Stroboscopic laser excitation system and method of using same

Abstract

Aspects of the present disclosure include systems for illuminating particles in a flow stream. Systems according to certain embodiments include a light source having a first laser configured for continuous illumination of the flow stream and a second laser configured for illumination of the flow stream at discrete intervals, wherein each discrete interval of illumination by the second laser is triggered by illumination of particles in the flow stream with the first laser. Methods of illuminating a sample in a flow stream with the subject light source are also described. Computer readable storage media for practicing the subject methods are provided. Kit-of-parts having one or more lasers are also provided.

Description

[0001] Cross-references to related applications

[0002] This application relates to U.S. Provisional Patent Application No. 62 / 978,751, filed February 19, 2020; the disclosure of which is incorporated herein by reference.

[0003] introduction

[0004] Characterization of analytes in biological fluids has become an important part of medical diagnosis and assessment of a patient's overall health. Detection of analytes in biological fluids (such as human blood or blood-derived products) can provide results that may play a role in determining treatment options for patients with multiple disease conditions.

[0005] Flow cytometry is a technique used to characterize and often classify biological materials, such as cells in a blood sample or target particles in another type of biological or chemical sample. A flow cytometer typically consists of a sample container for receiving a fluid sample (e.g., a blood sample) and a sheath container containing sheath fluid. The flow cytometer transports particles (including cells) in the fluid sample as a flow stream to a flow cell, while also guiding the sheath fluid into the flow cell. To characterize the composition of the flow stream, it is illuminated. Changes in the material within the flow stream, such as the presence of morphology or fluorescent labels, can cause changes in the observed light, which allow for characterization and separation.

[0006] To characterize the components in a flow stream, light must be incident on and collected from it. The light source in a flow cytometer can be a broad-spectrum lamp, a light-emitting diode (LED), or a single-wavelength laser. The light source is aligned with the flow stream and collects and quantifies the optical response from the incident particles. Summary of the Invention

[0007] This disclosure includes a system for irradiating particles in a flowing stream. A system according to some embodiments includes a first laser configured for continuously irradiating the flowing stream and a second laser configured for irradiating the flowing stream at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by particles in the flowing stream with the first laser. In some embodiments, the second laser is configured to irradiate the flowing stream at a location downstream of the first laser. In some embodiments, the first laser is a laser configured to irradiate the flowing stream with a light wavelength smaller than that of the second laser. In some cases, the first laser irradiates the flowing stream with light smaller than the light wavelength emitted from particles in the flowing stream. For example, the light wavelength of the first laser may be smaller than the fluorescence of particles (e.g., cells) in the flowing stream.

[0008] In some embodiments, the system includes a first laser configured for continuously irradiating a flowing stream and a plurality of lasers configured for irradiating the flowing stream at discrete intervals, such as two or more lasers, three or more lasers, and including four or more lasers for irradiating the flowing stream at discrete intervals. In some cases, the plurality of lasers configured for irradiating the flowing stream at discrete intervals are positioned to irradiate the flowing stream downstream of the first laser. In some cases, each of the lasers is configured to independently irradiate the flowing stream at intervals of 10 μm or less, such as 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, including positions on the flowing stream spaced 5 μm or less. In embodiments, each of the lasers is configured to independently irradiate the flowing stream at discrete intervals from 0.001 μs to 500 ms, such as 1 μs to 5000 μs.

[0009] In some embodiments, the system is configured to determine the irradiation time and duration of each laser. In some cases, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the irradiation time (i.e., discrete intervals at which irradiation begins) of each pair of lasers in the flow stream. In other cases, the memory includes instructions that, when executed by the processor, cause the processor to calculate the irradiation duration (i.e., the length of each discrete interval) of each pair of lasers in the flow stream. In still other cases, the memory includes instructions that, when executed by the processor, cause the processor to calculate the time interval between irradiations of each laser pair of lasers in the flow stream.

[0010] In some embodiments, the system is configured to activate one or more downstream lasers in response to the irradiation of particles in the flow stream by a first laser. In some embodiments, the system includes a beam stop located in the beam path between one or more downstream lasers and the flow stream, wherein the beam stop is configured to move in response to the irradiation of particles by the first laser. In some embodiments, the system includes a beam deflector located in the beam path between one or more downstream lasers and the flow stream, wherein the beam deflector is configured to guide light into the flow stream in response to the irradiation of particles by the first laser. In some cases, the beam deflector is an acousto-optic device, such as an acousto-optic deflector (AOD) or an acousto-optic modulator (AOM). In some cases, the beam deflector is an electro-optic device, such as an electro-optic deflector (EOD) or an electro-optic modulator (EOM).

[0011] In some embodiments, the system includes a light detection system for detecting light from particles in the illumination stream. The light detection system according to some embodiments includes a photodetector and an optical adjustment assembly configured to reduce the amount of light transmitted from the first laser to the photodetector. In some cases, the optical adjustment assembly is a bandpass filter, such as a long pass filter that transmits a spectral range of light longer than the illumination wavelength of the first laser. In other cases, the optical adjustment assembly is a dichroic mirror, such as a dichroic mirror that transmits a spectral range longer than the illumination wavelength of the first laser and reflects a spectral range including the illumination wavelength of the first laser.

[0012] In some embodiments, the photodetector is configured to detect light from each irradiated flow in the laser. In some embodiments, the photodetector includes only one photosensor assembly. In other embodiments, the photodetector is an array of photodetectors. In some embodiments, the light detection system includes a light propagation assembly that directs light from the flow to the photodetector. In some cases, the light propagation assembly includes an optical fiber. In some cases, the light propagation assembly includes a single optical fiber.

[0013] This disclosure also includes methods for irradiating a sample in a flowing stream. According to some embodiments, the method includes continuously irradiating the flowing stream with a first laser and irradiating the flowing stream with a second laser at discrete intervals. In embodiments, each discrete interval irradiated by the second laser is triggered by irradiating particles in the flowing stream with the first laser. In some embodiments, the method includes irradiating a location in the flowing stream downstream of the first laser with the second laser. In some embodiments, the wavelength of the first laser is shorter than the wavelength of the second laser.

[0014] In some embodiments, the method includes continuously irradiating the flow with a first laser and with multiple lasers, such as two or more lasers, for example, three or more lasers, and includes irradiating the flow with four or more lasers at discrete intervals. In some cases, the flow is irradiated with multiple lasers at a location downstream of the flow irradiated with the first laser. In some cases, the flow is irradiated with multiple lasers at locations spaced 10 μm or less apart from each other, for example, 5 μm or less. In some cases, each of the multiple lasers irradiates the flow at discrete intervals of 0.001 microseconds to 500 milliseconds, for example, 1 microsecond to 5000 microseconds.

[0015] In some embodiments, the method includes determining one or more of the time and duration of irradiation of each pair of particles in the laser stream. In some cases, the irradiation time of one or more laser streams is determined (i.e., when irradiation begins). In other cases, the irradiation duration of one or more laser streams is determined (i.e., the length of each discrete interval). In still other cases, the time interval between each pair of particles in the laser stream is determined. In some cases, the method includes irradiating particles in the flow stream with each successive pair of particles in the laser stream; detecting light from the flow stream in response to each pair of particles in the laser stream; and calculating the time interval for each particle irradiation in the laser stream.

[0016] In some embodiments, the method includes activating one or more downstream lasers in response to the illumination of particles in a flow stream by a first laser. In some embodiments, the method includes moving a beam stop in response to the illumination of particles by the first laser, the beam stop being located in a beam path between one or more downstream lasers and the flow stream. In some embodiments, the method includes directing light from one or more downstream lasers into the flow stream using a beam deflector in response to the illumination of particles by the first laser. In some cases, the beam deflector is an acousto-optic device, such as an acousto-optic deflector (AOD) or an acousto-optic modulator (AOM). In some cases, the beam deflector is an electro-optic device, such as an electro-optic deflector (EOD) or an electro-optic modulator (EOM).

[0017] The method disclosed herein also includes detecting light from particles in a flowing stream using a light detection system. In some embodiments, the light detection system includes a photodetector and an optical adjustment assembly configured to reduce the amount of light transmitted from a first laser to the photodetector. In some embodiments, light from the flowing stream is detected by the photodetector through a bandpass filter. In other embodiments, light from the flowing stream is detected by the photodetector through a dichroic mirror.

[0018] Non-transitory computer-readable storage media for practicing the subject method are also described. According to certain embodiments, the non-transitory computer-readable storage medium includes instructions stored thereon having an algorithm for continuously irradiating a flow with a first laser, an algorithm for irradiating the flow with a second laser at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with the first laser, an algorithm for detecting light from each irradiated particle in the flow with the laser using a single photodetector coupled to a single optical fiber; and an algorithm for calculating the irradiation time of each laser on the flow. In some embodiments, the non-transitory computer-readable storage medium includes instructions having an algorithm for continuously irradiating a flow with a first laser; and an algorithm for irradiating a flow with multiple lasers at discrete intervals, wherein each discrete interval irradiated by each of the multiple lasers is triggered by irradiating particles in the flow with the first laser. In some cases, a non-transitory computer-readable storage medium includes instructions having an algorithm for irradiating a particle in a flow stream with each successive irradiation of a particle in a laser, an algorithm for detecting light from the flow stream in response to each irradiation of a particle in the laser, and an algorithm for calculating the time interval between each irradiated particle in the laser.

[0019] In some embodiments, the non-transitory computer-readable storage medium includes instructions having an algorithm for activating a second laser in response to irradiation of a particle by a first laser. In some embodiments, the non-transitory computer-readable storage medium includes instructions having an algorithm for moving a beam stop located in a beam path between the second laser and the flow stream in response to irradiation of a particle by the first laser. In some embodiments, the non-transitory computer-readable storage medium includes instructions having an algorithm for guiding light from the second laser to the flow stream using a beam deflector in response to irradiation of a particle by the first laser.

[0020] A kit comprising one or more components of a subject system is also provided. According to some embodiments, the kit includes one or more lasers, such as lasers configured for continuous illumination and lasers configured for illumination at discrete intervals. In some embodiments, the kit may include switches (e.g., pulsed lasers) for operating one or more lasers at discrete intervals. The kit may also include optical adjustment components configured to reduce the passage of light wavelengths from one or more lasers. In some cases, the optical adjustment component is a bandpass filter, such as a longpass filter that transmits a spectral range of light longer than the illumination wavelengths of one or more lasers. In other cases, the optical adjustment component is a dichroic mirror, such as a dichroic mirror that transmits a spectral range of light longer than the illumination wavelengths of one or more lasers and reflects a spectral range including the wavelengths of one or more lasers.

[0021] The kit may also include an array of photodetectors for detecting light from the flowing stream. In some embodiments, the support stage includes a motor, such as a stepper motor. The kit may also include an optical relay system, such as an optical fiber (e.g., a single fiber), for propagating light from the sample in the flowing stream to the detector. Attached Figure Description

[0022] The invention can be best understood by reading the following detailed description in conjunction with the accompanying drawings, including the following figures:

[0023] Figure 1A A light source is depicted according to certain embodiments, having a laser configured for continuous illumination and three lasers configured for illumination at discrete intervals. Figure 1B A light source according to certain embodiments is depicted having a first laser configured to continuously irradiate a flow stream and a second laser configured to activate in response to irradiation of particles by the first laser. Figure 1C A first laser configured to continuously irradiate a flow stream, and a second laser configured to irradiate the flow stream in response to a movement of the irradiation beam aperture of the first laser on a particle, are described according to certain embodiments. Figure 1D A first laser configured to continuously irradiate a flow stream, according to certain embodiments, and a second laser configured to irradiate the flow stream when a beam deflector redirects light to the flow stream in response to irradiation of particles by the first laser, are described. Figure 2 A light source comprising a laser configured for continuous illumination and three lasers configured for illumination at discrete intervals, according to certain embodiments, is described, as well as a light detection system for transmitting and measuring light using a single photodetector.

[0024] Figure 3 The illustration depicts laser irradiation by three lasers at discrete intervals in response to a trigger signal from a continuously irradiating laser, according to certain embodiments.

[0025] Figure 4A A functional block diagram of a particle analysis system according to certain embodiments is depicted. Figure 4B A flow cytometer according to certain embodiments is described.

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

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

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

[0029] Figure 7 A block diagram of a computing system according to certain embodiments is depicted. Detailed Implementation

[0030] This disclosure includes a system for irradiating particles in a flowing stream. A system according to some embodiments includes a light source having a first laser configured for continuously irradiating the flowing stream and a second laser configured for irradiating the flowing stream at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flowing stream with the first laser. A method for irradiating a sample in a flowing stream with a subject light source is also described. A computer-readable storage medium for practicing the subject method is provided. An assembly having one or more lasers is also provided.

[0031] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as variations are naturally possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not restrictive, as the scope of the invention will be limited only by the appended claims.

[0032] Where numerical ranges are provided, it should be understood that, unless the context explicitly specifies otherwise, each intermediate value to one-tenth of the lower limit unit, the range between the upper and lower limits of that range and any other specified or intermediate value, is included in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also within this invention, but are subject to any specific exclusions within the ranges. Where the range includes one or both limitations, the range excluding one or both of those included limitations is also included in this invention.

[0033] Certain ranges are presented in this document with numerical values ​​beginning with the term “about”. The term “about” is used in this document to provide literal support for the exact number preceding it, as well as for numbers that are close to or approximate to the number preceding the term. In determining whether a number is close to or approximate to a specifically listed number, an unlisted number that is close to or approximates may be a substantially equivalent number to the specifically listed number provided in its presented context.

[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 this invention pertains. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, representative illustrative methods and materials are described hereafter.

[0035] All publications and patents referenced in this specification are incorporated herein by reference, just as each individual publication or patent is specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the methods and / or materials relating to the publication reference. References to any publication are for those published prior to the filing date and should not be construed as an admission that the invention is not entitled to precede such publication by virtue of a prior invention. Furthermore, the publication dates provided may differ from the actual publication dates that may require independent verification.

[0036] Note that, as used herein and in the appended claims, elements without quantifiers include plural indicators unless the context clearly specifies otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a preliminary basis for the use of exclusive terms such as "unique," "only," etc., when referencing claim elements or using the "negative" limitation.

[0037] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has its own components and features, which can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any enumerated methods may be performed in the order of the enumerated events or in any other logically possible order.

[0038] Although the apparatus and method have been or will be described for the purposes of grammatical fluency and functional interpretation, it should be clearly understood that, except as provided in 35 U.SC §112, they should not be construed as necessarily being limited in any way to the interpretation of “method” or “step,” but rather should be given the full meaning and scope of the definitions provided by the claims under the principle of equivalence, if the claims are to conform to 35 U.SC §112.

[0039] As summarized above, this disclosure provides systems and methods for irradiating particles in a flowing stream. In other embodiments described in this disclosure, a system having a first laser configured for continuous irradiation and a second laser configured for irradiation at discrete intervals is first described in more detail. Next, a method for irradiating a sample in a flowing stream and detecting light from particles in the flowing stream is described. An assembly having one or more components of the subject system is also provided.

[0040] A system for irradiating particles in a flowing stream

[0041] This disclosure includes a system for irradiating particles in a flowing stream. A system according to certain embodiments includes a first laser configured for continuously irradiating the flowing stream and a second laser configured for irradiating the flowing stream at discrete intervals, wherein each discrete interval of irradiation by the second laser is triggered by particles in the flowing stream from the first laser. As described herein, the term “continuous” is used in its conventional sense to refer to laser irradiation of a flowing stream that is constant and uninterrupted for the duration during which a target sample flows through the flowing stream. In some embodiments, the laser configured for continuous irradiation is an unshielded laser (i.e., not intermittently blocked by a beam stop or shielding assembly). In some embodiments, continuously irradiating the flow with a laser includes maintaining a constant laser irradiation intensity, for example, varying by 5% or less, such as 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, and includes changing the laser irradiation intensity by 0.0001% or less. In some embodiments, the laser configured for continuous irradiation of the flow does not exhibit intensity variation during the duration the sample flows through the flow. The light output intensity of the laser configured for continuous irradiation can be measured using any convenient device, including but not limited to scanning slit profilometers, charge-coupled devices (CCDs, such as enhanced charge-coupled devices, ICCDs), positioning sensors, power sensors (e.g., thermopile power sensors), optical power sensors, energy meters, digital laser photometers, laser diode detectors, and other types of photodetectors.

