Strobe laser excitation system and method of use

Abstract

Aspects of the present disclosure include a system for illuminating particles in a flowstream. The system, according to certain embodiments, includes a light source having a first laser configured for continuous illumination of the flowstream and a second laser configured for illumination of the flowstream at discrete intervals, each discrete interval of illumination by the second laser being triggered by illumination of a particle in the flowstream with the first laser. Methods for illuminating a sample in a flowstream with the subject light source are also described. Computer-readable storage media for implementing the subject methods are provided. Kits having one or more lasers are also provided.

Description

[Background technology]

[0001] Characterization of analytes in biological fluids has become an important part of medical diagnosis and evaluation of a patient's overall health and wellness. Detecting analytes in biological fluids, such as human blood or blood-derived products, can provide results that can play a role in determining treatment protocols for patients with various disease states.

[0002] Flow cytometry is a technique used to characterize, and often sort, biological materials, such as cells in a blood sample, or particles of interest within another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir that contains a sheath fluid.

[0003] A flow cytometer transports particles (including cells) in a fluid sample as a cell stream into a flow cell while directing a sheath fluid toward the flow cell. To characterize components of the flow stream, the flow stream is illuminated with light. Changes in materials within the flow stream, such as morphology or the presence of fluorescent labels, can cause changes in the observed light, and these changes allow for characterization and separation.

[0004] To characterize components within a flow stream, light must be impinged on the flow stream and collected. Light sources in flow cytometers can vary from broad spectrum lamps to light emitting diodes as well as single wavelength lasers. The light source is aligned with the flow stream and the optical response from the illuminated particles is collected and quantified. Summary of the Invention

[0005] Aspects of the present disclosure include a system for illuminating particles in a flow stream. The system according to certain embodiments includes 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, each discrete interval of illumination by the second laser being triggered by illumination of a particle in the flow stream with the first laser. In some embodiments, the second laser is configured to illuminate a location on the flow stream downstream from the first laser. In certain embodiments, the first laser is a laser configured to illuminate the flow stream with light having a wavelength shorter than the wavelength of light of the second laser. In certain examples, the first laser illuminates the flow stream with light having a wavelength shorter than the light emitted from the particles in the flow stream. For example, the wavelength of light of the first laser may be less than the fluorescence of the particles (e.g., cells) in the flow stream.

[0006] In some embodiments, the system includes a first laser configured for continuous illumination of the flow stream and a plurality of lasers configured for illumination of the flow stream at discrete intervals, e.g., two or more lasers for illuminating the flow stream at discrete intervals, e.g., three or more lasers, and four or more lasers. In some examples, the plurality of lasers configured to illuminate the flow stream at discrete intervals are positioned to illuminate the flow stream downstream from the first laser. In particular examples, each of the lasers is configured to independently illuminate locations on the flow stream that are spaced apart from one another by 10 μm or less, including locations on the flow stream that are spaced apart by 9 μm or less, e.g., 8 μm or less, e.g., 7 μm or less, e.g., 6 μm or less, and 5 μm or less. In embodiments, each of the lasers is configured to independently illuminate the flow stream over a discrete interval ranging from 0.001 μs to 500 ms, e.g., 1 μs to 5000 μs.

[0007] In some embodiments, the system is configured to determine the timing and duration of illumination by each of the lasers. In some examples, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to calculate the timing of illumination of the flow stream by each of the lasers (i.e., when to begin discrete intervals of illumination). In other examples, the memory includes instructions that, when executed by the processor, cause the processor to calculate the duration of illumination of the flow stream by each of the lasers (i.e., the length of each discrete interval). In yet other examples, the memory includes instructions that, when executed by the processor, cause the processor to calculate the time interval between illumination of the flow stream by each of the lasers.

[0008] In some embodiments, the system is configured to turn on one or more downstream lasers in response to illumination of particles in the flow stream by the first laser. In some embodiments, the system includes a beam stop positioned in a beam path between the one or more downstream lasers and the flow stream, the beam stop configured to be moved in response to illumination of particles by the first laser. In some embodiments, the system includes a beam diverter positioned in a beam path between the one or more downstream lasers and the flow stream, the beam diverter configured to direct light into the flow stream in response to illumination of particles by the first laser. In some examples, the beam diverter is an acousto-optical device, such as an acousto-optical deflector (AOD) or an acousto-optical modulator (AOM). In some examples, the beam diverter is an electro-optical device, such as an electro-optical deflector (EOD) or an electro-optical modulator (EOM).

[0009] In some embodiments, the system includes a light detection system for detecting light from particles in the illuminated flow stream. The light detection system according to certain embodiments includes a light detector and an optical conditioning component configured to reduce the amount of light transmitted from the first laser to the light detector. In some examples, the optical conditioning component is a bandpass filter, such as a longpass filter, that transmits a spectral range of light longer than the illumination wavelength of the first laser. In other examples, the optical conditioning component is a dichroic mirror, such as a dichroic mirror, that transmits a spectral range of light longer than the illumination wavelength of the first laser and reflects a spectral range of light that includes the illumination wavelength of the first laser.

[0010] In embodiments, the photodetector is configured to detect light from the flow stream illuminated by each of the lasers. In some embodiments, the photodetector includes only a single light sensor component. In other embodiments, the photodetector is a photodetector array. In certain embodiments, the light detection system includes a light propagating component for transmitting light from the flow stream to the photodetector. In some examples, the light propagating component includes an optical fiber. In certain examples, the light propagating component includes a single optical fiber.

[0011] Aspects of the present disclosure also include methods for illuminating a sample in a flowstream. The method according to certain embodiments includes continuously illuminating the flowstream with a first laser and illuminating the flowstream with a second laser at discrete intervals. In embodiments, each discrete interval of illumination with the second laser is triggered by illumination of a particle in the flowstream with the first laser. In some embodiments, the method includes illuminating a location on the flowstream downstream from the first laser with the second laser. In certain embodiments, the wavelength of light of the first laser is shorter than the wavelength of light of the second laser.

[0012] In some embodiments, the method includes continuously irradiating the flow stream with a first laser and irradiating the flow stream with a plurality of lasers at discrete intervals, e.g., with two or more lasers, e.g., with three or more lasers, and four or more lasers. In certain examples, the flow stream is irradiated with the plurality of lasers at locations downstream from the irradiation of the flow stream by the first laser. In some examples, the stream is irradiated with the plurality of lasers at locations spaced apart from each other by 10 μm or less, e.g., 5 μm or less. In some examples, the stream is irradiated with each of the plurality of lasers at discrete intervals ranging from 0.001 μs to 500 ms, e.g., 1 μs to 5000 μs.

[0013] In certain embodiments, the method includes determining one or more of a timing and duration of illumination of the flow stream by each of the lasers. In some examples, the timing of illumination by one or more of the lasers (i.e., when illumination should begin) is determined. In other examples, the duration of illumination by one or more of the lasers (i.e., the length of each discrete interval) is determined. In still other examples, the time interval between illumination of the flow stream by each of the lasers is determined. In certain examples, the method includes sequentially illuminating a particle in the flow stream with each of the lasers, detecting light from the flow stream in response to illumination of the particle with each of the lasers, and calculating the time interval between illumination of the particle by each of the lasers.

[0014] In some embodiments, the method includes turning on one or more downstream lasers in response to the illumination of the particle in the flow stream by the first laser. In some embodiments, the method includes moving a beam stop positioned in a beam path between the one or more downstream lasers and the flow stream in response to the illumination of the particle by the first laser. In some embodiments, the method includes directing light from one or more of the downstream lasers into the flow stream with a beam diverter in response to the illumination of the particle by the first laser. In some examples, the beam diverter is an acousto-optical device such as an acousto-optical deflector (AOD) or an acousto-optical modulator (AOM). In some examples, the beam diverter is an electro-optical device such as an electro-optical deflector (EOD) or an electro-optical modulator (EOM).

[0015] The disclosed method also includes detecting light from the particles in the flow stream with a light detection system. In some embodiments, the light detection system includes a light detector and an optical conditioning component configured to reduce an amount of light transmitted from the first laser to the light detector. In some embodiments, the light from the flow stream is detected by the light detector through a band pass filter. In other embodiments, the light from the flow stream is detected by the light detector through a dichroic mirror.

[0016] A non-transitory computer readable storage medium for implementing the subject methods is also described. A non-transitory computer readable storage medium according to certain embodiments includes instructions stored thereon having an algorithm for continuously irradiating the flow stream with a first laser, an algorithm for irradiating the flow stream with a second laser at discrete intervals, where each discrete interval for irradiation by the second laser is triggered by irradiation of a particle in the flow stream with the first laser, an algorithm for detecting light from particles in the flow stream illuminated by each of the lasers with a single photodetector coupled to a single optical fiber, and an algorithm for calculating the timing of irradiation of the flow stream by each of the lasers. In some embodiments, the non-transitory computer readable storage medium includes instructions having an algorithm for continuously irradiating the flow stream with a first laser, and an algorithm for irradiating the flow stream with multiple lasers at discrete intervals, where each discrete interval for irradiation by each of the multiple lasers is triggered by irradiation of a particle in the flow stream with the first laser. In some examples, the non-transitory computer readable storage medium includes instructions having an algorithm for sequentially irradiating a particle in the flow stream with each of the lasers, an algorithm for detecting light from the flow stream in response to irradiating the particle with each of the lasers, and an algorithm for calculating a time interval between irradiating the particle with each of the lasers.

[0017] In some embodiments, the non-transitory computer readable storage medium includes instructions having an algorithm for turning on a second laser 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 moving a beam stop positioned 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 directing light from the second laser with a beam diverter into the flow stream in response to irradiation of a particle by the first laser.

[0018] Kits including one or more components of the subject system are also provided. Kits according to certain embodiments include one or more lasers, such as a laser configured for continuous illumination and a laser configured for illumination at discrete intervals. In some embodiments, the kit may include a switch for operating one or more of the lasers at discrete intervals (e.g., pulsing the laser). The kit may also include an optical conditioning component configured to reduce the passage of wavelengths of light of one or more of the lasers. In some examples, the optical conditioning component is a bandpass filter, such as a longpass filter, that transmits a spectral range of light longer than the irradiating wavelength of one or more of the lasers. In other examples, the optical conditioning component is a dichroic mirror, such as a dichroic mirror that transmits a spectral range of light longer than the irradiating wavelength of one or more of the lasers and reflects a spectral range of light including the wavelength of one or more of the lasers.

[0019] The kits may also include a photodetector array for detecting light from the flowstream. In certain embodiments, the support stage includes a motor, such as a stepper motor. The subject kits may also include an optical relay system, such as an optical fiber (e.g., a single optical fiber), for transmitting light from the sample in the flowstream to the detector. [Brief description of the drawings]

[0020] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, which include:

[0021] [Figure 1A] FIG. 1 illustrates a light source having a laser configured for continuous illumination and three lasers configured for illumination at discrete intervals, according to certain embodiments. [Figure 1B] FIG. 1 illustrates a light source having a first laser configured for continuous illumination of a flow stream and a second laser configured to be turned on in response to illumination of a particle by the first laser, in accordance with certain embodiments. [Figure 1C] FIG. 1 illustrates a light source having a first laser configured for continuous illumination of a flow stream, in accordance with certain embodiments, and a second laser configured to illuminate the flow stream when a beam stop is moved in response to illumination of a particle by the first laser. [Figure 1D] FIG. 1 illustrates a light source having a first laser configured for continuous illumination of a flow stream, in accordance with certain embodiments, and a second laser configured to illuminate the flow stream when a beam diverter redirects light to the flow stream in response to illumination of a particle by the first laser. [Diagram 2] FIG. 1 illustrates a light source having a laser configured for continuous illumination, three lasers configured for illumination at discrete intervals, and a light detection system for transmitting and measuring light with a single photodetector, in accordance with certain embodiments. [Diagram 3] FIG. 13 illustrates laser firing at discrete intervals by three lasers in response to a trigger signal from a continuously firing laser, according to certain embodiments. [Figure 4A] FIG. 1 is a functional block diagram illustrating a particle analysis system, in accordance with certain embodiments. [Figure 4B] FIG. 1 illustrates a flow cytometer, in accordance with certain embodiments. [Diagram 5]FIG. 2 illustrates the functionality of an example particle analyzer control system, in accordance with certain embodiments. [Figure 6A] FIG. 1 is a schematic diagram illustrating a particle sorter system, in accordance with certain embodiments. [Figure 6B] FIG. 1 is a schematic diagram illustrating a particle sorter system, in accordance with certain embodiments. [Figure 7] FIG. 1 is a block diagram illustrating a computing system in accordance with certain embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Aspects of the present disclosure include a system for illuminating particles in a flow stream. The system according to certain embodiments includes 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, each discrete interval of illumination by the second laser being triggered by illumination of a particle in the flow stream with the first laser. Methods for illuminating a sample in a flow stream with the subject light source are also described. Computer readable storage media for implementing the subject methods are provided. Kits having one or more lasers are also provided.

[0023] Before the present invention is described in more detail, it should be understood that the invention is not limited to particular embodiments described, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0024] Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within this stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0025] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically stated number, the number that is close to or approximately the unstated number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically stated number.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described below.

[0027] All publications and patents cited herein are incorporated by reference herein to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0028] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for use of a "negative" limitation.

[0029] As will be apparent to those skilled in the art upon reading this disclosure, each of the separate embodiments described and illustrated herein has separate components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0030] Although the apparatus and methods are described with functional descriptions for grammatical fluidity, it is expressly understood that unless expressly recited under 35 U.S.C. 35 U.S.C. 112, the claims should not necessarily be construed as limited by construction of "means" or "step" limitations, but should be accorded the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of legal equivalents, and if the claims are expressly recited under 35 U.S.C. 35 U.S.C. 112, they should be accorded the full legal equivalents under 35 U.S.C. 35 U.S.C.

