particles; as well as Based on the detected light emissions from the irradiated particles, the positions of the particles in the core flow are determined.

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

3. The method of any one of claims 1-2, wherein the irradiation power density sensitive compound is covalently bonded to the particles in the composition.

4. The method according to any one of claims 1 to 3, wherein the irradiation power density sensitive compound is covalently bonded to the particles in the composition via a linker. The method of claim 4 , wherein the linker is a cleavable linker. The method of claim 4 , wherein the linker is a non-cleavable linker.

7. The method according to any one of claims 1-2, wherein the irradiation power density-sensitive compound is associated with the particle via a specific binding member.

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

9. The method according to any one of claims 1-8, wherein the irradiation power density sensitive compound comprises upconversion nanoparticles (UCNPs).

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

12. The method according to claim 11, wherein the upconversion nanoparticles comprise NaYF4:Yb,Er nanoparticles or NaYF4:YbTm nanoparticles.

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

14. A system comprising: a flow cell configured to deliver a composition comprising particles in a flow stream comprising a core flow and a sheath flow stream; a light source for irradiating said particles of said composition; a light detection system for detecting light from the irradiated particles, wherein the particles are stably associated with an irradiation power density-sensitive compound that emits light, the intensity of which depends on the irradiation power density of the light source incident on the particles; as well as A processor comprising a memory operably coupled to the processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to determine a position of the particle in the flow stream based on the detected light emissions from the irradiated particle.

15. A kit for determining the position of particles in a core stream of a flow stream passing through a flow cell of a flow cytometer, the kit comprising a composition comprising particles stably associated with an irradiance power density sensitive compound that emits light, the intensity of which depends on the irradiance power density of light incident on the particles.

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