[0042] In embodiments, the laser configured for continuous irradiation can vary and can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the laser configured for continuous irradiation is a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, the laser configured for continuous irradiation of interest includes metal vapor lasers, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, or a combination thereof. In other cases, the system includes ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, or combinations thereof. In other cases, the system includes semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs), or frequency-doubled or third-doubled versions of any of the above lasers.

[0043] The system of interest according to embodiments also includes one or more lasers configured to irradiate the flow at discrete intervals. The term "discrete interval" is used herein in its conventional sense, referring to irradiation of the flow for a predetermined duration followed by a period of time during which the flow is not irradiated by the lasers (e.g., by turning off the lasers or by blocking the lasers, for example, with a chopper, beam stop, etc.). In some embodiments, the lasers are configured to irradiate the flow at discrete intervals of 0.001 μs or longer, such as 0.005 μs or longer, such as 0.01 μs or longer, such as 0.05 μs or longer, such as 0.1 μs or longer, such as 0.5 μs or longer, such as 1 μs or longer, such as 5 μs or longer, such as 10 μs or longer, such as 50 μs or longer, such as 100 μs or longer, including 500 μs or longer. In some cases, the discrete intervals used for irradiating the flow can be 0.0001 μs to 500 ms, for example 0.0005 μs to 250 ms, for example 0.001 μs to 50 ms, for example 0.005 μs to 5 ms, for example 0.01 μs to 1000 μs, for example 0.05 to 750 μs, for example 0.1 μs to 500 μs, for example 0.5 μs to 250 μs, for example 1 μs to 100 μs, and include 10 μs to 100 μs. The interval between each laser irradiation can be 0.001 μs or longer, for example, 0.005 μs or longer, for example, 0.01 μs or longer, for example, 0.05 μs or longer, for example, 0.1 μs or longer, for example, 0.5 μs or longer, for example, 1 μs or longer, for example, 5 μs or longer, for example, 10 μs or longer, for example, 50 μs or longer, for example, 100 μs or longer, including 500 μs or longer. For example, each discrete interval irradiated by each laser can be 0.0001 μs to 500 ms, for example 0.0005 μs to 250 ms, for example 0.001 μs to 50 ms, for example 0.005 μs to 5 ms, for example 0.01 μs to 1000 μs, for example 0.05 to 750 μs, for example 0.1 μs to 500 μs, for example 0.5 μs to 250 μs, for example 1 μs to 100 μs, and includes 10 μs to 100 μs.

[0044] In some embodiments, the target light source includes two or more lasers configured to irradiate the flow at discrete intervals, such as three or more, four or more, five or more, ten or more, fifteen or more, twenty-five or more, and even fifty or more lasers configured to irradiate the flow at discrete intervals. To irradiate the flow at discrete intervals, each laser is operatively coupled to one or more components to provide intermittent irradiation with each laser. As described in more detail below, any convenient means can be used to provide intermittent irradiation, such as electronic switches for turning the lasers on and off, for example, computer-controlled switches triggered based on data signals (e.g., received or input data signals). In some embodiments, the lasers are configured to irradiate at discrete intervals by intermittently exposing the laser beam of each laser to a beam chopper or beam stop.

[0045] In some embodiments, each of the lasers is configured to irradiate the flow at discrete intervals at locations on the flow, the locations being downstream of locations irradiated by lasers configured for continuous irradiation. For example, in one instance, the target light source includes a first laser configured for continuous irradiation of the flow; a second laser configured to irradiate the flow at a location downstream of the first laser; a third laser configured to irradiate the flow at a location downstream of the second laser; and a fourth laser configured to irradiate the flow at a location downstream of the third laser. Depending on the flow velocity and the distance between the irradiation positions of each laser, each laser is configured to independently irradiate the flow at a downstream position of 5 μm or more, such as 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, 10 μm or more, 15 μm or more, 25 μm or more, 50 μm or more, 100 μm or more, 250 μm or more, or 500 μm or more, including wherein each laser is independently configured to irradiate the flow at discrete intervals at a position 1000 μm or more downstream of the irradiation position of the first laser. For example, the irradiation position of each laser on the flow can be 5 μm to 5000 μm, for example 10 μm to 2500 μm, for example 25 μm to 1000 μm, for example 50 μm to 750 μm, for example 75 μm to 500 μm, and including 100 μm to 250 μm downstream of the irradiation position of the first laser on the flow.

[0046] Figure 1AA light source according to certain embodiments is depicted having a laser configured for continuous illumination and three lasers configured for illumination at discrete intervals. Light source 100a includes a laser 101 configured for continuous illumination and lasers 102, 103, and 104, each located downstream of laser 101 along a flow stream 107 and configured for illumination at discrete intervals. Light source 100 includes a bandpass filter 105 configured to reduce the amount of light transmitted from the continuously illuminating laser 101 to a photodetector 106.

[0047] The distance between each pair of lasers irradiating the flow can vary, wherein the irradiation interval is independently 0.0001 μm or greater, for example 0.0005 μm or greater, for example 0.001 μm or greater, for example 0.005 μm or greater, for example 0.01 μm or greater, for example 0.05 μm or greater, for example 0.1 μm or greater, for example 0.5 μm or greater, for example 1 μm or greater, for example 2 μm or greater, for example 3 μm or greater, for example 4 μm or greater, for example 5 μm or greater, for example 6 μm or greater, for example 7 μm or greater, for example 8 μm or greater, for example 9 μm or greater, and includes 10 μm or greater. In some cases, the lasers in the target light source are configured to irradiate positions on the flow that are directly adjacent to each other (i.e., no irradiation spacing).

[0048] In embodiments, the laser configured for irradiating the flow at discrete intervals can vary and can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the laser configured for continuous irradiation is a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, the laser configured for continuous target irradiation includes metal vapor lasers, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, or a combination thereof. In other cases, the system includes ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, or combinations thereof. In other cases, the system includes semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs), or frequency-doubled or third-doubled implementations of any of the above lasers. The lasers can include any combination of laser types. For example, in some embodiments, the target system includes an array of lasers configured for irradiation at discrete intervals, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers. In some embodiments, the target system includes a continuous-wave diode laser array.

[0049] Depending on the desired wavelength of light produced in the output laser beam (e.g., for illuminating a sample in a flowing stream), each laser may have a specific wavelength varying from 200 nm to 1500 nm, such as 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, and including 400 nm to 800 nm. In some embodiments, the target laser may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser. In some embodiments, each of the lasers outputs light of a different wavelength. In some cases, the lasers in the system are positioned such that each downstream laser outputs light of a longer wavelength. For example, in a system comprising four lasers, the wavelength of light from the second laser is longer than that from the first laser, the wavelength of light from the third laser is longer than that from the second laser, and the wavelength of light from the fourth laser is longer than that from the third laser.

[0050] The light source may also include one or more optical adjustment components. The term "optical adjustment," used herein in its conventional sense, refers to any means capable of altering the spatial width of illumination or some other characteristic of illumination from one or more lasers, such as illumination direction, wavelength, beam width, beam intensity, focus, and pulse width. Optical adjustment means can be any convenient means of adjusting one or more characteristics of the lasers, including but not limited to lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimating devices, and combinations thereof. In some embodiments, the target system includes one or more focusing lenses. In one instance, the focusing lens may be a reducing lens. In another instance, the focusing lens is a magnifying lens. In other embodiments, the target system includes one or more mirrors. In other embodiments, the target system includes optical fibers. In some embodiments, the beams from each laser are combined by a beam combiner, such as a dichroic mirror beam combiner. In these embodiments, the beam combiner combines the beams from each laser and propagates the light to a beam shaping component.

[0051] In some embodiments, light from each laser is propagated to the flow stream via a mirror assembly. In some cases, the mirror assembly may include a first mirror and a second mirror, the second mirror being positioned to propagate light from the first mirror to the flow stream. In embodiments, the second mirror is positioned to propagate light from the first mirror at angles varying relative to the first mirror, for example from 1° to 90°, from 5° to 85°, from 10° to 80°, from 15° to 75°, from 20° to 70°, from 25° to 65°, and including angles from 30° to 60°. In some cases, the second mirror is positioned to propagate light from the first mirror orthogonally. In other embodiments, the second reflector is positioned to propagate light from the first reflector at varying angles relative to the laser, such as from 1° to 90°, from 5° to 85°, from 10° to 80°, from 15° to 75°, from 20° to 70°, from 25° to 65°, and including angles from 30° to 60°. In some cases, the second reflector is positioned to propagate light orthogonally relative to the laser. In some embodiments, the second reflector is also a beam combiner configured to combine beams from two or more lasers. In these embodiments, the second reflector may be a dichroic mirror that selectively transmits light of a desired wavelength.

[0052] In some embodiments, optical adjustment components (e.g., beamstops or beam chopper components) are movable. In some cases, the optical adjustment components can move in two dimensions, such as in the XY plane. In other cases, the optical adjustment components can move in three dimensions. In some embodiments, one or more optical adjustment components are configured to change angles, such as tilting relative to the laser. For example, the system can be configured to change the illumination position on the flow stream by changing the angle of the mirror relative to the laser by 5° or more, such as 10° or more, such as 15° or more, such as 20° or more, such as 30° or more, such as 45° or more, such as 60° or more, including 75° or more.

[0053] When optical adjustment components (e.g., one or more beam stops, beam choppers, etc.) are configured to move, the optical adjustment components can be configured to move continuously or at discrete intervals. In some embodiments, the movement of the optical adjustment components is continuous. In other embodiments, the optical adjustment components can move at discrete intervals, for example, in increments of 0.01 micrometers or greater, such as 0.05 micrometers or greater, such as 0.1 micrometers or greater, such as 0.5 micrometers or greater, such as 1 micrometer or greater, such as 10 micrometers or greater, such as 100 micrometers or greater, such as 500 micrometers or greater, such as 1 millimeter or greater, such as 5 millimeters or greater, such as 10 millimeters or greater, and including increments of 25 millimeters or greater.

[0054] The optical adjustment component structure can be moved using any displacement device, such as coupled to a movable support stage or directly to a motor-driven translation stage, lead screw translation component, gear translation device, such as stepper motor, servo motor, brushless motor, brushed DC motor, microstepper drive motor, high-resolution stepper motor, and other types of motor.

[0055] In some embodiments, the laser configured for continuous illumination and one or more lasers configured for illumination at discrete intervals are provided by a beam generator that generates two or more frequency-shifted beams. In some cases, the beam generator includes a laser, and a radio frequency generator is configured to apply a radio frequency drive signal to an acousto-optic device to generate laser beams with two or more angle deflections. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the laser in the target beam generator can be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, or a helium-selenium (He) laser. Se 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:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titania-sapphire lasers, Turing YAG lasers, ytterbium YAG lasers, Yb2O3 lasers or cerium-doped lasers and combinations thereof.

[0056] The acousto-optic device can be any convenient acousto-optic protocol configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angle-deflected laser beam from light from the laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0057] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams in the output laser beam, for example, to apply 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 includes being configured to apply 100 or more radio frequency drive signals.

[0058] In some cases, in order to generate an intensity distribution of an angularly deflected laser beam in the output laser beam, the controller is configured to apply an RF drive signal having an amplitude such as from about 0.001V to about 500V, for example from about 0.005V to about 400V, for example from about 0.01V to about 300V, for example from about 0.05V to about 200V, for example from about 0.1V to about 100V, for example from about 0.5V to about 75V, for example from about 1V to about 50V, for example from about 2V to 40V, for example from about 3V to about 30V, and including a variation from about 5V to about 25V. In some embodiments, each applied radio frequency drive signal frequency is from about 0.001 MHz to about 500 MHz, for example from about 0.005 MHz to about 400 MHz, for example from about 0.01 MHz to about 300 MHz, for example from about 0.05 MHz to about 200 MHz, for example from about 0.1 MHz to about 100 MHz, for example from about 0.5 MHz to about 90 MHz, for example from about 1 MHz to about 75 MHz, for example from about 2 MHz to about 70 MHz, for example from about 3 MHz to about 65 MHz, for example from about 4 MHz to about 60 MHz, and includes about 5 MHz to about 50 MHz.

[0059] In some embodiments, the controller has a processor having a memory operatively 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 profile with angled deflections of a desired intensity. For example, the memory may include instructions to generate two or more laser beams with the same angle deflection intensity, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and the memory may include instructions to generate 100 or more laser beams with the same angle deflection intensity. In other embodiments, the memory may include instructions to generate two or more laser beams with different angle deflections, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and the memory may include instructions to generate 100 or more laser beams with different angle deflections.

[0060] In some embodiments, the controller has a processor with a memory operatively 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 with increasing intensity along a horizontal axis from its edge to its center. In these cases, the intensity of the laser beam deflected at the center of the output beam can be from 0.1% to about 99% of the intensity of the laser beam deflected at the edge of the output laser beam along the horizontal axis, for example, from 0.5% to about 95%, for example, from 1% to about 90%, for example, from about 2% to about 85%, for example, from about 3% to about 80%, for example, from about 4% to about 75%, for example, from about 5% to about 70%, for example, from about 6% to about 65%, for example, from about 7% to about 60%, for example, from about 8% to about 55%, and includes about 10% to about 50% of the intensity of the laser beam deflected at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor with a memory operatively 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 with increasing intensity along a horizontal axis from its edge to its center. In these cases, the intensity of the laser beam deflected at the edge of the output beam can be from 0.1% to about 99%, for example from 0.5% to about 95%, for example from 1% to about 90%, for example from about 2% to about 85%, for example from about 3% to about 80%, for example from about 4% to about 75%, for example from about 5% to about 70%, for example from about 6% to about 65%, for example from about 7% to about 60%, for example from about 8% to about 55%, and includes about 10% to about 50% of the intensity of the laser beam deflected at the center of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor with a memory operatively 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 with a Gaussian intensity distribution along a horizontal axis. In other embodiments, the controller has a processor with a memory operatively 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 with a top-hat intensity distribution along a horizontal axis.

[0061] In embodiments, the target beam generator can be configured to generate spatially separated, angle-deflected laser beams within the output laser beam. Depending on the applied radio frequency drive signal and the desired illumination distribution of the output laser beam, the angle-deflected laser beams can be spaced 0.001 micrometers or greater, for example, 0.005 micrometers or greater, for example, 0.01 micrometers or greater, for example, 0.05 micrometers or greater, for example, 0.1 micrometers or greater, for example, 0.5 micrometers or greater, for example, 1 micrometer or greater, for example, 5 micrometers or greater, for example, 10 micrometers or greater, for example, 100 micrometers or greater, for example, 500 micrometers or greater, for example, 1000 micrometers or greater, including 5000 micrometers or greater. In some embodiments, the system is configured to generate angle-deflected laser beams within the output laser beam, which overlap, for example, with adjacent angle-deflected laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., overlap of beam points) can be 0.001 micrometers or larger, such as 0.005 micrometers or larger, such as 0.01 micrometers or larger, such as 0.05 micrometers or larger, such as 0.1 micrometers or larger, such as 0.5 micrometers or larger, such as 1 micrometer or larger, such as 5 micrometers or larger, such as 10 micrometers or larger, and includes 100 micrometers or larger.

[0062] In some cases, beam generators configured to generate two or more frequency-shifted beams include laser excitation modules as described in U.S. Patent Nos. 9,423,353, 9,784,661, 1,000,6852, 2017 / 0133,857, and 2017 / 0350,803, the disclosures of which are incorporated herein by reference.

[0063] Each laser in the subject system can be positioned at any suitable distance from the flow stream, such as 0.001 mm or greater, 0.005 mm or greater, 0.01 mm or greater, 0.05 mm or greater, 0.1 mm or greater, 0.5 mm or greater, 1 mm or greater, 5 mm or greater, 10 mm or greater, 25 mm or greater, and including 100 mm or greater. Furthermore, the light source can be configured to illuminate the sample at any suitable angle (e.g., relative to the vertical axis of the flow stream), such as 10° to 90°, 15° to 85°, 20° to 80°, 25° to 75°, and including angles from 30° to 60°, such as 90°.