[0031] As summarized above, the present disclosure provides a system and method for illuminating particles in a flow stream. In further describing the embodiments of the present disclosure, a system having a first laser configured for continuous illumination and a second laser configured for illumination at discrete intervals is first described in more detail. Then, a method for illuminating a sample in a flow stream and detecting light from particles in the flow stream is described. Kits having one or more components of the subject system are also provided.

[0032] System for irradiating particles in a flow stream - Patents.com Aspects of the present disclosure include a system for illuminating particles in a flowstream. The system according to certain embodiments includes a first laser configured for continuous illumination of the flowstream and a second laser configured for illumination of the flowstream at discrete intervals, each discrete interval of illumination by the second laser being triggered by illumination of particles in the flowstream with the first laser. As described herein, the term "continuous" is used herein in its conventional sense to refer to laser illumination of the flowstream that is constant and otherwise uninterrupted for the duration that a sample of interest is flowed through the flowstream. In some embodiments, the laser configured for continuous illumination is a laser that is not obscured (i.e., not intermittently blocked with a beam stop or obscuring component). In certain examples, continuous illumination of the flow stream with a laser includes maintaining a constant laser illumination intensity, including, for example, a laser illumination intensity that varies by 5% or less during the duration that the sample of interest is flowed through the flow stream, such as a laser illumination intensity that varies by 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 0.0001% or less. In certain embodiments, a laser configured for continuous illumination of a flow stream does not exhibit a change in intensity during the duration that the sample is flowed through the flow stream. The intensity of light output by a laser configured for continuous illumination can be measured with any convenient protocol, including, but not limited to, a scanning slit profiler, a charge-coupled device (CCD, e.g., an intensified charge-coupled device, ICCD), a positioning sensor, a power sensor (e.g., a thermopile power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector, among other types of light detectors.

[0033] In embodiments, the laser configured for continuous irradiation may be different and may 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 CO2 laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In another example, the laser configured for continuous irradiation is a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In yet another example, the laser for continuous irradiation of the target includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet another example, the system includes a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, a Y2O3 laser, or a cerium doped laser, and combinations thereof. In yet another example, the system includes a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or a frequency doubled or frequency tripled implementation of any of the above lasers.

[0034] The subject systems according to embodiments also include one or more lasers configured to irradiate the flow stream at discrete intervals. The term "discrete interval" is used herein in its conventional sense to refer to irradiation of the flow stream for a predetermined duration followed by a period during which the flow stream is not irradiated by the laser (e.g., by turning off the laser or by blocking the laser with a chopper, beam stop, etc.). In some embodiments, the laser is configured to irradiate the flow stream at discrete intervals of 0.001 μs or more, including, 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, for example, 1 μs or more, for example, 5 μs or more, for example, 10 μs or more, for example, 50 μs or more, for example, 100 μs or more, and 500 μs or more. In certain examples, the discrete intervals for irradiating the flow stream may be in the range of 0.0001 μs to 500 ms, including, 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 10 μs to 100 μs. The duration between each discrete interval of irradiation by each laser may be 0.001 μs or more, including, for example, 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, such as 1 μs or more, such as 5 μs or more, such as 10 μs or more, such as 50 μs or more, such as 100 μs or more, and 500 μs or more. For example, the duration between each discrete interval of irradiation by each laser may be in the range of 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 10 μs to 100 μs.

[0035] In some embodiments, the subject light source includes two or more lasers configured to irradiate the flow stream at discrete intervals, including, for example, three or more lasers, for example, four or more lasers, for example, five or more lasers, for example, ten or more lasers, for example, fifteen or more lasers, for example, twenty-five or more lasers, and fifty or more lasers configured to irradiate the flow stream at discrete intervals. To irradiate the flow stream at discrete intervals, each laser may be operatively coupled to one or more components to provide intermittent irradiation with each laser. Intermittent irradiation can be provided using any convenient protocol, such as an electronic switch for turning the laser on and off, such as a switch that is computer controlled and triggered based on a data signal (e.g., a received or input data signal), as described in more detail below. In some embodiments, the lasers are configured for irradiation at discrete intervals by intermittently exposing the laser beam of each laser to a beam chopper or beam stop.

[0036] In certain embodiments, each of the lasers is configured to irradiate the flowstream at discrete intervals at a location on the flowstream downstream from a location of irradiation by the laser configured for continuous irradiation. For example, in one example, the subject light source includes a first laser configured for continuous irradiation of the flowstream, a second laser configured to irradiate the flowstream at a location downstream from the first laser, a third laser configured to irradiate the flowstream at a location downstream from the second laser, and a fourth laser configured to irradiate the flowstream at a location downstream from the third laser. Depending on the flow rate of the flow stream and the distance between the locations of illumination by each laser, each of the lasers may be configured to independently illuminate the flow stream at a location that is 5 μm or more downstream from the location of illumination by the first laser, including cases where each laser is independently configured to illuminate the flow stream at discrete intervals, such as 6 μm or more, such as 7 μm or more, for example 8 μm or more, such as 9 μm or more, for example 10 μm or more, such as 15 μm or more, such as 25 μm or more, for example 50 μm or more, such as 100 μm or more, for example 250 μm or more, such as 500 μm or more, and where each laser is independently configured to illuminate the flow stream at a location that is 1000 μm or more downstream from the location of illumination by the first laser. For example, the location of irradiation of the flow stream by each laser may be a distance of 5 μm to 5000 μm downstream from the location of irradiation of the flow stream by the first laser, including, for example, 10 μm to 2500 μm, such as 25 μm to 1000 μm, such as 50 μm to 750 μm, such as 75 μm to 500 μm, and 100 μm to 250 μm.

[0037] 1A illustrates a light source having a laser configured for continuous illumination and three lasers configured for illumination at discrete intervals, according to certain embodiments. Light source 100a includes laser 101 configured for continuous illumination and lasers 102, 103, and 104 each positioned downstream from laser 101 along flow stream 107 and configured for illumination at discrete intervals. Light source 100 includes bandpass filter 105 configured to reduce the amount of light transmitted from continuously illuminating laser 101 to photodetector 106.

[0038] The distance between the illuminations on the flow stream by each of the lasers can vary, with the inter-illumination spacing being independently 0.0001 μm or more, including, for example, 0.0005 μm or more, such as 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 2 μm or more, such as 3 μm or more, such as 4 μm or more, such as 5 μm or more, such as 6 μm or more, such as 7 μm or more, such as 8 μm or more, such as 9 μm or more, and 10 μm or more. In certain examples, the lasers in the subject light source are configured to illuminate locations on the flow stream that are directly adjacent to one another (i.e., there is no inter-illumination space).

[0039] In embodiments, the laser configured for irradiation of the flow stream at discrete intervals may be different and may 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 CO2 laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In another example, the laser configured for continuous irradiation is a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In yet another example, the laser configured for continuous irradiation of the object includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet another example, the system includes a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a Y2O3 laser, or a cerium doped laser, and combinations thereof. In yet another example, the system includes a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or a frequency doubled or frequency tripled implementation of any of the above mentioned lasers. The laser may include any combination of several types of lasers. For example, in some embodiments, the subject 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 certain embodiments, the subject system includes an array of continuous wave diode lasers.

[0040] Depending on the desired wavelength of light to be generated in the output laser beam (e.g., for use in illuminating a sample in a flow stream), each laser may have a particular wavelength that varies from 200 nm to 1500 nm, including, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and 400 nm to 800 nm. In certain embodiments, the lasers of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser. In some embodiments, each of the lasers outputs a different wavelength of light. In certain instances, the lasers of the system are positioned such that each downstream laser outputs a longer wavelength of light. For example, if the system includes four lasers, the wavelength of light from the second laser is longer than the wavelength of light from the first laser, the wavelength of light from the third laser is longer than the wavelength of light from the second laser, and the wavelength of light from the fourth laser is longer than the wavelength of light from the third laser.

[0041] The light source may also include one or more optical conditioning components. The term "optical conditioning" is used herein in its conventional sense to refer to any device that can change the spatial width of the illumination or some other characteristic of the illumination from one or more of the lasers, such as, for example, the illumination direction, wavelength, beam width, beam intensity, focus, and pulse width. The optical conditioning protocol may be any convenient device that adjusts one or more characteristics of the laser, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimating protocols, and combinations thereof. In certain embodiments, the subject system includes one or more focusing lenses. In one example, the focusing lens may be a demagnifying lens. In another example, the focusing lens is a magnifying lens. In other embodiments, the subject system includes one or more mirrors. In yet other embodiments, the subject system includes an optical fiber. In some embodiments, the beams of light from each laser are combined by a beam combiner, such as a dichroic mirror beam combiner. In these embodiments, the beam combiner combines the light beams from each laser and propagates the light to the beam shaping component.

[0042] In certain embodiments, the light from each laser is propagated to the flow stream via a mirror component. In some examples, the mirror component may include a first mirror and a second mirror positioned to propagate the light from the first mirror to the flow stream. In embodiments, the second mirror is positioned to propagate the light from the first mirror at varying angles relative to the first mirror, including, for example, between 1° and 90°, for example, between 5° and 85°, for example, between 10° and 80°, for example, between 15° and 75°, for example, between 20° and 70°, for example, between 25° and 65°, and between 30° and 60°. In certain examples, the second mirror is positioned to propagate the light from the first mirror at a right angle. In other embodiments, the second mirror is positioned to propagate light from the first mirror at varying angles relative to the laser, including, for example, 1°-90°, for example, 5°-85°, for example, 10°-80°, for example, 15°-75°, for example, 20°-70°, for example, 25°-65°, and 30°-60°. In certain examples, the second mirror is positioned to propagate light perpendicular to the laser. In some embodiments, the second mirror is also a beam combiner configured to combine beams of light from two or more lasers. In these embodiments, the second mirror can be a dichroic mirror that selectively passes wavelengths of light, as desired.

[0043] In some embodiments, the optical conditioning components (e.g., beam stopper or beam chopper components) are movable. In some examples, the optical conditioning components are movable in two dimensions, such as in the XY plane. In other examples, the optical conditioning components are movable in three dimensions. In some embodiments, one or more optical conditioning components are configured to change an angle, such as tilting, relative to the laser. For example, the system may be configured to change the position of illumination on the flow stream by changing the angle of the mirror relative to the laser by 5° or more, including, for example, 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, and 75° or more.

[0044] When an optical conditioning component (e.g., one or more beam stoppers, beam choppers, etc.) is configured to move, the optical conditioning component may be configured to move continuously or in discrete intervals. In some embodiments, the movement of the optical conditioning component is continuous. In other embodiments, the optical conditioning component can be moved in discrete intervals, such as in increments of 0.01 microns or more, including increments of, for example, 0.05 microns or more, such as 0.1 microns or more, such as 0.5 microns or more, such as 1 micron or more, such as 10 microns or more, such as 100 microns or more, such as 500 microns or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, and 25 mm or more.

[0045] Any displacement protocol can be used to move the optical adjustment component structure, such as coupled to a movable support stage or directly coupled to a motor-actuated translation stage, a lead screw translation assembly, a geared translation device, such as those using stepper motors, servo motors, brushless electric motors, brushed DC motors, microstep drive motors, high resolution stepper motors, among other types of motors.

[0046] In certain embodiments, the laser configured for continuous illumination and one or more lasers configured for illumination at discrete intervals are provided by an optical beam generator that generates two or more beams of frequency-shifted light. In some examples, the optical beam generator includes a laser and a radio frequency generator configured to apply a radio frequency drive signal to an acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous wave laser. For example, the laser in the subject optical 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 CO2 laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof, a dye laser, such as a stilbene, coumarin, or rhodamine laser, a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe The laser may be a metal vapor laser, such as a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulim YAG laser, a ytterbium YAG laser, a Y2O3 laser, or a cerium doped laser, and combinations thereof.

[0047] The acousto-optic device can be any convenient acousto-optic protocol configured to frequency shift laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the subject system is configured to generate an angle-deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal can be applied to the acousto-optic device with any suitable high-frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0048] In an embodiment, the controller is configured to apply high frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams in the output laser beam, including being configured to apply, for example, three or more high frequency drive signals, for example, four or more high frequency drive signals, for example, five or more high frequency drive signals, for example, six or more high frequency drive signals, for example, seven or more high frequency drive signals, for example, eight or more high frequency drive signals, for example, nine or more high frequency drive signals, for example, ten or more high frequency drive signals, for example, fifteen or more high frequency drive signals, for example, twenty-five or more high frequency drive signals, for example, fifty or more high frequency drive signals, and one hundred or more high frequency drive signals.

[0049] In some examples, to generate an intensity profile of the angularly deflected laser beam within the output laser beam, the controller is configured to apply a high frequency drive signal having an amplitude that varies from about 0.005V to about 400V, such as from about 0.01V to about 300V, such as from about 0.05V to about 200V, such as from about 0.1V to about 100V, such as from about 0.5V to about 75V, such as from about 1V to about 50V, such as from about 2V to about 40V, such as from about 3V to about 30V, and from about 5V to about 25V. In some embodiments, each applied high frequency drive signal has a frequency of 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 from about 5 MHz to about 50 MHz.

[0050] In certain embodiments, the controller includes a processor having a memory operatively coupled thereto, the memory including instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam with a desired intensity profile. For example, the memory may include instructions for generating two or more angularly deflected laser beams having the same intensity, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more, and may include instructions for generating one hundred or more angularly deflected laser beams having the same intensity. In other embodiments, the memory may include instructions for generating two or more angularly deflected laser beams having different intensities, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more, and may include instructions for generating one hundred or more angularly deflected laser beams having different intensities.

[0051] In certain embodiments, the controller includes a processor having a memory operatively coupled thereto, the memory including instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center of the output laser beam along a horizontal axis. In these examples, the intensity of the angularly deflected laser beam at the center of the output beam can range from 0.1% to about 99% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis, including, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, and about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller includes a processor having a memory operatively coupled thereto, the memory including stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center of the output laser beam along a horizontal axis. In these examples, the intensity of the angularly deflected laser beam at the edge of the output beam can range from 0.1% to about 99% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis, including, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, and about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In yet another embodiment, the controller includes a processor having a memory operably coupled thereto, the memory including stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile that has a Gaussian distribution along a horizontal axis.In yet another embodiment, the controller includes a processor having a memory operatively coupled thereto, the memory including stored instructions that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0052] In embodiments, the subject optical beam generator may be configured to generate angularly polarized laser beams within the spatially separated output laser beam. Depending on the applied high frequency drive signal and the desired illumination profile of the output laser beam, the angularly polarized laser beams may be separated by 0.001 μm or more, including, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more, for example, 500 μm or more, for example, 1000 μm or more, and 5000 μm or more. In some embodiments, the system is configured to generate angularly polarized laser beams within the output laser beam that overlap adjacent angularly polarized laser beams along, for example, the horizontal axis of the output laser beam. The overlap between adjacent angularly deflected laser beams (e.g., beam spot overlap) can be 0.001 μm or more, including, for example, 0.005 μm or more overlap, for example, 0.01 μm or more overlap, for example, 0.05 μm or more overlap, for example, 0.1 μm or more overlap, for example, 0.5 μm or more overlap, for example, 1 μm or more overlap, for example, 5 μm or more overlap, for example, 10 μm or more overlap, and 100 μm or more overlap.

[0053] In particular examples, the light beam generator configured to generate two or more beams of frequency-shifted light includes a laser excitation module as described in U.S. Pat. Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0054] Each of the lasers in the subject systems may be positioned at any suitable distance from the flow stream, including, for example, at a distance of 0.001 mm or more from the flow stream, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, and 100 mm or more. Further, the light source may be configured to illuminate the sample at any suitable angle (e.g., relative to a normal axis of the flow stream), for example, at an angle ranging from 10° to 90°, including, for example, from 15° to 85°, for example, from 20° to 80°, for example, from 25° to 75°, and from 30° to 60°, for example, at an angle of 90°.

[0055] As discussed above, the light source of the subject system includes a first laser configured for continuous illumination of the flow stream and one or more lasers configured for illumination of the flow stream at discrete intervals, with each discrete interval for illumination by each of the lasers being triggered by illumination of a particle in the flow stream with a laser configured for continuous illumination. The term "triggered" is used herein in its conventional sense to refer to initiating illumination by one or more of the lasers configured for illumination at discrete intervals. In some embodiments, triggering illumination by the laser includes turning the laser from an off setting to an on setting and illuminating the flow stream with the laser. In other embodiments, triggering illumination by the laser includes moving a position or setting of an optical adjustment component, such as a beam stopper, to illuminate the flow stream with the laser. One or more of the lasers can be triggered using any convenient protocol, such as by detecting a particle being illuminated by the first laser with a photodetector (i.e., passing a continuous light beam of the first laser). In certain embodiments, the system includes a separate trigger detector operatively coupled to each of the other lasers such that when a particle is detected as being illuminated by the continuous laser, a trigger signal is output to each of the configured lasers for sufficient discrete intervals to initiate discrete intervals of illumination by one or more of the lasers.

[0056] In embodiments, the duration between when a particle is detected as being illuminated by the continuously illuminating laser and the beginning of a discrete interval of illumination by one or more of the lasers may vary depending on the flow rate of the flow stream and the distance between the locations of illumination on the flow stream. In some embodiments, the duration between illumination by the continuously illuminating laser and the beginning of a discrete interval of laser illumination may be 0.0001 μs or more, including, for example, 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, such as 1 μs or more, such as 2 μs or more, such as 3 μs or more, such as 4 μs or more, such as 5 μs or more, such as 6 μs or more, such as 7 μs or more, such as 8 μs or more, such as 9 μs or more, and 10 μs or more. In certain embodiments, the system is configured to delay the start of the discrete interval in response to output of the trigger signal, for example the delay is 0.0005 μs or more, such as 0.001 μs or more, for example 0.005 μs or more, such as 0.01 μs or more, for example 0.05 μs or more, such as 0.1 μs or more, for example 0.5 μs or more, such as 1 μs or more, for example 2 μs or more, such as 3 μs or more, for example 4 μs or more, such as 5 μs or more, for example 6 μs or more, such as 7 μs or more, for example 8 μs or more, such as 9 μs or more, and 10 μs or more.

[0057] In some embodiments, the system includes a processor having a memory operatively coupled thereto, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to calculate a timing to begin irradiating the flow stream with each of the lasers (i.e., when to begin discrete intervals of irradiation). In some examples, to calculate the timing of irradiation, the system is configured to sequentially irradiate particles flowing within the flow stream with all of the lasers of the light source, detect light from the flow stream in response to irradiating the particle with each of the lasers, and calculate a time of irradiation of the particle by each of the lasers. In certain examples, calculating the time to begin irradiation includes calculating a time interval between irradiation by each of the lasers.

[0058] In other embodiments, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to calculate a duration of illumination of the flowstream by each of the lasers (i.e., the length of each discrete interval). In some examples, to calculate the duration of illumination, the system is configured to sequentially illuminate particles flowing in the flowstream with all of the lasers of the light source, detect light from the flowstream in response to illumination of the particle with each of the lasers, and calculate the duration of the discrete interval of illumination by each laser. In these examples, the duration of each discrete interval may be calculated based on a location of illumination on the flowstream and a flow rate of the particle in the flowstream.

[0059] As described above, to illuminate the flow stream at discrete intervals, each laser may be operatively coupled to one or more components to provide intermittent illumination at each laser. In some embodiments, the system is configured to turn on one or more downstream lasers in response to illumination of particles in the flow stream by the first laser. Depending on the distance between the first laser and the one or more downstream lasers, each laser is configured to be independently turned on within 0.00001 μs or more, including, for example, within 0.00005 μs or more, for example, within 0.0001 μs or more, for example, within 0.0005 μs or more, for example, within 0.001 μs or more, for example, within 0.005 μs or more, for example, within 0.01 μs or more, for example, within 0.05 μs or more, for example, within 0.1 μs or more, for example, within 0.5 μs or more, and within 1 μs or more, in response to illumination by the first laser, depending on the distance between the first laser and the one or more downstream lasers. In certain examples, each laser is configured to be independently turned on within 0.00001 μs to 100 μs, including, for example, 0.00005 μs to 90 μs, for example, 0.0001 μs to 80 μs, for example, 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 1 μs to 10 μs, after irradiation of the particle by the first laser.

[0060] 1B shows a light source having a first laser configured for continuous illumination of a flow stream and a second laser configured to be turned on in response to illumination of a particle by the first laser, according to certain embodiments. Light source 100b includes laser 101a configured for continuous illumination of a flow stream in flow cell 107a and laser 102a positioned downstream of laser 101a. Laser 102a is in electrical communication with switch 102a1 that turns laser 102a on and off. Switch 102a1 is configured to turn laser 102a on when particle 110 is illuminated by laser 101a.

[0061] In some embodiments, the system includes a beam stop positioned in a beam path between one or more downstream lasers and the flow stream, the beam stop configured to be moved in response to irradiation of the particle by the first laser. Any displacement protocol can be used to move the beam stop, such as coupled to a movable support stage or directly coupled to a motorized translation stage, a lead screw translation assembly, a geared translation device, such as those using stepper motors, servo motors, brushless electric motors, brushed DC motors, microstep drive motors, high resolution stepper motors, among other types of motors. The beam stop may be moved within 0.00001 μs or more in response to illumination by the first laser, including within 0.00005 μs or more, such as within 0.0001 μs or more, such as within 0.0005 μs or more, such as within 0.001 μs or more, such as within 0.005 μs or more, such as within 0.01 μs or more, such as within 0.05 μs or more, such as within 0.1 μs or more, such as within 0.5 μs or more, and within 1 μs or more. In certain examples, the beam stop is moved out of the beam path of one or more downstream lasers within 0.00001 μs to 100 μs, including, for example, 0.00005 μs to 90 μs, for example, 0.0001 μs to 80 μs, for example, 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 1 μs to 10 μs, after irradiation of the particle by the first laser.

[0062] 1C illustrates a light source having a first laser configured for continuous illumination of the flow stream and a second laser configured to illuminate the flow stream when the beam stop is moved in response to illumination of the particle by the first laser, according to certain embodiments. Light source 100c includes laser 101a configured for continuous illumination of the flow stream in flow cell 107a and laser 103a positioned downstream of laser 101a. Light source 100c includes beam stop 103a1 positioned in the beam path between laser 103a and the flow stream. Beam stop 103a1 is configured to be moved out of the beam path between laser 103a and the flow stream when particle 110 is illuminated by laser 101a.

[0063] In some embodiments, the system includes a beam diverter positioned in a beam path between one or more downstream lasers. In embodiments, the beam diverter is configured to divert light from one or more downstream lasers away from the flow stream until triggered to redirect the light from the lasers toward the flow stream. In some examples, the beam diverter is configured to direct light toward the flow stream in response to illumination of a particle by the first laser. For example, a data signal may be generated in response to illumination of a particle in the flow stream by the first laser, and the beam diverter is configured to direct the diverted laser light toward the flow stream in response to the generated data signal.

[0064] In some examples, the beam diverter 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 direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator sufficient to direct light from the laser into the flow stream. In other examples, the beam diverter 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 a current or an applied voltage to an electro-optic device sufficient to direct light from the laser into the flow stream. In some embodiments, the beam diverter is configured to redirect the beam path of one or more downstream lasers in response to illumination by the first laser to within 0.00001 μs or more, including, for example, within 0.00005 μs or more, for example, within 0.0001 μs or more, for example, within 0.0005 μs or more, for example, within 0.001 μs or more, for example, within 0.005 μs or more, for example, within 0.01 μs or more, for example, within 0.05 μs or more, for example, within 0.1 μs or more, for example, within 0.5 μs or more, and within 1 μs or more. In certain examples, the beam diverter is configured to redirect the beam path of one or more downstream lasers within 0.00001 μs to 100 μs, including, for example, 0.00005 μs to 90 μs, for example, 0.0001 μs to 80 μs, for example, 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 1 μs to 10 μs, after irradiation of the particle by the first laser.

[0065] FIG. 1D shows a light source having a first laser configured for continuous illumination of the flow stream and a second laser configured to illuminate the flow stream when a beam diverter redirects light to the flow stream in response to illumination of the particle by the first laser, according to certain embodiments. Light source 100d includes laser 101a configured for continuous illumination of the flow stream in flow cell 107a and laser 104a positioned downstream of laser 101a. Light source 100d includes beam diverter 104a1 positioned in a beam path between laser 104a and the flow stream. The beam diverter is configured to divert the beam path of laser 104a from the flow stream, for example, by acousto-optical deflection (e.g., with an AOD) or electro-optical deflection (e.g., with an EOM). When particle 110 is illuminated by laser 101a, the beam diverter is configured to redirect the beam path of laser 104a to the flow stream.

[0066] In embodiments, light from particles in the flow stream illuminated with the subject light source is transmitted to a light detection system. The light detection system may include one or more light detectors. The light detectors in the subject systems may be any convenient light detection protocol, including, but not limited to, light sensors or light detectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or photodiodes, as well as combinations thereof, among other light detectors. The light detection system for measuring light from particles in the flow stream may include one or more light detectors, including, for example, two or more light detectors, for example, three or more light detectors, for example, four or more light detectors, for example, five or more light detectors, for example, ten or more light detectors, for example, twenty-five or more light detectors, and fifty or more light detectors.

[0067] In certain embodiments, the subject systems include a photodiode array having two or more photodiodes, e.g., two or more photodiodes, e.g., three or more, e.g., five or more, and ten or more photodiodes, each photodiode having a size of, e.g., 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2 Including 0.01cm 2 ~10cm 2 The active detection surface area of ​​each region may range from 0.1 to 1.0 mm.

[0068] In embodiments of the present disclosure, the subject photodetectors are configured to measure light collected at one or more wavelengths, including measuring light from particles in the flow stream at, for example, two or more wavelengths, for example, at five or more different wavelengths, for example, at 10 or more different wavelengths, for example, at 25 or more different wavelengths, for example, at 50 or more different wavelengths, for example, at 100 or more different wavelengths, for example, at 200 or more different wavelengths, for example, at 300 or more different wavelengths, and at 400 or more different wavelengths.

[0069] In embodiments, the light detectors are configured to measure light continuously or at discrete intervals. In some examples, the subject detectors are configured to make measurements of the collected light continuously. In other examples, the subject detectors are configured to make measurements at discrete intervals, such as measuring light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at some other interval.

[0070] Each optical detector may be positioned at any suitable distance from the flow stream so long as a usable optical signal is detectable. For example, detectors in the subject systems may be positioned 1 mm or more from the flow stream, including, for example, 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, such as 50 mm or more, such as 100 mm or more, such as 150 mm or more, such as 250 mm or more, and 500 mm or more from the flow stream. Detectors may also be positioned at any angle from the flow stream. For example, detectors may be angled from 10° to 90°, including, for example, 15° to 85°, such as 20° to 80°, such as 25° to 75°, and 30° to 60°, relative to the normal axis of the flow stream. In some examples, one or more detectors are positioned at 30° to 60° relative to the normal axis of the flow stream.