[0064] As described above, the light source of the subject system includes a first laser configured for continuously irradiating a flowing stream and one or more lasers configured for irradiating the flowing stream at discrete intervals, wherein each discrete interval of irradiation by each laser is triggered by particles in the flowing stream irradiated by the laser configured for continuous irradiation. The term "triggered" is used herein in its conventional sense, referring to initiating irradiation by one or more lasers configured for irradiation at discrete intervals. In some embodiments, triggering irradiation by lasers includes switching the laser from an off setting to an on setting and irradiating the flowing stream with the laser. In other embodiments, triggering irradiation by lasers includes moving the position of, for example, an optical adjustment component such as a beamstop, or setting, for example, an optical adjustment component such as a beamstop to irradiate the flowing stream with the laser. Any convenient means can be used to trigger one or more lasers, for example, by detecting particles irradiated by the first laser (i.e., the continuous beam of light from the first laser) using a photodetector. In some embodiments, the system includes a separate trigger detector operatively coupled to each of the other lasers, such that when a particle is detected by a successive laser irradiation, a trigger signal is output to each of the lasers, each of the lasers being configured to be discretely spaced enough to initiate discrete intervals of irradiation by one or more lasers.

[0065] In embodiments, the duration between when a particle is detected as being irradiated by a continuously irradiated laser and the start of discrete interval irradiation by one or more lasers can vary depending on the flow velocity of the flow and the distance of the irradiation location on the flow. In some embodiments, the duration between continuous irradiation by the laser and the start of discrete intervals of laser irradiation can be 0.0001 μs or greater, for example 0.0005 s or greater, for example 0.001 μs or greater, for example 0.005 μs or greater, for example 0.01 μs or greater, for example 0.05 μs or greater, for example 0.1 μs or greater, for example 0.5 μs or greater, for example 1 μs or greater, for example 2 μs or greater, for example 3 μs or greater, for example 4 μs or greater, for example 5 μs or greater, for example 6 μs or greater, for example 7 μs or greater, for example 8 μs or greater, for example 9 μs or greater, and includes 10 μs or greater. In some embodiments, the system is configured to respond to the activation of a discrete interval of output delay of a trigger signal. For example, the delay may be 0.0001 μs or greater, 0.0005 μs or greater, such as 0.001 μs or greater, 0.005 μs or greater, 0.01 μs or greater, 0.05 μs or greater, 0.1 μs or greater, 0.5 μs or greater, 1 μs or greater, 2 μs or greater, 3 μs or greater, 4 μs or greater, 5 μs or greater, 6 μs or greater, 7 μs or greater, 8 μs or greater, 9 μs or greater, and includes 10 μs or greater.

[0066] In some embodiments, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the start irradiation time (i.e., discrete intervals at which irradiation begins) for each pair of lasers in the flow stream. In some cases, to calculate the irradiation time, the system is configured to continuously irradiate particles in the flow stream with all lasers of the light source, detect lasers from the flow stream in response to particle irradiation, and calculate the time for each laser to irradiate the particles. In some cases, calculating the start irradiation time includes calculating the time interval between each irradiation of the lasers.

[0067] In some embodiments, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the duration of each pair of flow streams in the lasers (i.e., the length of each discrete interval). In some cases, to calculate the duration of irradiation, the system is configured to continuously irradiate particles in the flow stream with all lasers of the light source, detect lasers from the flow stream in response to particle irradiation, and calculate the duration of the discrete interval of irradiation for each laser. In these cases, the duration of each discrete interval can be calculated based on the irradiation position on the flow stream and the flow velocity of the particles in the flow stream.

[0068] As described above, in order to irradiate the flow at discrete intervals, each laser is operatively coupled to one or more components to provide intermittent irradiation with each laser. In some embodiments, the system is configured to activate one or more downstream lasers in response to irradiation of particles in the flow by a first laser. Depending on the distance between the first laser and one or more downstream lasers, each laser is configured to activate independently in response to irradiation by the first laser for the following time periods: 0.00001 μs or greater, for example, 0.00005 μs or greater, for example, 0.0001 μs or greater, for example, 0.0005 μs or greater, for example, 0.001 μs or greater, for example, 0.005 μs or greater, for example, 0.01 μs or greater, for example, 0.05 μs or greater, for example, 0.1 μs or greater, for example, 0.5 μs or greater, and including 1 μs or greater. In some cases, each laser is configured to turn on independently for 0.00001 μs to 100 μs after the particle is irradiated by the first laser, for example, 0.00005 μs to 90 μs, for example, 0.0001 μs to 80 μs, for example, from 0.0005 μs to 70 μs, for example, 0.001 μs to 60 μs, for example, 0.005 μs to 50 μs, for example, 0.01 μs to 40 μs, for example, 0.05 μs to 30 μs, for example, 0.1 μs to 20 μs, and including 1 μs to 10 μs.

[0069] Figure 1B A light source according to certain embodiments is depicted having a first laser configured to continuously irradiate a flowing stream and a second laser configured to turn on in response to irradiation of particles by the first laser. The light source 100a includes a laser 101a configured to continuously irradiate the flowing stream in a flow cell 107a and a laser 102a located downstream of the laser 101a. The laser 102a is electrically connected to a switch 102a1, which turns the laser on and off. The switch 102a1 is configured to turn on the laser 102a when particles 110 are irradiated by the laser 101a.

[0070] In some embodiments, the system includes a beamstop located in the beam path between one or more downstream lasers and the flow stream, wherein the beamstop is configured to move in response to irradiation of particles by the first laser. Any displacement device can be used to move the beamstop, such as a translation stage coupled to a movable support or directly coupled to a motor-driven translation stage, lead screw translation assembly, gear translation device, such as a stepper motor, servo motor, brushless motor, brushed DC motor, microstepper drive motor, high-resolution stepper motor, and other types of motors. The beamstop can move in response to irradiation by the first laser for the following durations: 0.00001 μs or more, for example, 0.00005 μs or more, for example, 0.0001 μs or more, for example, 0.0005 μs or more, for example, 0.001 μs or more, for example, 0.005 μs or more, for example, 0.01 μs or more, for example, 0.05 μs or more, for example, 0.1 μs or more, for example, 0.5 μs or more, and including 1 μs or more. In some cases, within 0.00001 μs to 100 μs after the particle is irradiated by the first laser, the beam stop moves along the beam path of one or more downstream lasers. This time period also includes, for example, 0.00005 μs to 90 μs, 0.0001 μs to 80 μs, 0.0005 μs to 70 μs, 0.001 μs to 60 μs, 0.005 μs to 50 μs, 0.01 μs to 40 μs, 0.05 μs to 30 μs, 0.1 μs to 20 μs, and includes 1 μs to 10 μs.

[0071] Figure 1C A first laser configured to continuously irradiate a flowing stream, and a second laser configured to irradiate the flowing stream when a beamstop moves in response to irradiation of a particle by the first laser, are depicted according to certain embodiments. A light source 100c includes a laser 101a configured to continuously irradiate the flowing stream in a flow cell 107a and a laser 103a located downstream of the laser 101a. The light source 100c includes a beamstop 103a1 located in the beam path between the laser 103a and the flowing stream. When a particle 110 is irradiated by the laser 101a, the beamstop 103a1 is configured to move from the beam path between the laser 103a and the flowing stream.

[0072] In some embodiments, the system includes a beam steering device located in the beam path between one or more downstream lasers. In embodiments, the beam steering device is configured to deflect light from one or more downstream lasers from the flow stream until triggered to redirect the light from the lasers back into the flow stream. In some cases, the beam steering device is configured to direct light into the flow stream in response to the irradiation of a particle by a first laser. For example, a data signal may be generated in response to the irradiation of a particle in the flow stream by the first laser, and the beam steering device is configured to direct the deflected laser into the flow stream in response to the generated data signal. In some cases, the beam steering device is an acousto-optic device, such as an acousto-optic deflector (AOD) or an acousto-optic modulator (AOM). In these embodiments, the data signal may include a change in a drive signal from a digital synthesizer (DDS), arbitrary waveform generator (AWG), or electrical pulse generator sufficient to direct light from the lasers into the flow stream. In other cases, the beam steering device is an electro-optic device, such as an electro-optic deflector (EOD) or an electro-optic modulator (EOM). In these embodiments, the data signal may include a change in current or applied voltage sufficient to guide light from the laser to the flow stream via an electro-optic device. In some embodiments, the beam steering is configured to redirect the beam path of one or more downstream lasers in response to irradiation by the first laser for a period of 0.00001 μs or longer, this period also being, for example, 0.00005 μs or more, such as 0.0001 μs or more, such as 0.0005 μs or more, such as 0.001 μs or more, such as 0.005 μs or more, such as 0.01 μs or more, such as 0.05 μs or more, such as 0.1 μs or more, such as 0.5 μs or more, and including 1 μs or longer. In some cases, within 0.00001 μs to 100 μs after irradiating the particle with the first laser, the beam deflector is configured to redirect the beam path of one or more downstream lasers. This time period also includes, for example, 0.00005 μs to 90 μs, 0.0001 μs to 80 μs, 0.0005 μs to 70 μs, 0.001 μs to 60 μs, 0.005 μs to 50 μs, 0.01 μs to 40 μs, 0.05 μs to 30 μs, 0.1 μs to 20 μs, and includes 1 μs to 10 μs.

[0073] Figure 1DA first laser and a second laser, configured to continuously irradiate a flowing stream according to certain embodiments, are depicted. The second laser is configured to irradiate the flowing stream when a beam deflector redirects light back to the flowing stream in response to irradiation of a particle by the first laser. A light source 100d includes a laser 101a configured to continuously irradiate the flowing stream in a flow cell 107a and a laser 104a located downstream of the laser 101a. The light source 100d includes a beam stop 104a1 located in a beam path between the laser 104a and the flowing stream. The beam deflector is configured to redirect the beam path of the laser 104a away from the flowing stream, for example, by acousto-optic deflection (e.g., using AOD) or electro-optic deflection (e.g., using EOM). When a particle 110 is irradiated by the laser 101a, the beam deflector is configured to redirect the beam path of the laser 104a back to the flowing stream.

[0074] In an embodiment, light from particles illuminating the flowing stream with a target light source is transmitted to a light detection system. The light detection system may include one or more photodetectors. The photodetectors in the system can be any convenient light detection device, including but not limited to photoelectric sensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-type charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or combinations thereof, and other photodetectors. The light detection system for measuring light from particles in the flowing stream may include one or more photodetectors, such as two or more, three or more, four or more, five or more, ten or more, twenty-five or more, or even fifty or more.

[0075] In some embodiments, the target system includes a photodiode array having more than one photodiode, such as two or more photodiodes, three or more, five or more, and including ten or more photodiodes, wherein the effective detection surface area of ​​each region of each photodiode can be 0.01 cm². 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 2up to 5cm 2 .

[0076] In embodiments of this disclosure, the target photodetector is configured to measure light collected at one or more wavelengths, such as at two or more wavelengths, such as at five or more wavelengths, such as at ten or more wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and includes measuring light from flowing stream particles at 400 or more different wavelengths.

[0077] In embodiments, the photodetector is configured to measure light continuously or at discrete intervals. In some cases, the target detector is configured to continuously measure the collected light. In other cases, the target detector is configured to measure at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0078] Each photodetector can be positioned at any suitable distance from the flow, provided a usable light signal can be detected. For example, detectors in a subject system can be positioned at distances of 1 mm or more, such as 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, 50 mm or more, 100 mm or more, 150 mm or more, 250 mm or more, and even 500 mm or more. Detectors can also be positioned at any angle to the flow. For example, detectors can be at angles of 10° to 90° relative to the vertical axis of the flow, such as 15° to 85°, 20° to 80°, 25° to 75°, and even 30° to 60°. In some cases, one or more detectors are positioned at 30° to 60° relative to the vertical axis of the flow.

[0079] In embodiments, the system is configured to detect forward-scattered light, side-scattered light, reflected light, transmitted light, or a combination thereof. In some embodiments, the light signal from the illumination flow can be detected by one or more detectors configured as forward-scattering detectors. In these embodiments, the forward-scattering detectors are positioned on one side of the flow flow opposite the light source and are positioned to collect and detect forward-propagating (e.g., scattered) light.

[0080] In some embodiments, the system includes a single photodetector configured to detect light from each laser of the light source. In these embodiments, the photodetector detects light from particles in a flowing stream, which are configured to continuously irradiate the lasers and to irradiate each laser at discrete intervals.

[0081] In some embodiments, the optical detection system includes an optical adjustment component configured to reduce the amount of light transmitted from a laser configured to continuously illuminate one or more photodetectors. In these embodiments, the optical adjustment component is configured to restrict, reduce, or limit at least one or more wavelengths of light (e.g., one or more wavelengths of light from a laser configured for continuous illumination) from the sample to the effective surface of the photodetector. The light transmitted to the photodetector may include optical components that restrict the propagation of one or more different wavelengths of light, such as 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, and including those restricting 500 or more different wavelengths of light. For example, in some embodiments, the optical adjustment component is a bandpass filter, such as a long-pass filter that transmits a spectral range of light longer than the illumination wavelength of the first laser. In other embodiments, the optical adjustment component is a dichroic mirror, such as a dichroic mirror that transmits a spectral range longer than the illumination wavelength of the first laser and reflects a spectral range including the illumination wavelength of the first laser.

[0082] In some embodiments, the system includes an optical collection system for collecting light from a flowing stream and directing it to a light detection system. The optical collection system may be physically coupled to the light detection system, for example, using an adhesive, co-molding together, or integrated into the light detection system. In some 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 via a connector, such as a hook and loop fastener, magnet, latch, slot, countersinks, counterbores, groove, pin, tether, hinge, Velcro, non-permanent adhesive, or a combination thereof.

[0083] In other embodiments, the optical detection system and the optical collection system are in optical communication but have no physical contact. For example, the optical collection system may be positioned at a distance of 0.001 mm or more from the optical detection system, 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 may be positioned at a distance of 100 mm or more from the optical detection system.

[0084] In some embodiments, the optical collection system includes optical fibers. For example, in some cases, the optical collection system may be a fiber optic repeater bundle, and light is transmitted through the fiber optic repeater bundle to the optical detection system. In some embodiments, the optical collection device is a single optical fiber configured to transmit light irradiated from each of the lasers to a single photodetector in the optical detection system. In these embodiments, the irradiation position spanned by the lasers on the flow stream is equal to or smaller than the diameter of the single optical fiber configured to collect light from the flow stream. For example, the laser in the subject system can be configured to illuminate locations on the flow spanning 100 μm or less, such as 90 μm or less, such as 80 μm or less, such as 70 μm or less, such as 60 μm or less, and including 50 μm or less, and a single optical fiber can have a diameter sufficient to collect light from each location illuminated by the laser, such as a diameter of 50 μm or greater, such as 60 μm or greater, such as 70 μm or greater, such as 80 μm or greater, such as 90 μm or greater, and including a single optical fiber configured to collect light from the illuminated flow having a diameter of 100 μm or greater.

[0085] Figure 2 A light source, according to certain embodiments, is depicted having a laser configured for continuous illumination and three lasers configured for illumination at discrete intervals, as well as a light detection system for transmitting and measuring light using a single photodetector. The light source 200 includes a laser 201 configured for continuous illumination and lasers 202, 203, and 204, respectively located downstream of the laser 101 along a flow stream 207 and configured for illumination at discrete intervals. The lasers 201, 202, 203, and 204 are placed approximately adjacent to each other, allowing light from the flow stream 207 to be collected using a single optical fiber 205 and propagated to a single photodetector 206, reducing the number of detector channels required to detect light from a sample.

[0086] In other embodiments, the optical acquisition system is a free-space optical relay system. For example, the free-space optical relay system may include a housing having a near end and a far end, with the near end coupled to the optical detection system. The free-space relay system may include any combination of different optical components, such as one or more lenses, mirrors, slits, pinholes, wavelength splitters, or combinations thereof.

[0087] In some embodiments, the system includes a flow cell configured to propagate particles in a flow stream. Any convenient flow cell that propagates a fluid sample to a sample interrogation region can be used, wherein in some embodiments, the flow cell includes a cylindrical flow cell, a truncated conical flow cell, or a flow cell including a proximal cylindrical portion defining a longitudinal axis and an end truncated conical portion terminating at a flat surface having an orifice transverse to the longitudinal axis.

[0088] In some embodiments, the sample flow exits from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow, the flow cell orifice can be of any suitable shape, wherein the target cross-sectional shape includes, but is not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses; and irregular shapes, such as parabolic bottoms coupled to the top of a plane. In some embodiments, the target flow cell has a circular orifice. In some embodiments, the nozzle orifice size can vary from 1 μm to 20000 μm, for example, 2 μm to 17500 μm, for example, 5 μm to 15000 μm, for example, 10 μm to 12500 μm, for example, 15 μm to 10000 μm, for example, 25 μm to 7500 μm, for example, 50 μm to 5000 μm, for example, 75 μm to 1000 μm, for example, 100 μm to 750 μm, and includes 150 μm to 500 μm. In some embodiments, the nozzle orifice is 100 μm.

[0089] In some embodiments, the flow cell includes a sample inlet configured to provide a sample to the flow cell. In embodiments, the sample inlet system is configured to provide a suitable sample flow into the flow cell chamber. Depending on the desired characteristics of the flow flow, the rate at which the sample is delivered to the flow cell chamber through the sample inlet can be 1 μL / min or more, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min or more, for example, 15 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, and includes 100 μL / min or more. In some cases, the rate at which the sample is delivered to the flow cell chamber through the sample inlet is 1 μL / sec or more, for example, 2 μL / sec or more, for example, 3 μL / sec or more, for example, 5 μL / sec or more, for example, 10 μL / sec or more, for example, 15 μL / sec or more, for example, 25 μL / sec or more, for example, 50 μL / sec or more, and includes 100 μL / sec or more.

[0090] The sample injection port can be an orifice located in the inner chamber wall or a conduit located proximal to the inner chamber. When the sample injection port is an orifice located in the inner chamber wall, the orifice can be of any suitable shape, wherein the target cross-sectional shape includes, but is not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses, etc.; and irregular shapes, such as parabolic bottoms coupled to the top of a plane. In some embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice can vary depending on the shape, and in some cases has an orifice size of 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as from 1 mm to 2 mm and including 1.25 mm to 1.75 mm, such as 1.5 mm.

[0091] In some cases, the sample injection port is a conduit located proximal to the flow cell chamber. For example, the sample injection port may be a conduit positioned such that its orifice aligns with the flow cell orifice. When the sample injection port is a conduit aligned with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, wherein the target cross-sectional shape includes, but is not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses, etc.; and irregular shapes, such as a parabola coupled to the top of a plane at the bottom. The orifice of the conduit varies depending on the shape, and in some cases has an orifice size of 0.1 mm to 5.0 mm, for example 0.2 mm to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example from 1 mm to 2 mm, and including 1.25 mm to 1.75 mm, for example 1.5 mm. The tip shape of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip with an angle of 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7°, including 5°.

[0092] In some embodiments, the flow cell further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid into the flow cell chamber, for example, in combination with a sample to generate a laminar sheath fluid flow surrounding the sample flow. Depending on the desired characteristics of the flow, the rate at which the sheath fluid is delivered to the flow cell chamber can be 25 μL / sec or more, for example 50 μL / sec or more, for example 75 μL / sec or more, for example 100 μL / sec or more, for example 250 μL / sec or more, for example 500 μL / sec or more, for example 750 μL / sec or more, for example 1000 μL / sec or more, and includes 2500 μL / sec or more.

[0093] In some embodiments, the sheath fluid injection port is an orifice located in the wall of the inner chamber. The sheath fluid injection port orifice can be any suitable shape, wherein the target cross-sectional shape includes, but is not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses; and irregular shapes, such as parabolic bottoms coupled to the top of a plane. The size of the sample injection port orifice can vary depending on the shape, and in some cases has an orifice size of 0.1 mm to 5.0 mm, such as 0.2 mm to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as from 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, such as 1.5 mm.

[0094] In some embodiments, the system further includes a pump in fluid communication with the flow cell to propagate the flow through the flow cell. Any convenient fluid pumping device can be used to control the flow rate 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 deliver fluid through the flow cell at a rate suitable for multiphoton counting of light from the sample in the flow stream. In some cases, the sample flow rate in the flow cell is 1 nL / min or greater, for example, 2 nL / min or greater, for example, 3 nL / min or greater, for example, 5 nL / min or greater, for example, 10 nL / min or greater, for example, 25 nL / min or greater, for example, 50 nL / min or greater, for example, 75 nL / min or greater, for example, 100 nL / min or greater, for example, 500 nL / min or greater, for example, 750 nL / min or greater, and includes 1000 nL / min or greater. For example, the system may include a pump configured to flow the sample through the flow cell at rates from 1 nL / min to 500 nL / min, such as 1 nL / min to 250 nL / min, such as 1 nL / min to 100 nL / min, such as 2 nL / min to 90 nL / min, such as 3 nL / min to 80 nL / min, such as 4 nL / min to 70 nL / min, such as 5 nL / min to 60 nL / min, including 10 nL / min to 50 nL / min. In some embodiments, the flow rate is 5 nL / min to 6 nL / min.

[0095] In some embodiments, the subject system is a flow cytometry system that employs the aforementioned photodetector system to detect light emitted by a sample in a flowing stream. In some embodiments, the subject system is a flow cytometer system. 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 ed., 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 The system described in Syst.24(3):203-255, the contents of which are incorporated herein by reference. In some cases, the target flow cytometer system includes BDBiosciences FACSCanto. TM II flow cytometer, BD Accuri TM 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 LSFortessa TM Flow cytometer, BD Biosciences LSRFortess TM X-20 flow cytometer and BDBiosciences FACSCalibur TM Cell sorting machine, BD Biosciences FACSCount TM Cell sorting machine, BDBiosciences FACSLyricTM Cell sorting machine and BD Biosciences via TM Cell sorting machine, BDBiosciences Influx TM Cell sorting machine, BD Biosciences Jazz TM Cell sorting machine, BD Biosciences Aria TM Cell sorting machine and BD Biosciences FACSMelody TM Cell sorting machines, etc.

[0096] In some embodiments, the subject particle sorting system is a flow cytometer system, such as those described in U.S. Patent Nos. 10,006,852, 9,952,076, 9,933,341, 9,784,661, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, and 8,235. Those described in 3,146, 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, and 5,602,039; all their public disclosures are incorporated herein by reference.

[0097] In some embodiments, the subject system is a flow cytometry system with an excitation module that uses radio frequency multiplexed excitation to generate multiple frequency-shifted beams. In these embodiments, the laser generator may include multiple lasers and one or more acousto-optic components (e.g., acousto-optic deflectors, acousto-optic frequency shifters) to generate multiple frequency-shifted comb beams. One or more of the frequency-shifted comb beams and the local oscillator beams may be configured to be received by beam-shaping components as described herein to generate one or more frequency-shifted beams having a substantially constant intensity distribution. In some cases, U.S. Patent Nos. 9,423,353, 9,784,661, 2017 / 0133,857, and 2017 / 0350,803 describe subject systems of flow cytometry systems with laser excitation modules, the disclosures of which are incorporated herein by reference.

[0098] In some embodiments, the target system includes a particle analysis system that can be used to analyze and characterize particles, regardless of whether the particles are physically sorted into a collection container. Figure 4AA principle block diagram of an example particle analysis system is shown. In some embodiments, particle analysis system 401 is a flow system. The particle analysis system 401 shown in Figure 4 can be configured to perform all or part of the methods described herein, for example. Particle analysis system 401 includes a jet system 402. Jet system 402 may include or be coupled to 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.

[0099] 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. Detection station 408 typically refers to a monitoring area 407 along the common sample path. In some embodiments, detection may include detecting light or one or more other characteristics of particle 403 as it passes through the monitored area 407. Figure 4A A detection station 408 with a monitoring area 407 is shown. Some embodiments of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations can monitor multiple areas.

[0100] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be referred to as event data. The data point can be a multi-dimensional data point, including values ​​for various properties measured for the particle. The detection system 404 is configured to collect a series of such data points within a first time interval.

[0101] The particle analysis system 401 may also include a control system 306. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The control system shown may be operationally associated with the jet system 402. The control system may be configured to generate a calculated signal frequency based on the number of data points collected by the detection system 404 during at least a portion of the first time interval. The control system 406 may also be configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may also compare the experimental signal frequency with the calculated signal frequency or a predetermined signal frequency.

[0102] Figure 4BA system 400 for flow cytometry according to an illustrative embodiment of the present invention is shown. 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 collecting 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.

[0103] The 115a-c laser is excited to emit light in the form of a laser beam. Figure 4B In the example system, the laser beams emitted from excitation lasers 415a-415c have wavelengths of 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first guided through one or more beam splitters 445a and 445b. Beam splitter 445a transmits 488 nm light and reflects 633 nm light. Beam splitter 445b transmits UV light (light with wavelengths between 10 nm and 400 nm) and reflects both 488 nm and 633 nm light.

[0104] The laser beam is then directed to a focusing lens 420, which focuses the beam onto the fluid flow portion containing the sample particles within a flow chamber 425. The flow chamber is part of a jet system that directs particles (typically one at a time) to the focused laser beam for interrogation. The flow chamber may include a flow cell in a benchtop cytometer or a nozzle tip in an airflow cytometer.

[0105] Light from the laser beam interacts with particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is absorbed and re-emitted at different wavelengths depending on the particle's characteristics, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. Fluorescence emission, as well as diffracted, refracted, reflected, and scattered light, can be transmitted to a forward scattering detector 430, a side scattering detector 435, and one or more fluorescence detectors 460a-460f via beam splitters 445a-445g, bandpass filters 450a-450e, long-pass filters 455a-455b, and a fluorescence collecting lens 440.

[0106] A fluorescence collecting lens 440 collects light emitted from particle-laser beam interactions and directs that 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. 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 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short-pass filters transmit light with wavelengths equal to or less than a specified wavelength. Long-pass filters, such as long-pass filters 455a-455b, transmit light with wavelengths equal to or longer than a specific wavelength. For example, long-pass filter 455a, as a 670 nm long-pass filter, transmits light equal to or longer than 670 nm. Filters are typically selected to optimize the detector's specificity for a particular fluorescent dye. A filter can be configured so that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0107] Beam splitters direct light of different wavelengths in different directions. Beam splitters can be characterized by filter properties, such as short-pass and long-pass filters. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 445g transmits light with wavelengths of 620 nm or shorter and reflects light with wavelengths greater than 620 nm in different directions. In one embodiment, beam splitters 445a-445g may include optical mirrors, such as dichroic mirrors.

[0108] A forward scattering detector 430 is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light, the excitation light which travels primarily forward through or around the particle. The intensity of the light detected by the forward scattering detector depends on the overall size of the particle. The forward scattering detector may include a photodiode. A side scattering detector 435 is configured to detect refracted and reflected light from the surface and internal structures of the particle and tends to increase with increasing particle structural complexity. One or more fluorescence detectors 460a-460f can detect fluorescence emission from fluorescent molecules associated with the particle. The side scattering detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected at the forward scattering detector 430, the side scattering detector 435, and the fluorescence detector can be converted into electronic signals (voltages) by the detectors. This data can provide information about the sample.

[0109] Those skilled in the art will recognize that the flow cytometer according to embodiments of the present invention is not limited to Figure 4B The flow cytometer shown may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors with various wavelengths and various different configurations.

[0110] During operation, the cytometer is controlled by a controller / processor 490, and measurement data from the detector can be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive output signals from it, and may 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) capabilities 497 may also be provided in the system. Memory 495, controller / processor 490, and I / O 497 may be provided entirely as part of the flow cytometer 410. In such an embodiment, a display may also form part of the I / O capability 497 for presenting experimental data to the user of the cytometer 400. Alternatively, some or all of memory 495, controller / processor 490, and I / O capabilities may be part of one or more external devices such as a general-purpose computer. In some embodiments, some or all of memory 495 and controller / processor 490 may communicate wirelessly or wiredly with the cytometer 410. The controller / processor 490, together with the memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0111] Figure 4BThe system shown comprises 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), as defined by the configuration of filters and / or separators in the beam path from flow cell 425 to each detector. Different fluorescent molecules used in flow cytometry experiments emit light in their own characteristic wavelength bands. Specific fluorescent labels and their associated fluorescence emission bands can be selected for the experiment to generally align with the filter windows of the detectors. However, as more detectors are provided and more labels are used, a perfect correspondence between filter windows and fluorescence emission spectra is not possible. While the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of a particular detector, some emission spectra of that label may also overlap with the filter windows of one or more other detectors, which is generally correct. This can be referred to as spillover. I / O497 can be configured to receive data on a flow cytometry experiment having a set of fluorescent labels and multiple cell populations with multiple labels, each cell population having a subset of multiple labels. I / O 497 can also be configured to receive biological data, marker density data, emission spectral data, data on marker assignment to one or more cell populations, and cytometer configuration data. Flow cytometry experimental data, such as tag spectral characteristics and flow cytometry configuration data, can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more tag-to-tag assignments.

[0112] Figure 5 A functional block diagram of an example particle analyzer control system, such as an analysis controller 500, for analyzing and displaying biological events is shown. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0113] 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 the biological event data to the analysis controller 500. A data communication channel may 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 through the data communication channel.

[0114] Analysis controller 500 may be configured to receive biological event data from particle analyzer or sorting system 502. The biological event data received from particle analyzer or sorting system 502 may include flow cytometry event data. Analysis controller 500 may be configured to provide a graphical display of a first graph including the biological event data to display device 506. For example, analysis controller 500 may also be configured to render regions of interest as gates surrounding the group of biological event data displayed by display device 506, overlaying the first graph. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest drawn on a single parameter histogram or binary graph. In some embodiments, the display may be used to display particle parameter or saturation detector data.

[0115] The analysis controller 500 can also be configured to display bio-event data on a display device 506 inside the door that differs from other events in the bio-event data outside the door. For example, the analysis controller 500 can be configured to make the color of the bio-event data contained inside the door different from the color of the bio-event data outside the door. The display device 506 can be a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0116] The analysis controller 500 can be configured to receive a door selection signal from a first input device to identify a door. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a door selection signal to the analysis controller 500 to identify a door to be displayed or manipulated on the display device 506 (e.g., by clicking on or within the desired door when the cursor is positioned there). In some embodiments, the first device can be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, a stylus, an optical detector, or a voice recognition system. Some input devices may include multiple input functions. In such an implementation, each of the input functions is considered to be an input device. For example, such as... Figure 5 As shown, the mouse 510 may include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0117] Triggering events can cause the analysis controller 500 to change the way data is displayed, which parts of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selecting a target population for particle sorting.

[0118] In some embodiments, the analysis controller 500 may be configured to detect when a door selection is initiated by the mouse 510. The analysis controller 500 may also be configured to automatically modify the plotting visualization to facilitate the selection process. This modification may be based on a specific distribution of the biological event data received by the analysis controller 500.

[0119] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can also be configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0120] Display device 506 can be configured to provide biological event data to analysis controller 500. The displayed data may include graphs of the biological event data and gates outlining portions of the graphs. Display device 506 can also be configured to change the presented information based on input received from analysis controller 500, along with input from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0121] In some implementations, the analysis controller 500 may generate a user interface to receive instance events for classification. For example, the user interface may include controls for receiving instance events or instance images. Instance events, images, or instance gates may be provided before collecting event data for the sample, or based on an initial set of events from a portion of the sample.

[0122] In some embodiments, the target system includes a particle sorting system. Figure 6A This is a schematic diagram of a particle sorter system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment of the present document. In some embodiments, the particle sorting system 600 is a cell sorting system. Figure 6A As shown, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to a nozzle 603, may include a nozzle 603, or may 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 flow). Within the moving fluid column 608, particles 609 (e.g., cells) align to pass through a monitoring area 611 irradiated by a radiation source 612 (e.g., a laser) (e.g., where laser flows intersect). Vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to split into multiple droplets 610, some of which contain particles 609.

[0123] In operation, detection station 614 (e.g., an event detector) identifies when a target particle (or target cell) crosses monitoring area 611. Detection station 614 is fed into timing circuit 628, which in turn feeds into flash charge circuit 630. At the drop interruption point notified by a timing drop delay (Δt), a flash charge can be applied to the moving fluid column 608, causing the target drop to carry a charge. The target drop may include one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect the droplet into a container, such as a collection tube or a porous or microporous sample plate, where the pores or micropores can be associated with the droplet of particular interest. Figure 6A As shown, the droplets can be collected in the discharge container 638.