[0071] In embodiments, the system is configured to detect forward scattered light, side scattered light, emitted light, transmitted light, or a combination thereof. In certain embodiments, the light signal from the illuminated flow stream may be detected by one or more detectors configured as forward scatter detectors. In these embodiments, the forward scatter detector is positioned on the opposite side of the flow stream from the light source and is positioned to collect and detect forward propagating (e.g., scattered) light.

[0072] In certain embodiments, the system includes a single photodetector configured to detect light from each of the lasers of the light source, in these embodiments, the photodetector detects light from particles in the flow stream illuminated with the laser configured for continuous illumination and with each laser configured for illumination at discrete intervals.

[0073] In some embodiments, the light detection system includes an optical conditioning component configured to reduce the amount of light transmitted from the laser configured for continuous illumination to one or more light detectors. In these embodiments, the optical conditioning component is configured to suppress, reduce, or limit the propagation of at least one or more wavelengths of light (e.g., one or more of the wavelengths of light of the laser configured for continuous illumination) from the sample to the active surface of the light detector. The optical conditioning component may include an optical component that limits the propagation of one or more different wavelengths of light, including limiting the propagation of, for example, 5 or more, for example 10 or more, for example 25 or more, for example 50 or more, for example 100 or more, for example 200 or more, for example 300 or more, and 500 or more different wavelengths of light. For example, in some embodiments, the optical conditioning component is a bandpass filter, such as a longpass filter that transmits a spectral range of light longer than the illumination wavelength of the first laser. In other embodiments, the optical conditioning component is a dichroic mirror, such as a dichroic mirror that transmits a spectral range of light longer than the illumination wavelength of the first laser and reflects a spectral range of light that includes the illumination wavelength of the first laser.

[0074] In some embodiments, the system includes an optical collection system for collecting light from the flow stream and directing it to the optical detection system. The optical collection system may be physically coupled to the optical detection system, for example with an adhesive, co-molded with the optical detection system, or integrated into the optical detection system. In certain embodiments, the optical collection system and the optical detection system are integrated into a single unit. In other embodiments, the optical collection system is coupled to the optical detection system with a connector, for example with a hook-and-loop fastener, a magnet, a latch, a notch, a countersink, a counterbore, a groove, a pin, a tether, a hinge, Velcro, a non-permanent adhesive, or a combination thereof.

[0075] In other embodiments, the optical detection system and the optical collection system are in optical communication but not in physical contact, for example, the optical collection system may be positioned 0.001 mm or more from the optical detection system, including, for example, 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 100 mm or more.

[0076] In some embodiments, the optical collection system includes an optical fiber. For example, in some instances, the optical collection system can be a fiber optic relay bundle, and the light is transmitted to the optical detection system through the fiber optic relay bundle. In certain embodiments, the optical collection unit is a single optical fiber configured to transmit light from illumination by each of the lasers to a single optical detector in the optical detection system. In these embodiments, the locations of illumination spanned by the lasers on the flow stream are equal to or less than the diameter of a single optical fiber configured to collect light from the flow stream. For example, the lasers in the subject systems can be configured to illuminate locations on the flow stream that span 100 μm or less, including, for example, 90 μm or less, such as 80 μm or less, such as 70 μm or less, such as 60 μm or less, and 50 μm or less, and the single optical fiber can have a diameter sufficient to collect light from each of the locations illuminated by the laser, including, for example, 50 μm or more in diameter, such as 60 μm or more, such as 70 μm or more, such as 80 μm or more, such as 90 μm or more, and where the single optical fiber configured to collect light from the illuminated flow stream is 100 μm or more.

[0077] 2 illustrates a light source having a laser configured for continuous illumination, three lasers configured for illumination at discrete intervals, and a light detection system for transmitting and measuring the light with a single photodetector, according to certain embodiments. Light source 200 includes laser 201 configured for continuous illumination, and lasers 202, 203, and 204, each positioned downstream from laser 201 along flow stream 207 and configured for illumination at discrete intervals. Lasers 201, 202, 203, and 204 are positioned approximately adjacent to one another such that light from flow stream 207 can be collected and transmitted to a single photodetector 206 using a single optical fiber 205, reducing the number of detector channels required to detect light from the sample.

[0078] In other embodiments, the optical collection system is a free space optical relay system. For example, the free space optical relay system may include a housing having a proximal end and a distal end, the proximal end being coupled to the optical detection system. The free space optical relay system may include any combination of different optical components, such as one or more lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof.

[0079] 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 testing region may be used, and in some embodiments, the flow cell is a cylindrical flow cell, a frusto-conical flow cell, or a flow cell that includes a proximal cylindrical portion defining a longitudinal axis and a distal frusto-conical portion terminating in a flat surface having an orifice that is transverse to the longitudinal axis.

[0080] In some embodiments, the sample flow stream originates from an orifice at the distal end of the flow cell. Depending on the desired characteristics of the flow stream, the flow cell orifice may be of any suitable shape, including, but not limited to, rectilinear cross-sectional shapes, such as, for example, square, rectangular, trapezoidal, triangular, hexagonal, curvilinear cross-sectional shapes, such as, for example, circular, elliptical, as well as irregular shapes, such as, for example, a parabolic bottom joined to a planar top. In certain embodiments, the subject flow cell has a circular orifice. The size of the nozzle orifice may vary in some embodiments from 1 μm to 20,000 μm, including, for example, from 2 μm to 17,500 μm, for example, from 5 μm to 15,000 μm, for example, from 10 μm to 12,500 μm, for example, from 15 μm to 10,000 μm, for example, from 25 μm to 7,500 μm, for example, from 50 μm to 5,000 μm, for example, from 75 μm to 1,000 μm, for example, from 100 μm to 750 μm, and from 150 μm to 500 μm. In a particular embodiment, the nozzle orifice is 100 μm.

[0081] In some embodiments, the flow cell includes a sample injection port configured to provide a sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable flow of the sample to the internal chamber of the flow cell. Depending on the desired characteristics of the flow stream, the rate of the sample delivered by the sample injection port to the flow cell chamber can be 1 μL / min or more, including, for example, 2 μL / min or more, such as 3 μL / min or more, such as 5 μL / min or more, such as 10 μL / min or more, such as 15 μL / min or more, such as 25 μL / min or more, such as 50 μL / min or more, and 100 μL / min or more, and in some examples, the rate of the sample delivered by the sample injection port to the flow cell chamber is 1 μL / sec or more, including, for example, 2 μL / sec or more, such as 3 μL / sec or more, such as 5 μL / sec or more, such as 10 μL / sec or more, such as 15 μL / sec or more, such as 25 μL / sec or more, such as 50 μL / sec or more, and 100 μL / sec or more.

[0082] The sample injection port may be an orifice located in the wall of the internal chamber or may be a conduit located at the proximal end of the internal chamber. When the sample injection port is an orifice located in the wall of the internal chamber, the sample injection port orifice may be of any suitable shape, including, but not limited to, rectilinear cross-sectional shapes, such as, for example, square, rectangular, trapezoidal, triangular, hexagonal, curved cross-sectional shapes, such as, for example, circular, elliptical, and irregular shapes, such as, for example, a parabolic bottom joined to a planar top. In certain embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in certain examples has an opening ranging from 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, such as, for example, 0.5 mm to 2.5 mm, such as, for example, 0.75 mm to 2.25 mm, such as, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, such as, for example, 1.5 mm.

[0083] In a particular example, the sample injection port is a conduit positioned at the proximal end of the flow cell internal chamber. For example, the sample injection port can be a conduit positioned to have the orifice of the sample injection port aligned with the flow cell orifice. When the sample injection port is a conduit positioned in line with the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, including, but not limited to, rectilinear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom joined to a planar top. The orifice of the conduit may vary in shape, with certain examples having openings ranging from 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm, and 1.25 mm to 1.75 mm, such as 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a beveled tip having a bevel angle ranging from 1° to 10°, including, for example, a bevel angle of 2° to 9°, such as 3° to 8°, such as 4° to 7°, and 5°.

[0084] In some embodiments, the flow cell also includes a sheath fluid injection port configured to provide a sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to provide a flow of sheath fluid to the flow cell interior chamber, e.g., in conjunction with the sample, to generate a laminated flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of the sheath fluid delivered by the sheath fluid injection system to the flow cell chamber can be 25 μL / sec or more, including, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, and 2500 μL / sec or more.

[0085] In some embodiments, the sheath fluid injection port is an orifice located in the wall of the internal chamber. The sheath fluid injection port orifice may be of any suitable shape, including, but not limited to, rectilinear cross-sectional shapes, such as, for example, square, rectangular, trapezoidal, triangular, hexagonal, curved cross-sectional shapes, such as, for example, circular, elliptical, and irregular shapes, such as, for example, a parabolic bottom coupled to a planar top. The size of the sample injection port orifice may vary depending on the shape, with certain examples having openings ranging from 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, such as, for example, 0.5 mm to 2.5 mm, such as, for example, 0.75 mm to 2.25 mm, such as, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, such as, for example, 1.5 mm.

[0086] In some embodiments, the system further includes a pump in fluid communication with the flow cell for propagating the flow stream through the flow cell. Any convenient fluid pumping protocol may be used to control the flow of the flow stream through the flow cell. In certain examples, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in the subject system is configured to convey fluid through the flow cell at a rate suitable for multiphoton counting of light from the sample in the flow stream. In some examples, the rate of sample flow in the flow cell is 1 nL / min or more, including, for example, 2 nL / min or more, such as 3 nL / min or more, such as 5 nL / min or more, such as 10 nL / min or more, such as 25 nL / min or more, such as 50 nL / min or more, such as 75 nL / min or more, such as 100 nL / min or more, such as 250 nL / min or more, such as 500 nL / min or more, such as 750 nL / min or more, and 1000 nL / min or more. For example, the system may include a pump configured to flow the sample through the flow cell at a rate ranging from 1 nL / min to 500 nL / min, including, for example, from 1 nL / min to 250 nL / min, for example, from 1 nL / min to 100 nL / min, for example, from 2 nL / min to 90 nL / min, for example, from 3 nL / min to 80 nL / min, for example, from 4 nL / min to 70 nL / min, for example, from 5 nL / min to 60 nL / min, and from 10 nL / min to 50 nL / min. In certain embodiments, the flow rate of the flow stream is between 5 nL / min to 6 nL / min.

[0087] In certain embodiments, the subject system is a flow cytometry system that uses the light detection system described above to detect light emitted by a sample in a flow stream. In certain embodiments, the subject system is a flow cytometry system. Suitable flow cytometry systems include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo,et al. (2012) Ann Clin Biochem. Jan; 49(pt1):17-28; Linden,et. al., Semin Throm Hemost. 2004 Oct; 30(5):502-11; Alison,et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig,et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are incorporated herein by reference.In particular examples, flow cytometry systems of interest include BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortess™ X-20 flow cytometer, and BD Biosciences FACSCalibur™ flow cytometer, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, and BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, and BD and the Biosciences FACSMelody™ cell sorter.

[0088] In some embodiments, the subject particle sorting systems are configured in accordance with U.S. Pat. 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, 8,233,146, 8,140,300, 8,250,410, 8,370,420, 8,470,430, 8,520,440, 8,670,450, 8,770,460, 8,870,470, 8,970,595, 8,870,490, 8,100,590, 8,120,590, 8,250,590, 8,370,420, 8,470,430, 8,520,590, 8,670,450, 8,770,460, 8,870,470, 8,970,590, 8,870,470, 8,970,590, 8,870,470, 8,100,590, 8,120,590, 8,130,590, 8,140,590, 8,150,590, 8,160,590, 8,170,590, 8,180,590, 8 Nos. 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, the disclosures of which are incorporated herein by reference in their entireties.

[0089] In certain embodiments, the subject system is a flow cytometry system having an excitation module that uses radio frequency multiplexing excitation to generate multiple frequency-shifted beams of light. In these embodiments, the laser light generator may include multiple lasers and one or more acousto-optical components (e.g., acousto-optical polarizers, acousto-optical frequency shifters) to generate multiple frequency-shifted comb beams. One or more of the frequency-shifted comb beams and the local oscillator beam may be configured to be received by a beam shaping component as described herein to generate one or more beams of frequency-shifted light having a substantially constant intensity profile. In some cases, the subject system is a flow cytometry system having a laser excitation module as described in U.S. Pat. Nos. 9,423,353, 9,784,661, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0090] In some embodiments, the subject system includes a particle analysis system that can be used to analyze and characterize particles with or without physical sorting of the particles into a collection vessel. FIG. 4A shows a functional block diagram of an example of a particle analysis system. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 shown in FIG. 4A can be configured to perform the methods described herein, in whole or in part. The particle analysis system 401 includes a fluidics system 402. The fluidics system 402 can include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, within which the particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0091] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. In some implementations, detection may include detecting light or one or more other characteristics of the particle 403 as it passes through the monitoring area 407. One detection station 408 with one monitoring area 407 is shown in FIG. 4A. Some implementations of the particle analysis system 401 may include multiple detection stations. Additionally, some detection stations may monitor more than one area.

[0092] Each signal is assigned a signal value to form a data point for each particle. As explained above, this data may be referred to as event data. The data points may be multi-dimensional data points that include values ​​of each property measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0093] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operatively associated with the fluidics system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the Poisson distribution and the number of data points collected by the detection system 404 during the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. Additionally, the control system 406 may compare the experimental signal frequency to that of the calculated signal frequency or a predetermined signal frequency.