[0124] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a target particle passes through a monitoring region 611. An exemplary droplet boundary detector is described in U.S. Patent Application No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 616 allows the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may be fed an amplitude signal 620 and / or a phase signal 618, which in turn are fed (via amplifier 622) to amplitude control circuitry 626 and / or frequency control circuitry 624. Amplitude control circuitry 626 and / or frequency control circuitry 624, in turn, control the droplet forming transducer 602. Amplitude control circuitry 626 and / or frequency control circuitry 624 may be included in a control system.

[0125] In some embodiments, the classification electronics (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory configured to store detected events and classification decisions based thereon. The classification decisions may be included in the event data of the particles. In some embodiments, detection system 616 and detection station 614 may be implemented as a single detection unit or communication coupling, such that event measurements can be collected by one of detection system 616 or detection station 614 and provided to non-collecting elements.

[0126] Figure 6B This is a schematic diagram of a particle sorting system according to an embodiment presented herein. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge can be applied via charging wires in the barbs. This generates 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. This can be achieved, for example, through sorting electronics or other detection systems. Figure 6B(Not shown in the image) to analyze particle information. Deflecting plates 652 and 654 can be independently controlled to attract or repel charged droplets to guide them to a destination collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflectors 652 and 654 can be controlled to guide particles along a first path 662 toward container 674 or along a second path 668 toward container 678. If the particles are not the target (e.g., do not exhibit scattering or illumination information within a specified classification range), the deflectors can allow the particles to continue along flow path 664. Such uncharged droplets can, for example, enter the waste container via a suction device 670.

[0127] It may include sorting electronics to initiate the collection of measurements, receive the fluorescence signal of the particles, and determine how to adjust the deflection plate to sort the particles. Figure 6B The illustrated implementation examples include BD FACSAria, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ). TM A series of flow cytometers.

[0128] Computer control system

[0129] This disclosure also includes computer-controlled systems, wherein the systems further include one or more computers for full or partial automation. In some embodiments, the system includes a computer having a computer-readable storage medium on which a computer program is stored, wherein the computer program, when loaded onto the computer, includes instructions for continuously irradiating the flow with a first laser; and instructions for irradiating the flow with a second laser at discrete intervals. In some embodiments, the computer program includes instructions for triggering the irradiation of the second laser at each discrete interval in response to irradiation of particles in the flow with the first laser.

[0130] In some embodiments, a computer control system configured for full or partial automation includes a memory storing instructions that, when executed by a processor, cause the processor to calculate the time at which each flow in the laser begins to irradiate (i.e., discrete intervals at which irradiation begins). In some embodiments, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the duration of irradiation of each flow in the laser (i.e., the length of each discrete interval).

[0131] In this embodiment, the system includes an input module, a processing module, and an output module. The system may include hardware and software components, wherein the hardware components may take the form of one or more platforms, such as servers, such that functional elements—that is, system elements that perform system-specific tasks (e.g., input and output of management information, processing information, etc.)—can be executed on one or more computer platforms represented by the system and across the execution of software applications.

[0132] The system may include display devices and operator input devices. For example, operator input devices may be keyboards, mice, or similar devices. The processing module includes a processor that can access memory storing instructions for performing steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup devices, and many other devices. The processor may be a commercially available processor, or it may be one of other processors that are existing or will become available. The processor executes the operating system and its interface with firmware and hardware in a well-known manner, and facilitates the processor's coordination and execution of the functions of various computer programs that can be written in various programming languages ​​known in the art, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof. The operating system, typically working with the processor, coordinates and executes the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all 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 based on a first optical signal and a second optical signal. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0133] System memory can be any of a variety of known or future memory storage devices. Examples include any commonly used random access memory (RAM), magnetic media (such as resident hard disks or magnetic tapes), optical media (such as optical discs), flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, removable hard disk drives, or floppy disk drives. This type of memory storage device typically reads from and / or writes to program storage media (not shown), such as compact discs, magnetic tapes, removable hard disks, or floppy disks, respectively. It is considered that any of these program storage media, or others currently in use or that may be developed in the future, can be a computer program product. As will be understood, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0134] In some embodiments, a computer program product is described, comprising a computer-usable medium in which control logic (computer software program, including program code) is stored. The control logic, when executed by a computer's processor, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. The implementation of a hardware state machine to perform the functions described herein will be apparent to those skilled in the art.

[0135] The memory can be any suitable device in which a processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including magnetic disks or optical discs or magnetic tapes or RAM, or any other suitable device, fixed or portable). The processor can include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel or pre-stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium using any of these devices connected to the memory. For example, a magnetic disk or optical disc can carry the program and can be read by a disk writer / reader. The system of the present invention also includes programming, for example, in the form of a computer program product, an algorithm for practicing the methods described above. The program according to the invention can be recorded on a computer-readable medium, such as any medium that a computer can directly read and access. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROMs; electronic storage media such as RAM and ROM; portable flash drives; and mixtures of these categories, such as magnetic / optical storage media.

[0136] The processor can also access communication channels to communicate with users in remote locations. A remote location refers to a user who does not have direct contact with the system and inputs information from external devices such as wide area networks (“WAN”), telephone networks, satellite networks, or any other suitable communication channel, including mobile phones (i.e., smartphones).

[0137] In some embodiments, the system according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or 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., RFID, Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB)). Communication protocols and cellular communications, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0138] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as USB ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the subject systems and other external devices, such as computer terminals configured for similar supplemental data communication (e.g., in a doctor's office or hospital environment).

[0139] In one embodiment, the communication interface is configured for infrared communication, Bluetooth communication, or any other suitable wireless communication protocol 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 the user can use in conjunction with them.

[0140] In one embodiment, the communication interface is configured to provide connectivity for data transmission by wirelessly connecting to a personal computer (PC) on a local area network (LAN) via the Internet or by connecting to the network via WiFi using a wireless hotspot, through a mobile network, the Internet Protocol (IP) of the Short Message Service (SMS).

[0141] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface, for example, using 802.11 or... A common standard for RF protocols or IrDA infrared protocols. The server device can be other portable devices, such as smartphones, personal digital assistants (PDAs), or laptops; in some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, keyboards, mice, or touchscreens.

[0142] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate with network or server devices the data stored in the subject system, for example, as described above in an optional data storage device, using one or more communication protocols and / or mechanisms.

[0143] The output controller may include a controller for any of a variety of known display devices used to present information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of picture elements. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. Functional elements of the computer may communicate with each other via a system bus. Some of these communications may be accomplished using networks or other types of remote communication in alternative embodiments. According to known technologies, the output manager may also provide information generated by the processing module to a user located at a remote location, for example, via the Internet, telephone, or satellite networks. The presentation of data by the output manager may be implemented according to a variety of known technologies. 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 Internet URL addresses so that the 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 type to be developed in the future, although they are typically a class of computers commonly referred to as servers. However, they can also be mainframes, workstations, or other computer types. They can be connected via any known or future type of cable or other communication system (including wireless systems), whether networked or otherwise. They can be in the same location or physically separated. A variety of operating systems can be used on any computer platform, depending on the type and / or brand of the chosen platform. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, etc.

[0144] Figure 7 The general structure of a computing device 600 according to certain embodiments is described. Figure 7 The general architecture of the computing device 700 shown includes the arrangement of computer hardware and software components. The computing device 700 may include... Figure 7The diagram shows more (or fewer) elements. However, it is not necessary to show all these conventional elements for the sake of providing an effective disclosure. As shown, computing device 700 includes processing unit 710, network interface 720, computer-readable media drive 730, input / output device interface 740, display 750, and input device 760, all of which can communicate with each other via a communication bus. Network interface 720 can provide connectivity to one or more networks or computing systems. Processing unit 710 can thus receive information and instructions from other computing systems or services via the network. Processing unit 710 can also communicate with memory 770 and further provide output information to optional display 750 via 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 devices.

[0145] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments) that are executed by processing device 710 to implement one or more embodiments. Memory 770 typically includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store operating system 772, which provides computer program instructions for use by processing device 710 in the general management and operation of computing device 700. Memory 770 may also include computer program instructions and other information for implementing aspects of this disclosure.

[0146] Method for irradiating particles in a flowing stream

[0147] This disclosure also includes methods for irradiating a sample containing particles in a flowing stream. According to some embodiments, the method includes continuously irradiating the flowing stream with a first laser and irradiating the flowing stream with one or more lasers at discrete intervals. In embodiments, each discrete interval irradiated by one or more lasers is triggered by irradiating particles in the flowing stream with the first laser. In some embodiments, the sample is a biological sample. The term "biological sample," used in its conventional sense, refers to a subset of tissues, cells, or components of a whole organism, plant, fungus, or animal, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sheep cord blood, urine, vaginal fluid, and semen that may be found in certain circumstances in blood, mucus, lymph, synovial fluid, etc. Thus, "biological sample" refers both to a natural organism or a subset of its tissues and to homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. Biological samples can be any type of organic tissue, including healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.). In some embodiments, biological samples are liquid samples, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., in which cases the sample is a blood sample, including whole blood, for example, blood obtained from venipuncture or finger prick (the blood may or may not be mixed with any reagents such as preservatives, anticoagulants, etc. before testing).

[0148] In some embodiments, the sample source is "mammal" or "milk animal," terms that are widely used to describe organisms belonging to the class Mammalia, including carnivora (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the object is a human. The method can be applied to samples obtained from human objects of both sexes and at any developmental stage (i.e., newborns, infants, adolescents, teenagers, and adults), wherein in some embodiments, the human object is an adolescent, teenager, or adult. While the invention can be applied to samples from human objects, it should be understood that these methods can also be performed on samples from other animal objects (i.e., in "non-human objects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0149] In embodiments, the method includes irradiating a sample in a flowing stream with a laser configured for continuous irradiation. As described above, the term "continuous" is used herein in its conventional sense, meaning irradiating the flowing stream with a laser in a constant manner without interruption during the duration the target sample flows through the flowing stream. In some embodiments, continuous irradiation of the sample includes irradiating the flowing stream with a laser configured not to be shielded (i.e., not intermittently shielded by beam-stopping or shielding components). In some embodiments, continuous irradiation of the flowing stream with a laser includes maintaining a constant laser irradiation intensity, for example, the laser irradiation intensity varying by 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, and includes laser irradiation intensity changes of 0.0001% or less. In some embodiments, continuous irradiation of the flowing stream includes irradiating the flowing stream with a laser whose intensity does not change during the sample flowing through the flowing stream. Light output intensity can be measured using any convenient device, including but not limited to scanning slit profilometers, charge-coupled devices (CCDs, such as enhanced charge-coupled devices, ICCDs), positioning sensors, power sensors (e.g., thermopile power sensors), optical power sensors, energy meters, digital laser photometers, laser diode detectors, and other types of photodetectors.

[0150] In practical approaches, lasers such as gas lasers, including helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon chloride (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof, can be used to continuously irradiate the flow. In other cases, lasers configured for continuous irradiation are dye lasers, such as stilbene lasers, coumarin lasers, or rhodamine lasers. In still other cases, lasers configured for continuous target irradiation include 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, or combinations thereof. In other cases, the system includes ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, or combinations thereof. In other cases, the system includes semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs), or frequency-doubled or third-doubled versions of any of the above lasers.

[0151] In embodiments, the method includes irradiating a flow with one or more lasers at discrete intervals, the lasers being triggered while irradiating particles during continuous irradiation of the flow as described above. The term "discrete interval" is used herein in its conventional sense, referring to irradiation of the flow for a predetermined duration, followed by a period of time during which the flow is not irradiated by the lasers (e.g., by turning off the lasers or by blocking the lasers, for example, with a chopper, beam stop, etc.). In some embodiments, the method includes irradiating the flow with one or more lasers at discrete intervals of 0.001 μs or greater, e.g., 0.005 μs or greater, e.g., 0.01 μs or greater, e.g., 0.05 μs or greater, e.g., 0.1 μs or greater, e.g., 0.5 μs or greater, e.g., 1 μs or greater, e.g., 5 μs or greater, e.g., 10 μs or greater, e.g., 50 μs or greater, e.g., 100 μs or greater, including 500 μs or greater. In some cases, the method includes irradiating the flow stream with one or more lasers at discrete intervals of 0.0001 μs to 500 ms, such as 0.0005 μs to 250 ms, such as 0.001 μs to 50 ms, such as 0.005 μs to 5 ms, such as 0.01 μs to 1000 μs, such as 0.05 μs to 750 μs, such as 0.1 μs to 500 μs, such as 0.5 μs to 250 μs, such as 1 μs to 100 μs, and including 10 μs to 100 μs. The interval between each laser irradiation can be 0.001 μs or greater, for example 0.005 μs or greater, for example 0.01 μs or greater, for example 0.05 μs or greater, for example 0.1 μs or greater, for example 0.5 μs or greater, for example 1 μs or greater, for example 5 μs or greater, for example 10 μs or greater, for example 50 μs or greater, for example 100 μs or greater, including 500 μs or greater. For example, each discrete interval irradiated by each laser can be 0.0001 μs to 500 ms, for example 0.0005 μs to 250 ms, for example 0.001 μs to 50 ms, for example 0.005 μs to 5 ms, for example 0.01 μs to 1000 μs, for example 0.05 to 750 μs, for example 0.1 μs to 500 μs, for example 0.5 μs to 250 μs, for example 1 μs to 100 μs, and includes 10 μs to 100 μs.

[0152] In practicing the subject method, one or more lasers configured to irradiate the flow at discrete intervals can be used to irradiate the flow at discrete intervals. The number of lasers can also be, for example, two or more, three or more, four or more, five or more, ten or more, fifteen or more, twenty-five or more, including fifty or more. Each of the lasers is operatively coupled to one or more components to provide intermittent irradiation with each laser. As described in more detail below, any convenient means can be used to provide intermittent irradiation, such as electronic switches for turning the lasers on and off, such as computer-controlled and data signal-based switches triggered by data signals (e.g., received or input data signals). In some embodiments, the lasers are configured to irradiate at discrete intervals by intermittently exposing the laser beam of each of the lasers to a beam chopper or beam stop.

[0153] In some embodiments, the method includes irradiating the flow at discrete intervals at locations on the flow downstream of the continuous laser irradiation positions. In one example, the method includes continuously irradiating a location on the flow with a first laser configured for continuous irradiation of the flow; irradiating the flow with a second laser at a location downstream of the first laser; irradiating the flow with a third laser at a location downstream of the second laser; and irradiating the flow with a fourth laser at a location downstream of the third laser. Depending on the flow velocity and the distance between the irradiation positions of each laser, the method may include independently irradiating the flow at locations 5 μm or more downstream of the first laser irradiation position, such as 6 μm or more, 7 μm or more, 8 μm or more, 9 μm or more, 10 μm or more, 15 μm or more, 25 μm or more, 50 μm or more, 100 μm or more, 250 μm or more, 500 μm or more, and includes irradiating the flow at discrete intervals at locations 1000 μm or more downstream of the first laser irradiation position. For example, the irradiation position of each laser on the flow can be 5 μm to 5000 μm, for example 10 μm to 2500 μm, for example 25 μm to 1000 μm, for example 50 μm to 750 μm, for example 75 μm to 500 μm, and including 100 μm to 250 μm downstream of the irradiation position of the first laser on the flow.

[0154] The distance between each laser irradiation of the flow can vary, wherein the irradiation spacing is independently 0.0001 μm or greater, for example 0.0005 μm or greater, for example 0.001 μm or greater, for example 0.005 μm or greater, for example 0.01 μm or greater, for example 0.05 μm or greater, for example 0.1 μm or greater, for example 0.5 μm or greater, for example 1 μm or greater, for example 2 μm or greater, for example 3 μm or greater, for example 4 μm or greater, for example 5 μm or greater, for example 6 μm or greater, for example 7 μm or greater, for example 8 μm or greater, for example 9 μm or greater, and includes 10 μm or greater. In some cases, the method involves irradiating with lasers located on the flow such that the lasers are directly adjacent to each other (i.e., there is no irradiation space).

[0155] In embodiments, the lasers used to irradiate the flowing stream at discrete intervals can each be independently a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or combinations thereof. In other cases, the lasers configured for continuous irradiation are dye lasers, such as stilbene lasers, coumarin lasers, or rhodamine lasers. In still other cases, the lasers configured for continuous target irradiation include 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, or combinations thereof. In other cases, the system includes ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, or combinations thereof. In other cases, the system includes semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs), or frequency-doubled or third-doubled implementations of any of the above lasers. The laser can include any combination of laser types.