[0094] 4B shows a system 400 for flow cytometry, according to an exemplary embodiment of the invention. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. Flow cytometer 410 includes one or more excitation lasers 415a-c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-g, one or more bandpass filters 450a-e, one or more longpass ("LP") filters 455a, 455b, and one or more fluorescence detectors 460a-f.

[0095] The pump lasers 415a-415c emit light in the form of laser beams. In the exemplary system of FIG. 4B, the wavelengths of the laser beams emitted from the pump lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first directed through one or more of the beam splitters 445a and 445b. The beam splitter 445a transmits 488 nm light and reflects 633 nm light. The beam splitter 445b transmits UV light (light having a wavelength in the range of 10-400 nm) and reflects 488 nm and 633 nm light.

[0096] The laser beam is then directed to a focusing lens 420, which focuses the beam into a portion of the fluid stream where the sample particles are located in a flow chamber 425. The flow chamber is the part of a fluidics system that directs particles in the stream, typically one at a time, into the focused laser beam for inspection. The flow chamber may comprise a flow cell in a benchtop cytometer or a nozzle tip in a stream-in-air cytometer.

[0097] Light from the laser beam interacts with particles in the sample by diffraction, refraction, reflection, scattering, and absorption, with re-emission at a variety of different wavelengths depending on the particle's properties, such as the particle's size, internal structure, and the presence of one or more fluorescent molecules bound to or naturally present on or within the particle. The fluorescent emission, as well as the diffracted, refracted, reflected, and scattered light, may be sent through one or more of beam splitters 445a-g, bandpass filters 450a-e, longpass filters 455a, 455b, and fluorescence collection lens 440 to one or more of a forward scatter detector 430, a side scatter detector 435, and one or more fluorescence detectors 460a-f.

[0098] The fluorescence collection lens 440 collects light emitted from the particle-laser beam interaction and sends the light towards one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths of light to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on each side of the center of the spectral band, i.e., from 500 nm to 520 nm. Shortpass filters pass light with wavelengths equal to or shorter than the specified wavelength. Longpass filters, such as longpass filters 455a-455b, pass light with wavelengths equal to or longer than the specified wavelength of light. For example, longpass filter 455a, a 670 nm longpass filter, passes light with wavelengths equal to or longer than 670 nm. Filters are often selected to optimize the specificity of the detector to a particular fluorochrome. The filters can be configured so that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0099] Beam splitters direct light of different wavelengths in different directions. Beam splitters may be characterized by filter properties such as short pass and long pass. For example, beam splitter 445g is a 620 SP beam splitter, meaning that beam splitter 445g passes light with wavelengths of 620 nm or shorter and reflects light with wavelengths longer than 620 nm in different directions. In one embodiment, beam splitters 445a-445g may comprise optical mirrors, such as dichroic mirrors.

[0100] The forward scatter detector 430 is positioned slightly off-axis from the direct beam through the flow cell and is configured to detect diffracted light, excitation light traveling mostly in a forward direction through or around the particle. The intensity of light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector may include a photodiode. The side scatter detector 435 is configured to detect refracted and reflected light from the surface and internal structure of the particle, which tends to increase with increasing structural complexity of the particle. Fluorescent emission from fluorescent molecules associated with the particle may be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected by the forward scatter detector 430, the side scatter detector 435, and the fluorescence detector may be converted to electronic signals (voltage) by the detectors. This data may provide information about the sample.

[0101] Those skilled in the art will recognize that a flow cytometer according to an embodiment of the present invention is not limited to the flow cytometer shown in Figure 4B, but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and in a variety of different configurations.

[0102] During operation, cytometer operation is controlled by controller / processor 490, and measurement data from the detectors may be stored in memory 495 and processed by controller / processor 490. Although not explicitly shown, controller / processor 190 is coupled to the detectors to receive output signals from the detectors, and may also be coupled to electrical and electromechanical components of flow cytometer 400 to control lasers, fluid flow parameters, and the like. Input / output (I / O) functionality 497 may also be provided within the system. Memory 495, controller / processor 490, and I / O 497 may be provided entirely as an integral part of flow cytometer 410. In such an embodiment, a display may also form part of I / O functionality 497 for presenting experimental data to a user of cytometer 400. Alternatively, memory 495 and controller / processor 490, as well as some or all of the I / O functionality, may be part of one or more external devices, such as a general purpose computer. In some embodiments, some or all of memory 495 and controller / processor 490 may be in wireless or wired communication with cytometer 410. Controller / processor 490, together with memory 495 and I / O 497, may be configured to perform a variety of functions related to the preparation and analysis of flow cytometer experiments.

[0103] The system shown in FIG. 4B includes six different detectors that detect fluorescent light in six different wavelength bands (sometimes referred to herein as “filter windows” for a given detector) defined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used for a flow cytometer experiment emit light in their own characteristic wavelength bands. The particular fluorescent labels and their associated fluorescent emission bands used in the experiment may be selected to generally match the filter windows of the detectors. However, as more detectors are provided and more labels are utilized, perfect correspondence between filter windows and fluorescent emission spectra is not possible. It is generally true that while the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of one particular detector, a portion of the emission spectrum of that label also overlaps with the filter window of one or more other detectors. This may be referred to as spillover. The I / O 497 may be configured to receive data regarding a flow cytometer experiment having a panel of fluorescent labels and a plurality of cell populations having multiple markers, each cell population having a subset of the multiple markers. I / O 497 may also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experimental data, such as label spectral characteristics and flow cytometer configuration data, may also be stored in memory 495. Controller / processor 490 may be configured to evaluate the assignment of one or more of the labels to markers.

[0104] 5 shows a functional block diagram of an example of a particle analyzer control system for analyzing and displaying biological events, such as an analysis controller 500. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of the biological events.

[0105] The particle analyzer or sorting system 502 may be configured to acquire biological event data. For example, a flow cytometer may generate flow cytometry event data. The particle analyzer 502 may be configured to provide the biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel.

[0106] The analysis controller 500 may be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 may include flow cytometry event data. The analysis controller 500 may be configured to provide a graphical display including a first plot of the biological event data on a display device 506. The analysis controller 500 may be further configured to render a region of interest as a gate around a population of the biological event data shown by the display device 506, e.g., overlaid on the first plot. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest plotted on a single parameter histogram or bivariate plot. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0107] Analysis controller 500 may further be configured to display biological event data on display device 506 within a gate differently than other events in the biological event data outside the gate. For example, analysis controller 500 may be configured to render the colors of biological event data contained within a gate differently than the colors of biological event data outside the gate. Display device 506 may be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0108] The analysis controller 500 may be configured to receive a gate selection signal from a first input device that identifies a gate. For example, the first input device may be implemented as a mouse 510. The mouse 510 may initiate a gate selection signal to the analysis controller 500 that identifies a gate to be displayed on or manipulated via the display device 506 (e.g., by clicking on or in the desired gate when the cursor is positioned at the desired gate). In some implementations, the first device may be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices may include multiple input functions. In such implementations, each input function may be considered an input device. For example, as shown in FIG. 5, the mouse 510 may include a right mouse button and a left mouse button, each of which may generate a trigger event.

[0109] The trigger event may cause the analysis controller 500 to change the way the data is displayed, what portion of the data is actually displayed on the display device 506, and / or provide input for further processing, such as selection of a population of interest for particle sorting.

[0110] In some embodiments, the analysis controller 500 may be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 may be further configured to automatically modify the visualization of the plot to facilitate the gating process. The modification may be based on a particular distribution of the biological event data received by the analysis controller 500.

[0111] The analysis controller 500 may be connected to a storage device 504. The storage device 504 may be configured to receive and store biological event data from the analysis controller 500. The storage device 504 may also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 may be further configured to enable retrieval of biological event data, such as flow cytometry event data, by the analysis controller 500.

[0112] The display device 506 may be configured to receive display data from the analysis controller 500. The display data may include a plot of the biological event data and a gate outlining an area of ​​the plot. The display device 506 may be further configured to modify the presented information according to input received from the analysis controller 500 in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0113] In some implementations, the analysis controller 500 may generate a user interface for receiving example events for filtering. For example, the user interface may include controls for receiving example events or example images. The example events or example images, or example gates, may be provided prior to collection of event data for the sample or based on an initial set of events for a portion of the sample.

[0114] In some embodiments, the subject system includes a particle sorter system. FIG. 6A is a schematic diagram of a particle sorter system 600 (e.g., particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorter system 600 is a cell sorter system. As shown in FIG. 6A, a droplet forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, may include, 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 stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are aligned in a single file across a monitoring area 611 (e.g., where a laser stream intersects) and are illuminated by an illumination source 612 (e.g., a laser). Vibration of the droplet forming transducer 602 causes the moving fluid column 608 to break up into a number of droplets 610 , some of which contain particles 609 .

[0115] In operation, the detection station 614 (e.g., an event detector) identifies when a particle of interest (or cell of interest) crosses the monitoring area 611. The detection station 614 feeds a timing circuit 628, which in turn feeds a flash charge circuit 630. At the droplet break-off point, signaled by a timed droplet delay (Δt), a flash charge is applied to the moving fluid column 608, resulting in the droplet of interest becoming charged. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplets are then sorted by activating a deflection plate (not shown) to deflect the droplets into a container such as a collection tube or a multi-well or microwell sample plate where a well or microwell may be associated with a particular droplet of interest. As shown in FIG. 6A, the droplets may be collected in a drain container 638.

[0116] The detection system 616 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal as a particle of interest passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Pat. No. 7,679,039, which is incorporated by reference herein in its entirety. The detection system 616 allows the instrument to accurately calculate the position of each detected particle in the droplet. The detection system 616 feeds the amplitude 620 and / or phase 618 signals, which then feed them (via amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included within a control system.

[0117] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled with a memory configured to store the detected events and the sorting decisions based thereon. The sorting decisions can be included in the particle event data. In some implementations, the detection system 616 and detection station 614 can be implemented as a single detection unit or can be communicatively coupled such that event measurements can be collected by one of the detection system 616 or detection station 614 and provided to a non-collection element.

[0118] FIG. 6B is a schematic diagram of a particle sorter system according to one embodiment presented herein. The particle sorter system 600 shown in FIG. 6B includes deflection plates 652 and 654. An electric charge is applied via a stream charging wire in the barb. This results in a stream of droplets 610 containing particles 610 for analysis. The particles may be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The particle information is analyzed, such as by sorting electronics or other detection systems (not shown in FIG. 6B). The deflection plates 652 and 654 may be independently controlled to attract or repel the charged droplets and direct the droplets toward a desired collection vessel (e.g., one of 672, 674, 676, or 678). As shown in FIG. 6B, the deflection plates 652 and 654 may be controlled to direct the particles along a first path 662 toward vessel 674 or along a second path 668 toward vessel 678. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflector may allow the particle to continue along flow path 664. Such uncharged droplets may be diverted into a waste container, such as via aspirator 670.

[0119] Sorting electronics may be included to initiate measurement collection, receive the fluorescent signal of the particles, and determine how to adjust the deflection plates to cause particle sorting. Exemplary implementations of the embodiment shown in Figure 6B include the BD FACSAria™ line of flow cytometers marketed by Becton, Dickinson and Company (Franklin Lakes, NJ).

[0120] Computer Control System Aspects of the present disclosure further include a computer control system, the system further including one or more computers for full or partial automation. In some embodiments, the system includes a computer having a computer readable storage medium having a computer program stored thereon, the computer program including instructions, when loaded into the computer, for continuously irradiating the flow stream with a first laser and irradiating the flow stream at discrete intervals with a second laser. In some embodiments, the computer program includes instructions for triggering each discrete interval for irradiation by the second laser in response to irradiating particles in the flow stream with the first laser.

[0121] In some embodiments, a computer control system configured for full or partial automation includes a memory having stored instructions that, when executed by a processor, cause the processor to calculate the timing to begin irradiating the flow stream with each of the lasers (i.e., when to begin a discrete interval of irradiation.) In other embodiments, the system includes a processor having a memory operatively coupled thereto, the memory having stored instructions that, when executed by the processor, cause the processor to calculate the duration of irradiating the flow stream with each of the lasers (i.e., the length of each discrete interval).

[0122] In an embodiment, the system includes an input module, a processing module, and an output module. The subject systems may include both hardware and software components, and the hardware components may take the form of one or more platforms, for example in the form of servers, such that functional elements, i.e., elements of the system that perform specific tasks (such as managing the input and output of information, processing information, etc.), may be implemented by the execution of software applications on and across one or more computer platforms represented by the system.

[0123] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, and the like. The processing module includes a processor that accesses a memory having instructions stored therein to perform the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are available or that become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor coordinating and executing the functions of various computer programs, which may be written in a variety of programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow a user to manually align the light source with the flow stream based on the first and second light signals, hi some embodiments, the processor includes analog electronics that provide feedback control, such as, for example, negative feedback control.

[0124] The system memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-write compact disk, flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices, including a compact disk drive, tape drive, removable hard disk drive, or disk drive. Such types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk, magnetic tape, removable hard disk, or floppy disk, respectively. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or in program storage devices used in conjunction with the memory storage devices.

[0125] In some embodiments, a computer program product is described comprising a computer usable medium having control logic (computer software program including program code) stored therein. The control logic, when executed by a processor, causes the computer, processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Examples of implementations of hardware state machines to perform the functions described herein will be apparent to those skilled in the relevant art.

[0126] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic or optical disk, or tape, or RAM, or any other suitable device, either fixed or portable). The processor may include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming may be provided remotely to the processor over a communication channel, or may be pre-stored in a computer program product, such as a memory or some other portable or fixed computer-readable storage medium, using any of those devices together with the memory. For example, a magnetic or optical disk may carry the programming 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 use in implementing the above-described method. The programming according to the present invention may be recorded on a computer-readable medium, for example any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROM, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0127] The processor may also have access to a communication channel for communicating with a user at a remote location, meaning that the user does not have direct contact with the system, but rather relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), a telephone network, a satellite network, or any other suitable communication channel, including a cellular phone (i.e., a smartphone).

[0128] In some embodiments, a system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0129] In one embodiment, the communications interface is configured to include one or more communications ports, e.g., a physical port or interface such as a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communications between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment) configured for similar complementary data communications.

[0130] In one embodiment, the communications interface is configured for infrared communications, Bluetooth® communications, or any other suitable wireless communications protocol to enable the subject system to communicate with other devices, such as a computer terminal and / or network, a communications-enabled mobile phone, a personal digital assistant, or any other communications device that a user may use in conjunction with.

[0131] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing the Internet Protocol (IP) via a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0132] In one embodiment, the subject system is configured to communicate wirelessly with the server device via a communications interface using common standards such as, for example, 802.11 or Bluetooth RF protocols, or IrDA infrared protocol. The server device may be another portable device, such as a smartphone, a personal digital assistant (PDA) or a notebook computer, or a larger device, such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touch screen.

[0133] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored within the subject system, e.g., within the optional data storage unit, with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0134] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. Where one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. A 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. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be accomplished in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network, in accordance with known techniques. Presentation of data by the output manager may be performed in accordance with a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses to allow the user to retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the subject system are typically of a class of computers commonly referred to as servers, but may be any type of known or future developed computer platform. However, they may be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cabling or other communication systems, including wireless systems, either networked or not. They may be co-located or physically separated.In some cases, depending on the type and / or configuration of the computer platform selected, various operating systems may be employed on any of the computer platforms. 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.

[0135] FIG. 7 illustrates a general architecture of an exemplary computing device 600, according to certain embodiments. The general architecture of the computing device 700 illustrated in FIG. 7 includes an arrangement of computer hardware and software components. The computing device 700 may include more (or less) elements than those illustrated in FIG. 7. However, not all of these typically conventional elements need to be illustrated to provide an enabling disclosure. As illustrated, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750, and input devices 760, all of which may communicate with each other via a communication bus. The network interface 720 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via a network. The processing unit 710 may also communicate with a memory 770, and may further provide output information for an optional display 750 via the input / output device interface 740. The input / output device interface 740 can also accept input from optional input devices 760, such as a keyboard, a mouse, a digital pen, a microphone, a touch screen, a gesture recognition system, a voice recognition system, a game pad, an accelerometer, a gyroscope, or other input devices.

[0136] Memory 770 may include computer program instructions (in some embodiments grouped as modules or components) that processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by processing unit 710 in the general management and operation of computing device 700. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0137] Method for irradiating particles in a flow stream - Patents.com Aspects of the present disclosure also include a method for irradiating a sample having particles in a flow stream. The method according to certain embodiments includes continuously irradiating the flow stream with a first laser and irradiating the flow stream with one or more lasers at discrete intervals. In embodiments, each discrete interval of irradiation with the one or more lasers is triggered by irradiation of a particle in the flow stream with the first laser. In some embodiments, the sample is a biological sample. The term "biological sample" is used in its conventional sense to refer to a whole organism, a plant, a fungus, or a subset of animal tissues, cells, or components that may be found in certain examples, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers to both a native organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, for example, plasma, serum, spinal fluid, lymphatic fluid, skin sections, respiratory, gastrointestinal, cardiovascular, and urinary tracts, tears, saliva, milk, blood cells, tumors, and organs. A biological sample can be any type of biological tissue, including both healthy and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or a derivative thereof, e.g., plasma, tears, urine, semen, and the like, and in some examples, the sample is a blood sample, including whole blood, such as blood obtained from a venipuncture or fingerstick (which may or may not be combined with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0138] In certain embodiments, the source of the sample is "mammal" or "mammalian," terms used broadly to refer to organisms within the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some examples, the subject is a human. The methods may be applied to samples obtained from human subjects of both genders, at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is a juvenile, adolescent, or adult. It should be understood that while the invention may be applied to samples from human subjects, it may also be practiced on samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0139] In an embodiment, the method includes irradiating a sample in a flowstream with a laser configured for continuous irradiation. As noted above, the term "continuous" is used herein in its conventional sense to refer to irradiating the flowstream with a laser in an otherwise uninterrupted, constant manner for the duration that the sample of interest is flowed through the flowstream. In some embodiments, continuously irradiating the sample includes irradiating the flowstream with a laser that is not obscured (i.e., not intermittently blocked by a beam stop or obscuring component). In certain examples, continuous irradiation of the flowstream with a laser includes maintaining a constant laser irradiation intensity, such as a laser irradiation intensity that varies by 5% or less, including a laser irradiation intensity that varies by 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 0.0001% or less, for the duration that the sample of interest is flowed through the flowstream. In certain embodiments, continuous illumination of the flow stream includes illuminating the flow stream with a laser that does not exhibit a change in intensity for the duration that the sample is flowed through the flow stream. The intensity of the output light may be measured with any convenient protocol, including, but not limited to, a scanning slit profiler, a charge coupled device (CCD, e.g., an intensified charge coupled device, ICCD), a positioning sensor, a power sensor (e.g., a thermopile power sensor), an optical power sensor, an energy meter, a digital laser photometer, a laser diode detector, among other types of photodetectors.

[0140] In carrying out the subject method, the slow stream can be continuously irradiated with a laser, such as 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 CO2 laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In another example, the laser configured for continuous irradiation is a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In yet another example, the laser for continuous irradiation of the subject includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet another example, the system includes a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, a Y2O3 laser, or a cerium doped laser, and combinations thereof. In yet another example, the system includes a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or a frequency doubled or frequency tripled implementation of any of the above lasers.

[0141] In embodiments, the method includes irradiating the flow stream at discrete intervals with one or more lasers that are triggered when particles are irradiated during continuous irradiation of the flow stream, as described above. The term "discrete interval" is used herein in its conventional sense to refer to irradiating the flow stream for a predetermined duration followed by a period during which the flow stream is not irradiated by the laser (e.g., by turning off the laser or by blocking the laser with a chopper, beam stop, etc.). In some embodiments, the method includes irradiating the flow stream with one or more lasers at discrete intervals of 0.001 μs or more, including, for example, 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, such as 1 μs or more, such as 5 μs or more, such as 10 μs or more, such as 50 μs or more, such as 100 μs or more, and 500 μs or more. In certain examples, the method includes irradiating the flow stream with one or more lasers at discrete intervals from 0.0001 μs to 500 ms, including, 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 10 μs to 100 μs. The duration between each discrete interval of irradiation by each laser may be 0.001 μs or more, including, for example, 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, such as 1 μs or more, such as 5 μs or more, such as 10 μs or more, such as 50 μs or more, such as 100 μs or more, and 500 μs or more. For example, the duration between each discrete interval of irradiation by each laser may be in the range of 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 10 μs to 100 μs.

[0142] In carrying out the subject methods, the flow stream may be irradiated at discrete intervals with one or more lasers configured to irradiate the flow stream at discrete intervals, including, for example, two or more, for example three or more, for example four or more, for example five or more, for example ten or more, for example fifteen or more, for example twenty-five or more, and fifty or more lasers. Each of the lasers may be operatively coupled to one or more components to provide intermittent irradiation with each laser. Intermittent irradiation may be provided using any convenient protocol, such as an electronic switch for turning the laser on and off, such as a switch that is computer controlled and triggered based on a data signal (e.g., a received or input data signal), as described in more detail below. In some embodiments, the lasers are configured for irradiation at discrete intervals by intermittently exposing the laser beam of each laser to a beam chopper or beam stop.

[0143] In certain embodiments, the method includes illuminating the flowstream at discrete intervals at locations on the flowstream that are downstream from the locations of continuous laser illumination. In one example, the method includes continuously illuminating locations on the flowstream with a first laser configured for continuous illumination of the flowstream, illuminating the flowstream with a second laser at a location downstream from the first laser, illuminating the flowstream with a third laser at a location downstream from the second laser, and illuminating the flowstream with a fourth laser at a location downstream from the third laser. Depending on the flow rate of the flow stream and the distance between the locations of irradiation by each laser, the method may include independently irradiating the flow stream at a location that is 5 μm or more downstream from the location of irradiation by the first laser, including irradiating the flow stream at discrete intervals, for example, at 6 μm or more, such as 7 μm or more, for example 8 μm or more, such as 9 μm or more, for example 10 μm or more, such as 15 μm or more, for example 25 μm or more, such as 50 μm or more, for example 100 μm or more, such as 250 μm or more, for example 500 μm or more, and at locations that are 1000 μm or more downstream from the location of irradiation by the first laser. For example, the location of irradiation of the flow stream by each laser may be a distance of 5 μm to 5000 μm downstream from the location of irradiation of the flow stream by the first laser, including, for example, 10 μm to 2500 μm, such as 25 μm to 1000 μm, such as 50 μm to 750 μm, such as 75 μm to 500 μm, and 100 μm to 250 μm.

[0144] The distance between the shots on the flow stream by each of the lasers can vary, with the inter-shot space being independently 0.0001 μm or more, including, for example, 0.0005 μm or more, such as 0.001 μm or more, for example 0.005 μm or more, such as 0.01 μm or more, for example 0.05 μm or more, such as 0.1 μm or more, for example 0.5 μm or more, such as 1 μm or more, for example 2 μm or more, such as 3 μm or more, for example 4 μm or more, such as 5 μm or more, for example 6 μm or more, such as 7 μm or more, for example 8 μm or more, such as 9 μm or more, and 10 μm or more. In certain examples, the method includes irradiating with lasers positioned on the flow stream such that the lasers are directly adjacent to one another (i.e., there is no inter-shot space).

[0145] In an embodiment, the lasers used to irradiate the flow stream at discrete intervals may each independently 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 CO2 laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In another example, the laser configured for continuous irradiation is a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In yet another example, the laser for continuous irradiation of the target includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet another example, the system includes a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, a Y2O3 laser, or a cerium doped laser, and combinations thereof. In yet another example, the system includes a semiconductor diode laser, an optically pumped semiconductor laser (OPSL), or a frequency doubled or frequency tripled implementation of any of the above lasers. The laser may include any combination of several types of lasers.

[0146] Depending on the desired wavelength of light to be generated in the output laser beam (e.g., for use in illuminating a sample in the flow stream), each laser may have a particular wavelength that varies from 200 nm to 1500 nm, including, for example, 250 nm to 1250 nm, for example, 300 nm to 1000 nm, for example, 350 nm to 900 nm, and 400 nm to 800 nm. In certain embodiments, the lasers of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser. In some embodiments, each of the lasers outputs a different wavelength of light. In certain examples, the lasers of the system are positioned such that each downstream laser outputs a longer wavelength of light. For example, if the method includes illuminating a flow stream with four lasers, the wavelength of light from the second laser is longer than the wavelength of light from the first laser, the wavelength of light from the third laser is longer than the wavelength of light from the second laser, and the wavelength of light from the fourth laser is longer than the wavelength of light from the third laser.

[0147] The flow stream may be illuminated by each of the lasers at any suitable distance from the flow stream, such as a distance of 0.001 mm or more from the flow stream, including a distance of 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, and 100 mm or more. Further, illumination of the flow stream may be at any suitable angle (e.g., relative to a normal axis of the flow stream), such as an angle in the range of 10° to 90°, such as an angle of 90°, including, for example, 15° to 85°, such as 20° to 80°, such as 25° to 75°, and 30° to 60°.

[0148] In embodiments, the illumination of particles in the flow stream at discrete intervals with one or more lasers is triggered by illumination of particles in the flow stream with a laser configured for continuous illumination. In some embodiments, triggering illumination with a laser includes turning a laser from an off setting to an on setting and illuminating the flow stream with the laser. In other embodiments, triggering illumination with a laser includes moving a position or setting of an optical adjustment component, such as a beam stopper, to illuminate the flow stream with the laser. One or more of the lasers can be triggered using any convenient protocol, such as by detecting particles being illuminated by a first laser with a photodetector (i.e., through a continuous light beam of the first laser). In certain embodiments, the system includes outputting a trigger signal from a trigger detector operably coupled to each of the lasers to initiate discrete intervals of illumination.

[0149] In some embodiments, the duration between when a particle is detected as being illuminated by the continuously illuminating laser and the beginning of a discrete interval of illumination by one or more of the lasers may vary depending on the flow rate of the flow stream and the distance between the locations of illumination on the flow stream. In some embodiments, the duration between illumination by the continuously illuminating laser and the beginning of a discrete interval of laser illumination may be 0.0001 μs or more, including, for example, 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, such as 1 μs or more, such as 2 μs or more, such as 3 μs or more, such as 4 μs or more, such as 5 μs or more, such as 6 μs or more, such as 7 μs or more, such as 8 μs or more, such as 9 μs or more, and 10 μs or more. In certain embodiments, the system is configured to delay the start of the discrete interval in response to output of the trigger signal, for example the delay is 0.0005 μs or more, such as 0.001 μs or more, for example 0.005 μs or more, such as 0.01 μs or more, for example 0.05 μs or more, such as 0.1 μs or more, for example 0.5 μs or more, such as 1 μs or more, for example 2 μs or more, such as 3 μs or more, for example 4 μs or more, such as 5 μs or more, for example 6 μs or more, such as 7 μs or more, for example 8 μs or more, such as 9 μs or more, and 10 μs or more.