[0156] Depending on the desired wavelength of light produced in the output laser beam (e.g., for irradiating a sample in a flowing stream), each laser may have a specific wavelength varying from 200 nm to 1500 nm, such as 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, and including 400 nm to 800 nm. In some embodiments, the target laser may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser. In some embodiments, each of the lasers outputs light of a different wavelength. In some cases, the lasers of the system are positioned such that each downstream laser outputs light of a longer wavelength. For example, in a method that includes irradiating a flowing stream with four lasers, the wavelength of light from the second laser is longer than that from the first laser, the wavelength of light from the third laser is longer than that from the second laser, and the wavelength of light from the fourth laser is longer than that from the third laser.

[0157] The flow can be illuminated by each of the lasers from any suitable distance from the flow, such as 0.001 mm or greater, 0.005 mm or greater, 0.01 mm or greater, 0.05 mm or greater, 0.1 mm or greater, 0.5 mm or greater, 1 mm or greater, 5 mm or greater, 10 mm or greater, 25 mm or greater, and including 100 mm or greater. Furthermore, the flow can be illuminated at any suitable angle (e.g., relative to the vertical axis of the flow), such as 10° to 90°, 15° to 85°, 20° to 80°, 25° to 75°, and including angles from 30° to 60°, such as 90°.

[0158] In embodiments, irradiating particles in a flowing stream with one or more lasers at discrete intervals is triggered by irradiating the particles in the flowing stream with lasers configured for continuous irradiation. In some embodiments, triggering irradiation by lasers includes switching a laser from an off setting to an on setting and irradiating the flowing stream with the laser. In other embodiments, triggering irradiation by lasers includes moving the position of, for example, an optical adjustment component such as a beamstop, or setting an optical adjustment component such as a beamstop to irradiate the flowing stream with a laser. Any convenient means can be used to trigger one or more lasers, for example, by detecting particles irradiated by a first laser (i.e., a continuous beam of light passing through the first laser) with a photodetector. In some embodiments, the system includes outputting a trigger signal from a trigger detector operatively coupled to each laser to initiate discrete irradiation intervals.

[0159] In some embodiments, the duration between when a particle is detected as being irradiated by a continuously irradiated laser and the discontinuous irradiation interval initiated by one or more lasers can vary depending on the flow velocity and the distance between the irradiated particles on the flow stream. In some embodiments, the duration between continuous irradiation by the laser and the start of the discrete irradiation interval can be 0.0001 μs or greater, for example 0.0005 s or greater, for example 0.001 μs or greater, for example 0.005 μs or greater, for example 0.01 μs or greater, for example 0.05 μs or greater, for example 0.1 μs or greater, for example 0.5 μs or greater, for example 1 μs or greater, for example 2 μs or greater, for example 3 μs or greater, for example 4 μs or greater, for example 5 μs or greater, for example 6 μs or greater, for example 7 μs or greater, for example 8 μs or greater, for example 9 μs or greater, and includes 10 μs or greater. In some embodiments, the system is configured to respond to the activation of a discrete interval of output delay of a trigger signal. For example, the delay may be 0.0001 μs or greater, 0.0005 μs or greater, such as 0.001 μs or greater, 0.005 μs or greater, 0.01 μs or greater, 0.05 μs or greater, 0.1 μs or greater, 0.5 μs or greater, 1 μs or greater, 2 μs or greater, 3 μs or greater, 4 μs or greater, 5 μs or greater, 6 μs or greater, 7 μs or greater, 8 μs or greater, 9 μs or greater, and includes 10 μs or greater.

[0160] In some embodiments, the method includes calculating the start-up irradiation time (i.e., discrete intervals at which irradiation begins) for each pair of lasers in the flow. In some cases, to calculate the irradiation time, the method includes continuously irradiating particles in the flow with all lasers of the light source, detecting light from the flow in response to irradiation of each pair of particles in the lasers, and calculating the time for each laser to irradiate the particles. In some cases, calculating the start-up irradiation time includes calculating the time interval between each laser irradiation.

[0161] Figure 3A laser irradiation at discrete intervals by three lasers in response to a trigger signal from a continuously irradiating laser is depicted according to certain embodiments. The light source includes a laser 301 configured for continuous irradiation and lasers 302, 303, and 304, respectively located downstream of laser 301 along a flow stream and configured for irradiation at discrete intervals. When laser 301 irradiates a particle at time window 301a, a trigger signal is output to lasers 302, 303, and 304 to irradiate the flow stream during time windows 302a, 303a, and 304a, during which the particle will pass through the irradiation path of lasers 302, 303, and 304. The lasers are configured to sequentially turn on in response to the trigger signal to follow the path of the particle in the flow stream.

[0162] In other embodiments, the method includes calculating the duration of illumination of each pair of particles in the flow stream (i.e., the length of each discrete interval). In some cases, to calculate the duration of illumination, the method includes continuously illuminating particles in the flow stream with all lasers of the light source, detecting light from the flow stream in response to illumination of each pair of particles in the lasers, and calculating the duration of the discrete interval of illumination by each laser. In these cases, the duration of each discrete interval can be calculated based on the illumination position on the flow stream and the flow velocity of the particles in the flow stream.

[0163] In some embodiments, the method includes activating one or more downstream lasers in response to irradiation of particles in a flow stream by a first laser. Depending on the distance between the first laser and the one or more downstream lasers, each laser may independently activate in response to irradiation by the first laser for a time of 0.00001 μs or longer, such time being, for example, 0.00005 μs or greater, such as 0.0001 μs or greater, such as 0.0005 μs or greater, such as 0.001 μs or greater, such as 0.005 μs or greater, such as 0.01 μs or greater, such as 0.05 μs or greater, such as 0.1 μs or greater, such as 0.5 μs or greater, and including 1 μs or greater. In some cases, each laser is configured to turn on independently for 0.00001 μs to 100 μs after the particle is irradiated by the first laser, for example, 0.00005 μs to 90 μs, for example, 0.0001 μs to 80 μs, for example, from 0.0005 μs to 70 μs, for example, 0.001 μs to 60 μs, for example, 0.005 μs to 50 μs, for example, 0.01 μs to 40 μs, for example, 0.05 μs to 30 μs, for example, 0.1 μs to 20 μs, and including 1 μs to 10 μs.

[0164] In some embodiments, the method includes moving a beamstop in response to irradiation of particles by a first laser, the beamstop being located in the beam path between one or more downstream lasers and the flow stream. Moving the beamstop can be achieved by connecting the beamstop to a movable support stage or directly to a motor-driven translation stage, lead screw translation assembly, gear translation device, such as a stepper motor, servo motor, brushless motor, brushed DC motor, microstepper drive motor, high-resolution stepper motor, and other types of motor. The beamstop can move in response to irradiation by the first laser for a time of 0.00001 μs or longer, for example, 0.00005 μs or greater, for example, 0.0001 μs or greater, for example, 0.0005 μs or greater, for example, 0.001 μs or greater, for example, 0.005 μs or greater, for example, 0.01 μs or greater, for example, 0.05 μs or greater, for example, 0.1 μs or greater, for example, 0.5 μs or greater, and including 1 μs or greater. In some cases, within 0.00001 μs to 100 μs after the particle is irradiated by the first laser, the beam stop moves along the beam path of one or more downstream lasers. This time period also includes, for example, 0.00005 μs to 90 μs, 0.0001 μs to 80 μs, 0.0005 μs to 70 μs, 0.001 μs to 60 μs, 0.005 μs to 50 μs, 0.01 μs to 40 μs, 0.05 μs to 30 μs, 0.1 μs to 20 μs, and includes 1 μs to 10 μs.

[0165] In some embodiments, the method includes directing light from one or more downstream lasers into a flow stream using a beam steering device in response to the irradiation of particles by a first laser. In some embodiments, in response to generating a data signal by irradiating particles in the flow stream with the first laser, the beam steering device redirects light from one or more downstream lasers into the flow stream in response to the generated data signal. In some cases, the beam steering device is an acousto-optic device, such as an acousto-optic deflector (AOD) or an acousto-optic modulator (AOM). In these embodiments, the data signal may include altering a drive signal from a digital frequency synthesizer (DDS), an arbitrary waveform generator (AWG), or an electropulse generator in a manner sufficient to redirect light from the lasers into the flow stream. In other cases, the beam steering device is an electro-optic device, such as an electro-optic deflector (EOD) or an electro-optic modulator (EOM). In these embodiments, the data signal may include altering an input current or applying a voltage to the electro-optic device in a manner sufficient to redirect light from the lasers into the flow stream. In some embodiments, the beam deflector is configured to redirect the beam path of one or more downstream lasers in response to irradiation by the first laser for a period of 0.00001 μs or longer, this period also being, for example, 0.00005 μs or longer, such as 0.0001 μs or longer, such as 0.0005 μs or longer, such as 0.001 μs or longer, such as 0.005 μs or longer, such as 0.01 μs or longer, such as 0.05 μs or longer, such as 0.1 μs or longer, such as 0.5 μs or longer, and including 1 μs or longer. In some cases, the beam deflector is configured to redirect the beam path of one or more downstream lasers within 0.00001 μs to 100 μs after the first laser irradiates the particle. This time period may also be, for example, 0.00005 μs to 90 μs, 0.0001 μs to 80 μs, 0.0005 μs to 70 μs, 0.001 μs to 60 μs, 0.005 μs to 50 μs, 0.01 μs to 40 μs, 0.05 μs to 30 μs, 0.1 μs to 20 μs, and may include 1 μs to 10 μs.

[0166] According to some embodiments, the method of this disclosure further includes detecting light from particles in the flow. In embodiments, light from the flowing flow is transmitted to and detected by a light detection system having one or more photodetectors. The photodetector used to practice the subject method can be any convenient light detection device, including but not limited to photosensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-type charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or combinations thereof, and other photodetectors. Light from particles in the flowing flow can be detected using one or more photodetectors, such as two or more, three or more, four or more, five or more, ten or more, twenty-five or more, including fifty or more.

[0167] In some embodiments, a photodiode array having more than one photodiode is used to detect light from particles in the flowing stream, such as two or more photodiodes, three or more, five or more, and including ten or more photodiodes, wherein the effective detection surface area of ​​each region of each photodiode can be 0.01 cm². 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 5cm 2 .

[0168] In embodiments of this disclosure, light from particles in a flowing stream can be measured at one or more wavelengths, such as at two or more wavelengths, such as at five or more wavelengths, such as at ten or more wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and includes measuring light from particles in a flowing stream at 400 or more different wavelengths.

[0169] In embodiments, light can be measured continuously or at discrete intervals using a photodetector. In some cases, the target detector is configured to continuously measure the collected light. In other cases, the target detector is configured to measure at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0170] As long as a usable light signal can be detected, the light from the flow can be measured at any suitable distance from the flow with each photodetector. For example, detectors can be positioned at a distance of 1 mm or greater from the flow, such as 5 mm or greater, 10 mm or greater, 15 mm or greater, 25 mm or greater, 50 mm or greater, 100 mm or greater, 150 mm or greater, 250 mm or greater, and including distances of 500 mm or greater. Detectors can also be placed at any angle to the flow. For example, detectors can be at angles of 10° to 90° relative to the vertical axis of the flow, such as 15° to 85°, 20° to 80°, 25° to 75°, and including 30° to 60°. In some cases, one or more detectors are positioned at 30° to 60° relative to the vertical axis of the flow.

[0171] In embodiments, the method may include detecting forward-scattered light, side-scattered light, reflected light, transmitted light, or a combination thereof. In some embodiments, the light signal from the illuminating flow may be detected by one or more detectors configured as forward-scattering detectors. In these embodiments, the forward-scattering detectors are positioned on one side of the flow opposite the light source and are positioned to collect and detect forward-propagating (e.g., scattered) light.

[0172] In some embodiments, the method includes detecting light from each laser using a single photodetector. In these embodiments, the photodetector detects light from particles in a flowing stream, which are configured to continuously irradiate the lasers and to irradiate each laser at discrete intervals.

[0173] In some embodiments, the method includes detecting light from a flowing stream, the light from the flowing stream being transmitted through an optical adjustment component configured to reduce the amount of light transmitted from a laser configured to continuously illuminate one or more photodetectors. In these embodiments, the optical adjustment component is configured to restrict, reduce, or limit the propagation of at least one or more wavelengths of light (e.g., one or more wavelengths of light from a laser configured for continuous illumination) from the sample to the effective surface of the photodetector. The light transmitted to the photodetector may include optical components that restrict the propagation of one or more different wavelengths of light, such as 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, and including restricting the propagation of 500 or more different wavelengths of light. For example, in some embodiments, the optical adjustment component is a bandpass filter, such as a long-pass filter that transmits a spectral range of light longer than the illumination wavelength of a first laser. In other embodiments, the optical adjustment component is a dichroic mirror, such as a dichroic mirror that transmits a spectral range longer than the illumination wavelength of the first laser and reflects a spectral range including the illumination wavelength of the first laser.

[0174] In some embodiments, the method includes collecting light from a flowing stream using an optical collection system and directing it to a photodetector. In some embodiments, light is transmitted from the flowing stream to a photodetector having an optical fiber. In some cases, the optical collection system may be an optical fiber repeater, and the light is transmitted to the photodetector via the optical fiber repeater. In some embodiments, the optical collection unit is a single optical fiber configured to transmit light irradiated from each of the lasers to a single photodetector in a light detection system. In these embodiments, the method includes irradiating the flowing stream with a laser at a location spanning a length equal to or less than the diameter of a single optical fiber configured to collect light from the flowing stream. For example, the method may include irradiating locations across the flow stream spanning 100 μm or less, such as 90 μm or less, such as 80 μm or less, such as 70 μm or less, such as 60 μm or less, and including locations of 50 μm or less, and a single optical fiber may have a diameter sufficient to collect light from each location irradiated by the laser, such as a diameter of 50 μm or greater, such as 60 μm or greater, such as 70 μm or greater, such as 80 μm or greater, such as 90 μm or greater, and includes a single optical fiber configured to collect light from the irradiated flow stream having a diameter of 100 μm or greater.

[0175] In other embodiments, light from the flowing stream is transmitted to a photodetector using a free-space optical relay system. For example, the free-space optical relay system may include a housing having a near end and a far end, with the near end coupled to the light detection system. The free-space relay system may include any combination of different optical components, such as one or more lenses, mirrors, slits, pinholes, wavelength splitters, or combinations thereof.

[0176] During the subject method, one or more measurements of the collected light may be performed, such as two or more, three or more, five or more, and including ten or more. In some embodiments, light propagation is measured two or more times, and in some cases the data are averaged.

[0177] In some embodiments, the method includes adjusting the light before detecting it with a light detection system. For example, light from a sample source may pass through one or more lenses, mirrors, 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 directed to the light detection system or optical collection system as described above. In other cases, emitted light from the sample passes through one or more collimators to reduce the beam divergence transmitted to the light detection system.

[0178] Computer-readable storage media

[0179] This disclosure also includes non-transitory computer-readable storage media having instructions for practicing the subject methods. Computer-readable storage media can be used on one or more computers to achieve full or partial automation of the system to implement the methods described herein. In some embodiments, instructions according to the methods described herein can be encoded in a “programmed” form onto 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, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network attached storage (NAS), whether these devices are internal or external to a computer. Files containing information can be “stored” on a computer-readable medium, where “stored” means recording the information so that a computer can access and retrieve it later. The computer-implemented methods described herein can be executed using programming that can be written in one or more of any number of computer programming languages. For example, these languages ​​include Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as many other languages.

[0180] In some embodiments, the target computer-readable storage medium includes a computer program stored thereon, wherein the computer program, when loaded onto a computer, includes instructions having: an algorithm for continuously irradiating a flow with a first laser; an algorithm for irradiating the flow with a second laser at discrete intervals, wherein each discrete interval of irradiation by the second laser is triggered by irradiating a particle in the flow with the first laser; an algorithm for detecting light from each irradiated particle in the flow with the laser using a single photodetector coupled to a single optical fiber; and an algorithm for calculating the irradiation time of each pair of irradiated particles in the flow with the laser. In some embodiments, the non-transitory computer-readable storage medium includes instructions having: an algorithm for continuously irradiating a flow with a first laser; and an algorithm for irradiating a flow with multiple lasers at discrete intervals, wherein each discrete interval of irradiation by each of the multiple lasers is triggered by irradiating a particle in the flow with the first laser. In some cases, the non-transitory computer-readable storage medium includes instructions having: an algorithm for continuously irradiating a particle in the flow with each of the lasers; an algorithm for detecting light from the flow in response to each pair of irradiated particles in the laser; and an algorithm for calculating the time interval between each irradiated particle in the laser.