[0150] In some embodiments, the method includes calculating a timing to begin irradiating the flow stream with each of the lasers (i.e., when to begin discrete intervals of irradiation). In some examples, to calculate the timing of irradiation, the method includes sequentially irradiating a particle flowing within the flow stream with all of the lasers of the light source, detecting light from the flow stream in response to irradiating the particle with each of the lasers, and calculating a time of irradiation of the particle by each of the lasers. In particular examples, calculating the time to begin irradiation includes calculating a time interval between irradiation by each of the lasers.

[0151] 3 illustrates laser illumination at discrete intervals by three lasers in response to a trigger signal from a laser that illuminates continuously, according to certain embodiments. The light source includes laser 301 configured for continuous illumination, and lasers 302, 303, and 304, each positioned downstream from laser 301 along the flow stream and configured for illumination at discrete intervals. When laser 301 illuminates a particle in time window 301a, trigger signals are output to lasers 302, 303, and 304 such that the particle illuminates the flow stream during time windows 302a, 303a, and 304a that intersect the path of illumination by lasers 302, 303, and 304. The lasers are configured to turn on in sequence in response to the trigger signal to follow the path of the particle in the flow stream.

[0152] In other embodiments, the method includes calculating a duration of illumination of the flowstream by each of the lasers (i.e., the length of each discrete interval). In some examples, to calculate the duration of illumination, the method includes sequentially illuminating a particle flowing in the flowstream with all of the lasers of the light source, detecting light from the flowstream in response to illumination of the particle with each of the lasers, and calculating the duration of the discrete interval of illumination by each laser. In these examples, the duration of each discrete interval may be calculated based on the location of illumination on the flowstream and the flow rate of the particle in the flowstream.

[0153] In some embodiments, the method includes turning on one or more downstream lasers in response to illumination of a particle in the flow stream by the first laser. Depending on the distance between the first laser and the one or more downstream lasers, each laser may be independently turned on within 0.00001 μs or more, including, for example, within 0.00005 μs or more, for example, within 0.0001 μs or more, for example, within 0.0005 μs or more, for example, within 0.001 μs or more, for example, within 0.005 μs or more, for example, within 0.01 μs or more, for example, within 0.05 μs or more, for example, within 0.1 μs or more, for example, within 0.5 μs or more, and within 1 μs or more, in response to illumination by the first laser. In certain examples, each laser is configured to be independently turned on within 0.00001 μs to 100 μs, including, for example, 0.00005 μs to 90 μs, for example, 0.0001 μs to 80 μs, for example, 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 1 μs to 10 μs, after irradiation of the particle by the first laser.

[0154] In some embodiments, the method includes moving a beam stop positioned in a beam path between one or more downstream lasers and the flow stream in response to irradiation of the particle by the first laser. The beam stop may be moved by coupling the beam stop to a movable support stage or directly coupling the beam stop to a motorized translation stage, a lead screw translation assembly, a geared translation device, such as those using stepper motors, servo motors, brushless electric motors, brushed DC motors, microstep drive motors, high resolution stepper motors, among other types of motors. The beam stop may be moved within 0.00001 μs or more in response to illumination by the first laser, including within 0.00005 μs or more, such as within 0.0001 μs or more, such as within 0.0005 μs or more, such as within 0.001 μs or more, such as within 0.005 μs or more, such as within 0.01 μs or more, such as within 0.05 μs or more, such as within 0.1 μs or more, such as within 0.5 μs or more, and within 1 μs or more. In certain examples, the beam stop is moved out of the beam path of one or more downstream lasers within 0.00001 μs to 100 μs, including, for example, 0.00005 μs to 90 μs, for example, 0.0001 μs to 80 μs, for example, 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 1 μs to 10 μs, after irradiation of the particle by the first laser.

[0155] In some embodiments, the method includes using a beam diverter to redirect light from one or more of the downstream lasers into the flow stream in response to illumination of the particle by the first laser. In some embodiments, a data signal is generated in response to illumination of the particle in the flow stream by the first laser, and light from the one or more downstream lasers is redirected into the flow stream by the beam diverter in response to the generated data signal. In some examples, the beam diverter 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 modifying a drive signal from a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator in a manner sufficient to redirect light from the laser into the flow stream. In other examples, the beam diverter 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 modifying an input current or applying a voltage to an electro-optical device in a manner sufficient to redirect light from the laser into the flow stream. In some embodiments, the beam diverter is configured to redirect the beam path of one or more downstream lasers within 0.00001 μs or more, including within 0.00005 μs or more, such as within 0.0001 μs or more, such as within 0.0005 μs or more, such as within 0.001 μs or more, such as within 0.005 μs or more, such as within 0.01 μs or more, such as within 0.05 μs or more, such as within 0.1 μs or more, such as within 0.5 μs or more, and within 1 μs or more, in response to illumination by the first laser. In certain examples, the beam diverter is configured to redirect the beam path of one or more downstream lasers within 0.00001 μs to 100 μs, including, for example, 0.00005 μs to 90 μs, for example, 0.0001 μs to 80 μs, for example, 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 1 μs to 10 μs, after irradiation of the particle by the first laser.

[0156] The disclosed method, according to certain embodiments, also includes detecting light from particles in the stream. In an embodiment, the light from the flow stream is transmitted to and detected by a light detection system having one or more light detectors. The light detector for implementing the subject method can be any convenient light detection protocol, including, but not limited to, light sensors or light detectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or photodiodes, as well as combinations thereof, among other light detectors. The light from particles in the flow stream can be detected using one or more light detectors, including, for example, two or more light detectors, for example, three or more light detectors, for example, four or more light detectors, for example, five or more light detectors, for example, ten or more light detectors, for example, twenty-five or more light detectors, and fifty or more light detectors.

[0157] In certain embodiments, light from particles in the flow stream is detected with a photodiode array having two or more photodiodes, e.g., two or more photodiodes, e.g., three or more, e.g., five or more, and ten or more photodiodes, each photodiode having a size of, e.g., 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2 Including 0.01cm 2 ~10cm 2 The active detection surface area of ​​each region may range from 0.1 to 1.0 mm.

[0158] In embodiments of the present disclosure, light from particles in the flow stream may be measured at one or more wavelengths, including measuring light from particles in the flow stream at, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., 25 or more different wavelengths, e.g., 50 or more different wavelengths, e.g., 100 or more different wavelengths, e.g., 200 or more different wavelengths, e.g., 300 or more different wavelengths, and 400 or more different wavelengths.

[0159] In embodiments, the light may be measured by the photodetector continuously or at discrete intervals. In some examples, the subject detector is configured to make measurements of the collected light continuously. In other examples, the subject detector is configured to make measurements at discrete intervals, such as detecting light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at some other interval.

[0160] Light from the flow stream can be measured at each optical detector at any suitable distance from the flow stream so long as a usable optical signal is detectable. For example, the detectors can be positioned 1 mm or more from the flow stream, including, for example, 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more, such as 50 mm or more, such as 100 mm or more, such as 150 mm or more, such as 250 mm or more, and 500 mm or more from the flow stream. The detectors can also be positioned at any angle from the flow stream. For example, the detectors can be angled from 10° to 90°, including, for example, 15° to 85°, such as 20° to 80°, such as 25° to 75°, and 30° to 60°, relative to the vertical axis of the flow stream. In some examples, one or more detectors are positioned at 30° to 60° relative to the vertical axis of the flow stream.

[0161] In embodiments, the method may include detecting forward scattered light, side scattered light, emitted light, transmitted light, or a combination thereof. In certain embodiments, the light signal from the illuminated flow stream may be detected by one or more detectors configured as forward scatter detectors. In these embodiments, the forward scatter detector is positioned on the opposite side of the flow stream from the light source and is positioned to collect and detect forward propagating (e.g., scattered) light.

[0162] In certain embodiments, the method includes detecting light from each of the lasers with a single photodetector, in these embodiments, the photodetector detects light from particles in the flow stream illuminated with the laser configured for continuous illumination and each laser configured for illumination at discrete intervals.

[0163] In some embodiments, the method includes detecting light from the flow stream transmitted through an optical conditioning component configured to reduce an amount of light transmitted from a laser configured for continuous illumination to one or more photodetectors. In these embodiments, the optical conditioning component is configured to inhibit, reduce, or limit the propagation of at least one or more wavelengths of light (e.g., one or more of the wavelengths of light of a laser configured for continuous illumination) from the sample to an active surface of the photodetector. The optical conditioning component may include an optical component that limits the propagation of one or more different wavelengths of light, including limiting the propagation of, for example, 5 or more, for example 10 or more, for example 25 or more, for example 50 or more, for example 100 or more, for example 200 or more, for example 300 or more, and 500 or more different wavelengths of light. For example, in some embodiments, the optical conditioning component is a bandpass filter, such as a longpass filter, that transmits a spectral range of light longer than the illumination wavelength of the first laser. In other embodiments, the optical conditioning component is a dichroic mirror, such as a dichroic mirror that transmits a spectral range of light longer than the illumination wavelength of the first laser and reflects a spectral range of light that includes the illumination wavelength of the first laser.

[0164] In some embodiments, the method includes collecting and directing light from the flow stream to a photodetector using an optical collection system.

[0165] In some embodiments, the light is transmitted from the flow stream to the photodetector with an optical fiber. In some examples, the optical collection system can be a fiber optic optical relay bundle, and the light is transmitted to the photodetector through the fiber optic optical relay bundle. In certain embodiments, the optical collection unit is a single optical fiber configured to collect light from each of the illuminations by the lasers to a single photodetector in the optical detection system. In these embodiments, the method includes illuminating the flow stream with the lasers at locations that span a length equal to or less than the diameter of a single optical fiber configured to collect light from the flow stream. For example, the method may include illuminating locations on the flow stream that span 100 μm or less, including, for example, 90 μm or less, such as 80 μm or less, such as 70 μm or less, such as 60 μm or less, and 50 μm or less, and a single optical fiber may have a diameter sufficient to collect light from each of the locations illuminated by the laser, including, for example, a diameter of 50 μm or more, such as 60 μm or more, such as 70 μm or more, such as 80 μm or more, such as 90 μm or more, and where a single optical fiber configured to collect light from the illuminated flow stream is 100 μm or more.

[0166] In other embodiments, the light from the flow stream is transmitted to the photodetector using a free space optical relay system. For example, the free space optical relay system may include a housing having a proximal end and a distal end, the proximal end being coupled to the optical detection system. The free space optical relay system may include any combination of different optical components, such as one or more lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof.

[0167] Measurements of the collected light may be made one or more times during the subject methods, including, for example, two or more times, for example, three or more times, for example, five or more times, and ten or more times. In certain embodiments, the light propagation is measured two or more times, and in certain instances, the data is averaged.

[0168] In some embodiments, the method includes conditioning the light before detecting the light with the light detection system. For example, the light from the sample source may be passed through one or more lenses, mirrors, pinholes, slits, gratings, optical refractors, and any combination thereof. In some examples, the collected light is passed through one or more focusing lenses to reduce the profile of the light directed to the light detection system or optical collection system, as described above. In other examples, the light emitted from the sample is passed through one or more collimators to reduce the divergence of the light beam transmitted to the light detection system.

[0169] Computer-readable storage medium Aspects of the present disclosure further include a non-transitory computer-readable storage medium having instructions for carrying out the subject methods. The computer-readable storage medium may be used on one or more computers for full or partial automation of a system for carrying out the methods described herein. In certain embodiments, instructions according to the methods described herein may be coded on a computer-readable medium in the form of "programming," and the term "computer-readable medium" as used herein refers to any non-transitory storage medium involved 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 disks, solid-state disks, and network-attached storage (NAS), regardless of whether the devices are internal or external to the computer. A file containing information may be "stored" on a computer-readable medium, where "storing" means recording information such that the information is accessible and retrievable by a computer at a later date. The computer-implemented methods described herein may be performed using programming that may be written in one or more of any number of computer programming languages, including, for example, Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as any of many others.

[0170] In some embodiments, a subject computer readable storage medium includes a computer program stored thereon, the computer program including instructions, when loaded into a computer, having an algorithm for continuously irradiating the flow stream with a first laser, an algorithm for irradiating the flow stream with a second laser at discrete intervals, where each discrete interval for irradiation by the second laser is triggered by irradiation of a particle in the flow stream with the first laser, an algorithm for detecting light from particles in the flow stream illuminated by each of the lasers with a single photodetector coupled to a single optical fiber, and an algorithm for calculating the timing of illumination of the flow stream by each of the lasers. In some embodiments, a non-transitory computer readable storage medium includes instructions having an algorithm for continuously irradiating the flow stream with a first laser, and an algorithm for irradiating the flow stream with a plurality of lasers at discrete intervals, where each discrete interval for irradiation by each of the plurality of lasers is triggered by irradiation of a particle in the flow stream with the first laser. In some examples, the non-transitory computer readable storage medium includes instructions having an algorithm for sequentially irradiating a particle in the flow stream with each of the lasers, an algorithm for detecting light from the flow stream in response to irradiating the particle with each of the lasers, and an algorithm for calculating a time interval between irradiating the particle with each of the lasers.

[0171] In some embodiments, the non-transitory computer readable storage medium includes instructions having an algorithm for turning on a second laser 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 moving a beam stop positioned 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 directing light from the second laser with a beam diverter into the flow stream in response to irradiation of a particle by the first laser.

[0172] The computer readable storage medium may be used on one or more computer systems having a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses a memory having stored instructions to perform the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are available or become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor coordinating and executing the functions of various computer programs, which may be written in a variety of programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

[0173] kit Kits including one or more components of the subject systems are also provided. Kits according to certain embodiments include one or more lasers, such as a laser configured for continuous illumination and a laser configured for illumination at discrete intervals. In some embodiments, the kit may include a switch for operating one or more of the lasers at discrete intervals (e.g., pulsing the laser). The kit may also include an optical conditioning component configured to reduce the passage of wavelengths of light of one or more of the lasers. In some examples, the optical conditioning component is a bandpass filter, such as a longpass filter, that transmits a spectral range of light longer than the irradiating wavelength of one or more of the lasers. In other examples, the optical conditioning component is a dichroic mirror, such as a dichroic mirror that transmits a spectral range of light longer than the irradiating wavelength of one or more of the lasers and reflects a spectral range of light including the wavelength of one or more of the lasers.