[0181] In some embodiments, the non-transitory computer-readable storage medium includes instructions having an algorithm for activating a second laser in response to irradiation of a particle by a first laser. In some embodiments, the non-transitory computer-readable storage medium includes instructions having an algorithm for moving a beam stop located in a beam path between the second laser and the flow stream in response to irradiation of a particle by the first laser. In some embodiments, the non-transitory computer-readable storage medium includes instructions having an algorithm for guiding light from the second laser to the flow stream using a beam deflector in response to irradiation of a particle by the first laser.

[0182] Computer-readable storage media can be used on one or more computer systems having displays and operator input devices. For example, the operator input device can be a keyboard, mouse, or similar device. The processing module includes a processor that can access memory storing instructions for performing the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup devices, and many other devices. The processor can be a commercially available processor, or it can be one of other processors that are currently available or will become available. The processor executes the operating system and its interface with firmware and hardware in a well-known manner, and facilitates the processor's coordination and execution of various computer programs written in various programming languages ​​known in the art, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof. The operating system, typically working with the processor, coordinates and executes the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all consistent with known technologies.

[0183] Complete sets of components

[0184] A kit comprising one or more components of a subject system is also provided. According to some embodiments, the kit includes one or more lasers, such as lasers configured for continuous illumination and lasers configured for illumination at discrete intervals. In some embodiments, switches (e.g., pulsed lasers) for operating one or more lasers at discrete intervals may be included. The kit may also include an optical adjustment component configured to reduce the passage of light wavelengths from one or more lasers. In some cases, the optical adjustment component is a bandpass filter, such as a longpass filter that transmits a spectral range of light longer than the illumination wavelengths of one or more lasers. In other cases, the optical adjustment component is a dichroic mirror, such as a dichroic mirror that transmits a spectral range of light longer than the illumination wavelengths of one or more lasers and reflects a spectral range including the wavelengths of one or more lasers.

[0185] The kit may also include an array of photodetectors for detecting light from the flowing stream. In some embodiments, the support stage includes a motor, such as a stepper motor. The kit may also include an optical relay system, such as an optical fiber (e.g., a single fiber), for propagating light from the sample in the flowing stream to the detector.

[0186] In addition to the components described above, the subject matter kit may also include (in some embodiments) instructions for implementing the subject matter method. These instructions may exist in various forms within the subject matter kit, with one or more forms present in the kit. One form of these instructions may be as printed information on a suitable medium or substrate, such as on one or more sheets of paper with the information printed thereon, in the packaging of the kit, in a packaging insert, etc. Another form of these instructions is as a computer-readable medium on which information has been recorded, such as a disk, optical disc (CD), portable flash drive, etc. Yet another possible form of these instructions is a website address that can be used to access information on a deleted site via the Internet.

[0187] use

[0188] The subject system, method, and computer system can be used in a variety of applications where it is necessary to analyze and classify the particle composition of samples (e.g., biological samples) in fluid media. This disclosure also reveals applications in flow cytometry, where it is desirable to provide a flow cytometer that offers improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, reduced particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting. In embodiments, this disclosure reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In some embodiments, the subject system provides a fully automated apparatus, requiring minimal manual adjustment of the flow cytometer during use.

[0189] This disclosure also reveals that cells prepared from biological samples may be needed for research, laboratory testing, or therapeutic applications. In some embodiments, the subject methods and apparatus can facilitate the preparation of single cells from a target fluid or tissue biological sample. For example, the subject methods and systems facilitate the acquisition of cells from fluid or tissue samples for use as research or diagnostic samples for diseases such as cancer. Similarly, the subject methods and systems facilitate the acquisition of cells from fluid or tissue samples for therapeutic purposes. Compared to conventional flow cytometry systems, the methods and apparatus of this disclosure allow for the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, fluids) with increased efficiency and low cost.

[0190] The aspects of the subject matter described herein, including embodiments, may be advantageous, either alone or in combination with one or more other aspects or embodiments. Without limiting the description, certain non-limiting aspects of the disclosure numbered 1-139 are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or hereinafter individually numbered aspects. This is intended to support combinations of all such aspects, but is not limited to, the combinations of aspects expressly specified below:

[0191] 1. A system comprising:

[0192] Light source, the light source comprising:

[0193] A first laser is configured to continuously irradiate the flowing stream;

[0194] A second laser is configured to irradiate the flow at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with the first laser.

[0195] An optical detection system, the optical detection system comprising:

[0196] A single optical fiber, configured to collect light from each particle in the irradiated flow of the laser; and

[0197] A single photodetector, configured to detect light transmitted by the single optical fiber; and

[0198] A processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the irradiation time for each pair of flow streams in the laser.

[0199] 2. The system according to 1, wherein the second laser is configured to irradiate the flow at a position downstream of the first laser.

[0200] 3. The system according to any one of 1 to 2, wherein the wavelength of light from the first laser is shorter than the wavelength of light from the second laser.

[0201] 4. The system according to any one of 1 to 3, wherein the system comprises:

[0202] A first laser configured for continuously irradiating a flowing stream; and

[0203] Multiple lasers are configured to irradiate the flow at discrete intervals.

[0204] 5. The system according to 4, wherein the plurality of lasers are configured to irradiate the flow at a position downstream of the first laser.

[0205] 6. The system according to 5, wherein the plurality of lasers are configured to irradiate the flow at positions spaced 10 μm or less from each other.

[0206] 7. The system according to any one of 4 to 6, wherein each of the plurality of lasers outputs light of a different wavelength.

[0207] 8. The system according to any one of 5 to 6, wherein the plurality of lasers comprises:

[0208] A second laser is configured to irradiate the flow at a position downstream of the first laser;

[0209] A third laser is configured to irradiate the flow at a position downstream of the second laser; and

[0210] A fourth laser is configured to irradiate the flow at a position downstream of the third laser;

[0211] 9. The system according to 8, wherein:

[0212] The wavelength of the second laser is greater than that of the first laser;

[0213] The wavelength of the third laser is greater than that of the second laser;

[0214] The wavelength of the fourth laser is greater than that of the third laser.

[0215] 10. The system according to any one of 4 to 9, wherein each discrete interval of irradiation of the flow by each of the plurality of lasers is independently triggered by irradiating particles in the flow with the first laser.

[0216] 11. The system according to any one of 1 to 10, wherein each discrete interval of each laser comprises a duration of 1 μs to 500 μs.

[0217] 12. The system according to any one of 1 to 11, wherein the memory includes instructions stored thereon, which, when executed by a processor, cause the processor to calculate the irradiation time of each pair of flow streams in the laser in such a way as:

[0218] Each laser beam illuminates a particle in the flowing stream;

[0219] In response to each pair of particles being illuminated in the laser, light from the flowing stream is detected; and

[0220] Calculate the time interval between each irradiated particle in the laser.

[0221] 13. The system according to any one of 1 to 12, wherein the system is configured to turn on the second laser in response to irradiation of a particle by the first laser.

[0222] 14. The system according to any one of 1 to 12, wherein the system further comprises a beam stop located in the beam path between the second laser and the flow stream.

[0223] 15. The system according to 14, wherein the processor’s memory includes instructions stored thereon that, when executed by the processor, cause the processor to move a beam stop from the beam path between the second laser and the flow stream in response to the irradiation of the particles by the first laser.

[0224] 16. The system according to any one of 1 to 12, wherein the system further comprises a beam deflector located in a beam path between the second laser and the flow stream, wherein the beam deflector is configured to deflect light from the second laser away from the flow stream.

[0225] 17. The system of claim 16, wherein the beam deflector is configured to direct light from the second laser into the flow stream in response to irradiation of the particles by the first laser.

[0226] 18. The system according to any one of 16 to 17, wherein the beam deflector includes an acousto-optic modulator.

[0227] 19. The system according to 18, wherein the acousto-optic device is an acousto-optic modulator (AOM).

[0228] 20. The system according to 18, wherein the acousto-optic device is an acousto-optic deflector (AOD).

[0229] 21. The system according to any one of 16 to 17, wherein the beam deflector comprises an electro-optic device.

[0230] 22. The system according to 21, wherein the electro-optic device is an electro-optic modulator (EOM).

[0231] 23. The system according to 21, wherein the electro-optic device is an electro-optic deflector (EOD).

[0232] 24. The system according to any one of 1 to 23 further includes a light detection system, wherein the light detection system comprises:

[0233] Single photodetector; and

[0234] An optical adjustment component configured to reduce the amount of light transmitted from the first laser to the photodetector.

[0235] 25. The system of claim 24, wherein the optical adjustment component includes a bandpass filter.

[0236] 26. The system according to any one of 1 to 15, wherein the single photodetector is a photodetector array.

[0237] 27. A system comprising:

[0238] A first laser configured for continuously irradiating a flowing stream; and

[0239] Configure a second laser for irradiating the flow at discrete intervals.

[0240] Each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with the first laser.

[0241] 28. The system according to 27, wherein the second laser is configured to irradiate the flow at a position downstream of the first laser.

[0242] 29. The system according to any one of 27 to 28, wherein the wavelength of light from the first laser is shorter than the wavelength of light from the second laser.

[0243] 30. The system according to any one of 27 to 29, wherein the system comprises:

[0244] A first laser configured for continuously irradiating a flowing stream; and

[0245] Multiple lasers are configured to irradiate the flow at discrete intervals.

[0246] 31. The system according to 30, wherein the plurality of lasers are configured to irradiate the flow at a position downstream of the first laser.

[0247] 32. The system according to 31, wherein the plurality of lasers are configured to irradiate the flow at positions spaced apart from each other by 10 μm or less.

[0248] 33. The system according to any one of 31 to 32, wherein the plurality of lasers comprises:

[0249] A second laser is configured to irradiate the flow at a position downstream of the first laser;

[0250] A third laser is configured to irradiate the flow at a position downstream of the second laser; and

[0251] A fourth laser is configured to irradiate the flow at a position downstream of the third laser.

[0252] 34. The system according to 33, wherein:

[0253] The wavelength of the second laser is greater than that of the first laser;

[0254] The wavelength of the third laser is greater than that of the second laser;

[0255] The wavelength of the fourth laser is greater than that of the third laser.

[0256] 35. The system according to any one of 30 to 34, wherein each discrete interval of irradiation of the flow by each of the plurality of lasers is independently triggered by irradiating particles in the flow with the first laser.

[0257] 36. The system according to any one of 27 to 35, wherein each discrete interval of each laser comprises a duration of 1 μs to 500 μs.

[0258] 37. The system according to any one of 27 to 36, wherein the system is configured to turn on the second laser in response to irradiation of a particle by the first laser.

[0259] 38. The system according to any one of 27 to 36, wherein the system further comprises a beam stop located in the beam path between the second laser and the flow stream.

[0260] 39. The system of claim 38, wherein the system includes a processor, the processor including a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to move a beam stop from a beam path between a second laser and a flow stream in response to irradiation of particles by a first laser.

[0261] 40. The system according to any one of 27 to 36, wherein the system further comprises a beam deflector located in a beam path between the second laser and the flow stream, wherein the beam deflector is configured to deflect light from the second laser away from the flow stream.

[0262] 41. The system according to 40, wherein the beam deflector is configured to direct light from the second laser into the flow stream in response to irradiation of the particles by the first laser.

[0263] 42. The system according to any one of 40 to 41, wherein the beam deflector includes an acousto-optic device.

[0264] 43. The system according to 42, wherein the acousto-optic device is an acousto-optic modulator (AOM).

[0265] 44. The system according to 42, wherein the acousto-optic device is an acousto-optic deflector (AOD).

[0266] 45. The system according to any one of 40 to 41, wherein the beam deflector comprises an electro-optic device.

[0267] 46. ​​The system according to 45, wherein the electro-optic device is an electro-optic modulator (EOM).

[0268] 47. The system according to 45, wherein the electro-optic device is an electro-optic deflector (EOD).

[0269] 48. The system according to any one of 27 to 47, further comprising a light detection system, wherein the light detection system includes:

[0270] Photodetector; and

[0271] An optical adjustment component configured to reduce the amount of light transmitted from the first laser to the photodetector.

[0272] 49. The system of claim 48, wherein the optical adjustment component includes a bandpass filter.

[0273] 50. The system according to any one of 48 to 49, wherein the photodetector is configured to detect light from each irradiated flow of a laser from a light source.

[0274] 51. According to the system of 50, the photodetector includes a photodetector array.

[0275] 52. The system according to any one of 48 to 51 further includes a light propagation component configured to transmit light from the flow stream to the photodetector.

[0276] 53. The system according to 52, wherein the optical propagation component includes an optical fiber.

[0277] 54. The system according to any one of 48 to 53, wherein the photodetector is a single photodetector configured to detect light from each of the lasers in the light source.

[0278] 55. The system according to 54, wherein the optical propagation component consists of a single optical fiber.

[0279] 56. A method comprising:

[0280] The flow is continuously irradiated with a first laser;

[0281] The flow is irradiated with a second laser at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with a first laser.

[0282] A single photodetector coupled to a single optical fiber is used to detect light from each particle in the irradiated flow of the laser; and

[0283] Calculate the irradiation time for each pair of flow streams in the laser.

[0284] 57. The method according to 56, wherein the second laser is configured to irradiate the flow at a position downstream of the first laser.

[0285] 58. The method according to any one of 56 to 57, wherein the wavelength of light from the first laser is shorter than the wavelength of light from the second laser.

[0286] 59. The method according to any one of 56 to 58, comprising:

[0287] The flow is continuously irradiated with a first laser; and

[0288] A flow is irradiated with multiple lasers at discrete intervals, wherein each discrete interval of irradiation by each of the multiple lasers is triggered by irradiating particles in the flow with a first laser.

[0289] 60. The method according to 59, wherein the plurality of lasers are configured to irradiate the flow at a position downstream of the first laser.

[0290] 61. The method according to 60, wherein the plurality of lasers are configured to irradiate the flow at positions spaced apart from each other by 10 μm or less.

[0291] 62. The method according to any one of 59 to 61, wherein each of the plurality of lasers outputs light of a different wavelength.

[0292] 63. The system according to any one of 59 to 62, wherein the plurality of lasers comprises:

[0293] A second laser is configured to irradiate the flow at a position downstream of the first laser;

[0294] A third laser is configured to irradiate the flow at a position downstream of the second laser; and

[0295] A fourth laser is configured to irradiate the flow at a position downstream of the third laser;

[0296] 64. According to the method described in 62, wherein:

[0297] The wavelength of the second laser is greater than that of the first laser;

[0298] The wavelength of the third laser is greater than that of the second laser;

[0299] The wavelength of the fourth laser is greater than that of the third laser.

[0300] 65. The method according to any one of 56 to 64, wherein each discrete interval of each laser comprises a duration of 1 μs to 500 μs.

[0301] 66. The method according to any one of 55 to 65, wherein calculating the irradiation time of each pair of flow streams in the laser comprises:

[0302] Each laser continuously illuminates the particles in the flowing stream;

[0303] In response to the illumination of each particle in the laser, light from the flowing stream is detected; and

[0304] Calculate the time interval between each irradiated particle in the laser.

[0305] 67. The method according to any one of 56 to 66, wherein the method includes activating the second laser in response to irradiation of the particles by the first laser.

[0306] 68. The method according to any one of 56 to 66, wherein the method includes moving a beam stop located in a beam path between a second laser and a flow stream in response to irradiation of particles by a first laser.

[0307] 69. The method according to any one of 56 to 66, wherein the method includes, in response to irradiation of the particles by the first laser, guiding light from the second laser to the flow stream using a beam deflector.

[0308] 70. The method according to 69, wherein the beam deflector includes an acousto-optic device.

[0309] 71. The method according to 70, wherein the acousto-optic device is an acousto-optic modulator (AOM).

[0310] 72. The method according to 70, wherein the acousto-optic device is an acousto-optic deflector (AOD).

[0311] 73. The method according to 69, wherein the beam deflector includes an electro-optic device.

[0312] 74. The method according to 73, wherein the electro-optic device is an electro-optic modulator (EOM).

[0313] 75. The method according to 73, wherein the electro-optic device is an electro-optic deflector (EOD).

[0314] 76. The method according to any one of 56 to 75 further comprises detecting light from the flowing stream using a light detection system, said light detection system comprising:

[0315] Photodetector; and

[0316] An optical adjustment component configured to reduce the amount of light transmitted from the first laser to the photodetector.

[0317] 77. The method according to 76, wherein the optical adjustment component is a bandpass filter.

[0318] 78. The system according to any one of 76 to 77, wherein the single detector is a photodetector array.

[0319] 79. The method according to any one of 76 to 78, wherein the light detection system includes a light propagation component configured to transmit light from the flow stream to the photodetector.

[0320] 80. A method comprising:

[0321] The flow is continuously irradiated with a first laser; and

[0322] The flow is irradiated with a second laser at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with a first laser.

[0323] 81. The method according to 80, wherein the second laser is configured to irradiate the flow at a position downstream of the first laser.

[0324] 82. The method according to any one of 80 to 81, wherein the wavelength of light from the first laser is shorter than the wavelength of light from the second laser.

[0325] 83. The method according to any one of 80 to 82, comprising:

[0326] The flow is continuously irradiated with a first laser; and

[0327] A flow is irradiated with multiple lasers at discrete intervals, wherein each discrete interval of irradiation by each of the multiple lasers is triggered by irradiating particles in the flow with a first laser.

[0328] 84. The method according to 83, wherein the plurality of lasers are configured to irradiate the flow at a position downstream of the first laser.

[0329] 85. The method according to 84, wherein the plurality of lasers are configured to irradiate the flow at positions spaced apart from each other by 10 μm or less.

[0330] 86. The method according to any one of 83 to 85, wherein each of the plurality of lasers outputs light of a different wavelength.

[0331] 87. The system according to any one of 84 to 86, wherein the plurality of lasers comprises:

[0332] A second laser is configured to irradiate the flow at a position downstream of the first laser;

[0333] A third laser is configured to irradiate the flow at a position downstream of the second laser; and

[0334] A fourth laser is configured to irradiate the flow at a position downstream of the third laser.

[0335] 88. According to the method described in 87, wherein:

[0336] The wavelength of the second laser is greater than that of the first laser;

[0337] The wavelength of the third laser is greater than that of the second laser;

[0338] The wavelength of the fourth laser is greater than that of the third laser.

[0339] 89. The method according to any one of 80 to 88, wherein each discrete interval of each laser comprises a duration of 1 μs to 500 μs.

[0340] 90. The method according to any one of 80 to 89 further includes calculating the irradiation time of each pair of flow streams in the laser.

[0341] 91. The method according to 90, wherein calculating the irradiation time of each pair of flow streams in the laser includes:

[0342] Each laser continuously illuminates the particles in the flowing stream;

[0343] In response to the illumination of each particle in the laser, light from the flowing stream is detected; and

[0344] Calculate the time interval between each irradiated particle in the laser.

[0345] 92. The method according to any one of 80 to 91, wherein the method includes activating the second laser in response to irradiation of the particles by the first laser.

[0346] 93. The method according to any one of 80 to 91, wherein the method includes moving a beam stop located in a beam path between a second laser and a flow stream in response to irradiation of particles by a first laser.

[0347] 94. The method according to any one of 80 to 91, wherein the method includes, in response to irradiation of the particles by the first laser, guiding light from the second laser to the flow stream using a beam deflector.

[0348] 95. The method according to 94, wherein the beam deflector includes an acousto-optic device.

[0349] 96. The method according to 95, wherein the acousto-optic device is an acousto-optic modulator (AOM).

[0350] 97. According to the method described in 95, the acousto-optic device is an acousto-optic deflector (AOD).

[0351] 98. The method according to 94, wherein the beam deflector includes an electro-optical device.

[0352] 99. The method according to 98, wherein the electro-optic device is an electro-optic modulator (EOM).

[0353] 100. The method according to 98, wherein the electro-optic device is an electro-optic deflector (EOD).

[0354] 101. The method according to any one of 80 to 100 further includes detecting light from the flowing stream using a light detection system, said light detection system comprising:

[0355] Photodetector; and

[0356] An optical adjustment component configured to reduce the amount of light transmitted from the first laser to the photodetector.

[0357] 102. The method according to 101, wherein the optical adjustment component is a bandpass filter.

[0358] 103. The method according to any one of 101 to 102, wherein the photodetector comprises a photodetector array.

[0359] 104. The method according to any one of 101 to 103, wherein the light detection system includes a light propagation component configured to transmit light from the flow stream to the photodetector.

[0360] 105. The method according to 104, wherein the optical propagation component includes an optical fiber.

[0361] 106. The method according to any one of 101 to 105, wherein the photodetector is a single photodetector configured to detect light from each of the lasers in the light source.

[0362] 107. The system according to any one of 105 to 106, wherein the optical propagation component comprises a single optical fiber.

[0363] 108. A non-transitory computer-readable storage medium, comprising instructions stored thereon, the instructions comprising:

[0364] An algorithm for continuously irradiating the flow with a first laser;

[0365] An algorithm that uses a second laser to irradiate the flow at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with a first laser;

[0366] An algorithm that uses a single photodetector coupled to a single optical fiber to detect light from each particle in an irradiated flow from a laser; and

[0367] An algorithm for calculating the irradiation time of each pair of flow streams in a laser.

[0368] 109. The non-transitory computer-readable storage medium according to 108, wherein the instructions include an algorithm for irradiating the flow with a second laser at a location downstream of the first laser.

[0369] 110. The non-transitory computer-readable storage medium according to 108, wherein the wavelength of light from the first laser is shorter than the wavelength of light from the second laser.

[0370] 111. The non-transitory computer-readable storage medium according to claim 108, wherein the instructions include:

[0371] An algorithm for continuously irradiating the flow with a first laser; and

[0372] An algorithm that uses multiple lasers to irradiate a flow at discrete intervals, wherein each discrete interval of irradiation by each of the multiple lasers is triggered by irradiating particles in the flow with a first laser.

[0373] 112. The non-transitory computer-readable storage medium according to 111, wherein the plurality of lasers are configured to irradiate the flow at a location downstream of the first laser.

[0374] 113. The non-transitory computer-readable storage medium according to 112, wherein the plurality of lasers are configured to irradiate the flow at positions spaced apart from each other by 10 μm or less.

[0375] 114. The non-transitory computer-readable storage medium according to 112, wherein each of the plurality of lasers outputs light of a different wavelength.

[0376] 115. The non-transitory computer-readable storage medium according to 112 to 114, wherein the instructions include:

[0377] An algorithm for irradiating the flow with a second laser at a position downstream of the first laser;

[0378] An algorithm for irradiating the flow with a third laser at a location downstream of the second laser;

[0379] And an algorithm for irradiating the flow with a fourth laser at a position downstream of the third laser.

[0380] 116. The non-transitory computer-readable storage medium according to 115, wherein:

[0381] The wavelength of the second laser is greater than that of the first laser;

[0382] The wavelength of the third laser is greater than that of the second laser;

[0383] The wavelength of the fourth laser is greater than that of the third laser.

[0384] 117. The non-transitory computer-readable storage medium according to any one of 108 to 116, wherein each discrete interval of each laser comprises a duration of 1 μs to 500 μs.

[0385] 118. The non-transitory computer-readable storage medium according to 108 to 117, wherein the instructions include:

[0386] An algorithm that uses each laser to continuously irradiate particles in a flowing stream;

[0387] An algorithm for detecting light from a flowing stream in response to each particle irradiation in a laser; and

[0388] An algorithm for calculating the time interval between each irradiated particle in a laser.

[0389] 119. The non-transitory computer-readable storage medium according to any one of 108 to 118, wherein the instructions include an algorithm for activating the second laser in response to irradiation of the particles by the first laser.

[0390] 120. The non-transitory computer-readable storage medium according to any one of 108 to 118, wherein the instructions include an algorithm for moving a beam stop in a beam path between a second laser and a flow stream in response to irradiation of a particle by a first laser.

[0391] 121. The non-transitory computer-readable storage medium according to any one of 108 to 118, wherein the instructions include an algorithm for guiding light from a second laser to a flow stream using a beam deflector in response to irradiation of particles by a first laser.

[0392] 122. The non-transitory computer-readable storage medium according to 121, wherein the optical steering device includes an acousto-optic device.

[0393] 123. The non-transitory computer-readable storage medium according to 122, wherein the acousto-optic device is an acousto-optic modulator (AOM).

[0394] 124. The non-transitory computer-readable storage medium according to 122, wherein the acousto-optic device is an acousto-optic deflector (AOD).

[0395] 125. The non-transitory computer-readable storage medium of claim 121, wherein the optical steering device comprises an electro-optical device.

[0396] 126. The non-transitory computer-readable storage medium according to 125, wherein the electro-optic device is an electro-optic modulator (EOM).

[0397] 127. The non-transitory computer-readable storage medium according to 125, wherein the electro-optic device is an electro-optic deflector (EOD).

[0398] 128. The non-transitory computer-readable storage medium according to any one of 108 to 127, wherein the instructions further include an algorithm for detecting light from a flowing stream using a light detection system, the light detection system comprising:

[0399] Photodetector; and

[0400] An optical adjustment component configured to reduce the amount of light transmitted from the first laser to the photodetector.

[0401] 129. A complete set of components, comprising:

[0402] Light sources including a first laser and a second laser;

[0403] Fiber optics; and

[0404] Photodetector.

[0405] 130. The assembly according to 129 further includes an optical adjustment component configured to reduce the transmission of the light wavelength of the first laser.

[0406] 131. The kit of components according to 130, wherein the optical adjustment assembly is a bandpass filter.

[0407] 132. The kit of components according to 130, wherein the optical adjustment component is a dichroic filter.

[0408] 133. The assembly according to any one of 129 to 132 further includes a plurality of lasers.

[0409] 134. The complete assembly according to 133, wherein the plurality of lasers includes a 405nm laser, a 488nm laser, a 561nm laser, and a 635nm laser.

[0410] 135. A complete set of components, comprising:

[0411] Light sources including the first laser and the second laser; and

[0412] An optical adjustment component configured to reduce the wavelength of light passing through the first laser.

[0413] 136. The assembly according to 135 further includes multiple lasers.

[0414] 137. The assembly according to 136, wherein the plurality of lasers includes a 405nm laser, a 488nm laser, a 561nm laser, and a 635nm laser.

[0415] 138. The kit of components according to any one of 135 to 137, wherein the optical adjustment assembly is a bandpass filter.

[0416] 139. The kit of components according to any one of 135 to 137, wherein the optical adjustment component is a dichroic filter.

[0417] Although the foregoing invention has been described in some detail by way of illustration and examples for the purpose of clear understanding, it will be apparent to those skilled in the art, based on the teachings of the present invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0418] Therefore, the foregoing merely illustrates the principles of the invention. It should be understood that those skilled in the art will be able to devise various arrangements that, while not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all embodiments and conditional language listed herein are primarily intended to aid the reader in understanding the principles of the invention and the inventors' concepts for contributing to the field, and should be interpreted as not being limited to these specifically listed embodiments and conditions. Moreover, all statements herein referencing the principles, aspects, and embodiments of the invention and specific examples thereof are intended to cover their structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any elements developed that perform the same function regardless of structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly recited in the claims.

[0419] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, when the exact phrase “for a means of…” or “for a step of…” is referenced at the beginning of such limitation of the claims, 35U.SC §112(f) or 35U.SC §112(6) is explicitly defined as being invoked only to limit the claims; if such an exact phrase is not used in the limitation of the claims, then 35U.SC §112(f) or 35U.SC §112(6) is not invoked.

Claims

1. A flow cytometer, comprising: A flow tank, configured to convey a flow of liquid through which a flow stream is passed; Light source, the light source comprising: A first laser is configured to continuously irradiate the flowing stream; A second laser is configured to irradiate the flow at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with the first laser. The wavelength of the light from the first laser is shorter than the wavelength of the light from the second laser; An optical detection system, the optical detection system comprising: A single optical fiber, configured to collect light from each particle in the irradiated flow of the laser; and A single photodetector, configured to detect light transmitted by the single optical fiber; and A processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the irradiation time of the second laser on the flowing stream; The processor's memory includes instructions stored thereon, which, when executed by the processor, cause the processor to perform the following operations: The second laser is activated in response to the irradiation of the particles by the first laser; In response to the irradiation of the particles by the first laser, the beam stop is moved from the beam path between the second laser and the flow stream; or In response to the irradiation of particles by the first laser, the light from the second laser is redirected into the flow using a beam deflector.

2. The flow cytometer according to claim 1, wherein, The second laser is configured to irradiate the flow at a location downstream of the first laser.

3. The flow cytometer according to any one of claims 1 to 2, wherein, The flow cytometer includes: A first laser configured for continuously irradiating a flowing stream; and Multiple second lasers configured to irradiate the flow at discrete intervals. Each discrete interval of the irradiation of the flow by each of the plurality of second lasers is independently triggered by irradiating particles in the flow with the first laser.

4. The flow cytometer according to any one of claims 1 to 2, wherein, The memory includes instructions stored thereon that, when executed by a processor, cause the processor to calculate the irradiation time of the second laser on the flowing stream in such a way as follows: The first laser is used to irradiate the particles in the flowing stream; In response to the illumination of particles by the first laser, light from the flowing stream is detected; and Calculate the time interval between particles irradiated by the second laser.

5. The flow cytometer according to claim 1, wherein, The beam deflector is selected from acousto-optic devices or electro-optic devices.

6. A method for use in a flow cytometer, comprising: The first laser continuously irradiates the flowing stream, which includes liquid. The flow is irradiated with a second laser at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with a first laser. A single photodetector coupled to a single optical fiber is used to detect light from each particle in the irradiated flow of the laser. and Calculate the irradiation time of the flow by the second laser; The wavelength of the light from the first laser is shorter than the wavelength of the light from the second laser; The method includes: The second laser is activated in response to the irradiation of the particles by the first laser; In response to the irradiation of particles by the first laser, the beam stop located in the beam path between the second laser and the flow stream is moved; or In response to the irradiation of particles by the first laser, a beam deflector is used to guide the light from the second laser into the flow.

7. The method of claim 6, comprising: The flow is continuously irradiated with a first laser; and The flow is irradiated with multiple second lasers at discrete intervals. Each discrete interval of the illumination by each of the plurality of second lasers is triggered by irradiating particles in the flow with the first laser.

8. The method according to claim 7, wherein, The plurality of second lasers are configured to irradiate the flow at a position downstream of the first laser, and each of the plurality of second lasers outputs light of a different wavelength.

9. The method according to any one of claims 7 to 8, wherein, The calculation of the irradiation time for each pair of flow streams in the second laser includes: The particles in the flowing stream are continuously irradiated by the first laser. In response to the illumination of particles by the first laser, light from the flowing stream is detected; and Calculate the time interval between each irradiated particle in the second laser.

10. The method according to claim 6, wherein, The beam deflector is selected from acousto-optic devices or electro-optic devices.

11. A non-transitory computer-readable storage medium comprising instructions stored thereon, the instructions comprising: An algorithm that continuously irradiates a flow containing liquid using a first laser; An algorithm that uses a second laser to irradiate the flow at discrete intervals, wherein each discrete interval irradiated by the second laser is triggered by irradiating particles in the flow with a first laser; An algorithm that uses a single photodetector coupled to a single optical fiber to detect light from each particle in an irradiated flow from a laser. and An algorithm for calculating the irradiation time of the second laser on the flowing stream; The wavelength of the light from the first laser is shorter than the wavelength of the light from the second laser; The instructions mentioned therein include: An algorithm for activating the second laser in response to the irradiation of particles by the first laser; An algorithm for moving a beam stop located in the beam path between the second laser and the flow stream in response to the irradiation of particles by the first laser; or An algorithm that guides light from a second laser into a flow stream using a beam deflector in response to irradiation of particles by a first laser.

12. The non-transitory computer-readable storage medium of claim 11, wherein the instructions comprise: An algorithm for continuously irradiating particles in a flowing stream with a first laser; An algorithm for detecting light from a flowing stream in response to the illumination of particles by a first laser; and An algorithm for calculating the time interval between particles irradiated by the second laser.

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