[0174] The kits may also include a photodetector array for detecting light from the flowstream. In certain embodiments, the support stage includes a motor, such as a stepper motor. The subject kits may also include an optical relay system, such as an optical fiber (e.g., a single optical fiber), for transmitting light from the sample in the flowstream to the detector.

[0175] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for carrying out the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, such as a sheet or sheets of paper on which the information is printed, in the kit packaging, in a package insert, etc. Yet another form in which these instructions may be present is a computer readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Yet another form in which these instructions may be present is a website address that may be used via the Internet to access the information at a remote site.

[0176] Utilities The subject systems, methods, and computer systems are used in a variety of applications where it is desirable to analyze and separate particle components within a sample in a fluid medium, such as a biological sample. The present disclosure is also used in flow cytometers where it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and improved particle deflection during cell sorting. In embodiments, the present disclosure reduces the need for user input or manual adjustments during sample analysis in a flow cytometer. In certain embodiments, the subject systems provide fully automated protocols, such that adjustments to the flow cytometer during use require little, if any, human input.

[0177] The present disclosure also finds use in applications where cells prepared from a biological sample may be desirable for research, laboratory testing, or therapeutic use. In some embodiments, the subject methods and devices may facilitate obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods and systems may facilitate obtaining cells from a fluid or tissue sample used as a research or diagnostic specimen for a disease such as cancer. Similarly, the subject methods and systems may facilitate obtaining cells from a fluid or tissue sample used in therapy. The methods and devices of the present disclosure allow for the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, bodily fluids) with improved efficiency and lower cost compared to conventional flow cytometry systems.

[0178] Aspects (including embodiments) of the subject matter described herein may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the description, certain non-limiting aspects (appendices) of the present disclosure, numbered 1 to 139, are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects (appendices) may be used or combined with any of the preceding aspects or any of the aspects following the individually numbered aspect (appendices). This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects (appendices) explicitly provided below.

[0179] 1. A first laser configured for continuous illumination of a flow stream; a second laser configured for irradiating the flow stream at discrete intervals, each discrete interval of irradiation by the second laser being triggered by irradiation of a particle in the flow stream with the first laser; A light source comprising: a single optical fiber configured to collect light from particles in the flow stream illuminated by each of the lasers; a single photodetector configured to detect light transmitted by the single optical fiber; An optical detection system comprising: a processor comprising a memory operatively coupled to the processor, the memory including instructions stored therein that, when executed by the processor, cause the processor to calculate timing of illumination of the flow stream by each of the lasers; A system comprising: 2. The system of claim 1, wherein a second laser is configured to irradiate the flow stream at a location downstream from the first laser. 3. The system of claim 1 or 2, wherein the wavelength of the light from the first laser is shorter than the wavelength of the light from the second laser. 4. The system a first laser configured for continuous illumination of the flow stream; a plurality of lasers configured for irradiating the flow stream at discrete intervals; The system according to any one of claims 1 to 3, comprising: 5. The system of claim 4, wherein the multiple lasers are configured to irradiate the flow stream at a location downstream from the first laser.

[0180] 6. The system of claim 5, wherein the multiple lasers are configured to irradiate the flow stream at locations spaced 10 μm or less apart from one another. 7. The system of any one of claims 4 to 6, wherein each of the multiple lasers outputs light of a different wavelength. 8. Multiple lasers a second laser configured to irradiate the flow stream at a location downstream from the first laser; a third laser configured to irradiate the flow stream at a location downstream from the second laser; a fourth laser configured to irradiate the flow stream at a location downstream from the third laser; and 7. The system of claim 5 or 6, comprising: 9. The wavelength of the light from the second laser is longer than the wavelength of the light from the first laser; the wavelength of the light from the third laser is longer than the wavelength of the light from the second laser; the wavelength of the light from the fourth laser is longer than the wavelength of the light from the third laser; 9. The system of claim 8. 10. A system according to any one of claims 4 to 9, wherein each discrete interval of irradiation of the flow stream by each of the plurality of lasers is independently triggered by irradiation of a particle in the flow stream with a first laser.

[0181] 11. The system of any one of claims 1 to 10, wherein each discrete interval of each laser has a duration between 1 μs and 500 μs. 12. The memory includes instructions stored in the memory that, when executed by the processor, cause the processor to: irradiating particles in the flow stream with each of the lasers; detecting light from the flow stream in response to illumination of the particle with each of the lasers; calculating a time interval between irradiation of the particle by each of the lasers; Let it be calculated by A system according to any one of claims 1 to 11. 13. The system of any one of claims 1 to 12, wherein the system is configured to turn on the second laser in response to irradiation of the particle by the first laser. 14. The system of any one of claims 1 to 12, wherein the system further comprises a beam stop positioned in a beam path between the second laser and the flow stream. 15. The system of claim 14, wherein the memory of the processor includes instructions stored in the memory that, when executed by the processor, cause the processor to move a beam stop out of a beam path between the second laser and the flow stream in response to irradiation of the particle by the first laser.

[0182] 16. The system of any one of claims 1 to 12, further comprising a beam diverter positioned in a beam path between the second laser and the flow stream, the beam diverter configured to divert light from the second laser away from the flow stream. 17. The system of claim 16, wherein the beam diverter is configured to direct light from the second laser into the flow stream in response to irradiation of the particle by the first laser. 18. The system of claim 16 or 17, wherein the beam diverter comprises an acousto-optical device. 19. The system of claim 18, wherein the acousto-optical device is an acousto-optical modulator (AOM). 20. The system of claim 18, wherein the acousto-optic device is an acousto-optic deflector (AOD).

[0183] 21. The system of claim 16 or 17, wherein the beam diverter comprises an electro-optical device. 22. The system of claim 21, wherein the electro-optical device is an electro-optical modulator (EOM). 23. The system of claim 21, wherein the electro-optical device is an electro-optical deflector (EOD). 24. The optical detector further comprises an optical detection system, a single photodetector; an optical adjustment component configured to reduce an amount of light transmitted from the first laser to the photodetector; Equipped with A system according to any one of appendixes 1 to 23. 25. The system of claim 24, wherein the optical adjustment component is a bandpass filter. 26. The system of any one of claims 1 to 15, wherein the single photodetector is a photodetector array.

[0184] 27. A first laser configured for continuous illumination of the flow stream; a second laser configured for irradiating the flow stream at discrete intervals; and Equipped with each discrete interval of illumination by the second laser is triggered by illumination of a particle in the flow stream with the first laser; system. 28. The system of claim 27, wherein a second laser is configured to irradiate the flow stream at a location downstream from the first laser. 29. The system of claim 27 or 28, wherein the wavelength of the light from the first laser is shorter than the wavelength of the light from the second laser. 30. The system a first laser configured for continuous illumination of the flow stream; a plurality of lasers configured for irradiating the flow stream at discrete intervals; Equipped with 30. A system according to any one of appendices 27 to 29. 31. The system of claim 30, wherein the multiple lasers are configured to irradiate the flow stream at a location downstream from the first laser.

[0185] 32. The system of claim 31, wherein the multiple lasers are configured to irradiate the flow stream at locations spaced 10 μm or less apart from one another. 33. Multiple lasers are a second laser configured to irradiate the flow stream at a location downstream from the first laser; a third laser configured to irradiate the flow stream at a location downstream from the second laser; a fourth laser configured to irradiate the flow stream at a location downstream from the third laser; and Equipped with 33. The system of claim 31 or 32. 34. The wavelength of the light from the second laser is longer than the wavelength of the light from the first laser; the wavelength of the light from the third laser is longer than the wavelength of the light from the second laser; the wavelength of the light from the fourth laser is longer than the wavelength of the light from the third laser; 34. The system of claim 33. 35. A system according to any one of claims 30 to 34, wherein each discrete interval of irradiation of the flow stream by each of the plurality of lasers is independently triggered by irradiation of a particle in the flow stream with a first laser. 36. The system of any one of claims 27 to 35, wherein each discrete interval of each laser has a duration of 1 μs to 500 μs.

[0186] 37. The system of any one of claims 27 to 36, wherein the system is configured to turn on a second laser in response to irradiation of the particle by the first laser. 38. The system of any one of claims 27 to 36, wherein the system further comprises a beam stop positioned in the beam path between the second laser and the flow stream. 39. The system of claim 38, wherein the system comprises a processor including a memory operatively coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to move a beam stop out of a beam path between the second laser and the flow stream in response to irradiation of a particle by the first laser. 40. The system of any one of claims 27 to 36, further comprising a beam diverter positioned in a beam path between the second laser and the flow stream, the beam diverter configured to divert light from the second laser away from the flow stream. 41. The system of claim 40, wherein the beam diverter is configured to direct light from the second laser into the flow stream in response to irradiation of the particle by the first laser.

[0187] 42. The system of claim 40 or 41, wherein the beam diverter comprises an acousto-optical device. 43. The system of claim 42, wherein the acousto-optical device is an acousto-optical modulator (AOM). 44. The system of claim 42, wherein the acousto-optic device is an acousto-optic deflector (AOD). 45. The system of claim 40 or 41, wherein the beam diverter comprises an electro-optical device. 46. ​​The system of claim 45, wherein the electro-optical device is an electro-optical modulator (EOM).

[0188] 47. The system of claim 45, wherein the electro-optical device is an electro-optical deflector (EOD). 48. The method according to claim 1, further comprising the steps of: A photodetector; an optical adjustment component configured to reduce an amount of light transmitted from the first laser to the photodetector; Equipped with A system according to any one of appendices 27 to 47. 49. The system of claim 48, wherein the optical adjustment component is a bandpass filter. 50. The system of claim 48 or 49, wherein the optical detector is configured to detect light from the flow stream illuminated by each of the lasers of the light source. 51. The system of claim 50, wherein the light detector comprises a light detector array.

[0189] 52. The system of any one of claims 48 to 51, further comprising a light propagating component configured to transmit light from the flow stream to the light detector. 53. The system of claim 52, wherein the light propagating component comprises an optical fiber. 54. The system of any one of notes 48 to 53, wherein the optical detector is a single optical detector configured to detect light from each of the lasers of the light source. 55. The system of claim 54, wherein the light propagating component comprises a single optical fiber.

[0190] 56. Continuously irradiating the flow stream with a first laser; irradiating the flow stream with a second laser at discrete intervals, each discrete interval for irradiation with the second laser being triggered by irradiation of a particle in the flow stream with the first laser; detecting light from particles in the flow stream illuminated by each of the lasers with a single photodetector coupled to a single optical fiber; calculating a timing for irradiating the flow stream by each of the lasers; A method comprising: 57. The method of claim 56, wherein a second laser is configured to irradiate the flow stream at a location downstream from the first laser. 58. The method of claim 56 or 57, wherein the wavelength of the light from the first laser is shorter than the wavelength of the light from the second laser. 59. Continuously irradiating the flow stream with a first laser; Irradiating the flow stream with multiple lasers at discrete intervals Including, each discrete interval for illumination by each of the plurality of lasers is triggered by illumination of a particle in the flow stream with a first laser; 59. The method according to any one of claims 56 to 58. 60. The method of claim 59, wherein multiple lasers are configured to irradiate the flow stream at a location downstream from the first laser.

[0191] 61. The method of claim 60, wherein multiple lasers are configured to irradiate the flow stream at locations spaced 10 μm or less apart from one another. 62. The method of any one of appendixes 59-61, wherein each of the plurality of lasers outputs light of a different wavelength. 63. Multiple lasers are a second laser configured to irradiate the flow stream at a location downstream from the first laser; a third laser configured to irradiate the flow stream at a location downstream from the second laser; a fourth laser configured to irradiate the flow stream at a location downstream from the third laser; and Equipped with 63. The method according to any one of appendices 59 to 62. 64. The wavelength of the light from the second laser is longer than the wavelength of the light from the first laser; the wavelength of the light from the third laser is longer than the wavelength of the light from the second laser; the wavelength of the light from the fourth laser is longer than the wavelength of the light from the third laser; 63. The method according to claim 62. 65. The method of any one of claims 56 to 64, wherein each discrete interval of each laser has a duration between 1 μs and 500 μs.

[0192] 66. Calculating the timing of illumination of the flow stream by each of the lasers; sequentially irradiating particles within the flow stream with each of the lasers; detecting light from the flow stream in response to illumination of the particle with each of the lasers; calculating a time interval between irradiation of the particle by each of the lasers; Including, 66. The method according to any one of claims 55 to 65. 67. The method of any one of claims 56-66, wherein the method includes turning on a second laser in response to irradiation of the particle by the first laser. 68. The method of any one of claims 56-66, wherein the method includes moving a beam stop positioned in a beam path between the second laser and the flow stream in response to irradiation of the particle by the first laser. 69. The method of any one of claims 56-66, wherein the method includes, in response to irradiation of the particle by the first laser, directing light from a second laser into the flow stream using a beam diverter. 70. The method of claim 69, wherein the beam diverter comprises an acousto-optical device.

[0193] 71. The method of claim 70, wherein the acousto-optical device is an acousto-optical modulator (AOM). 72. The method of claim 70, wherein the acousto-optical device is an acousto-optical deflector (AOD). 73. The method of claim 69, wherein the beam diverter comprises an electro-optical device. 74. The method of claim 73, wherein the electro-optical device is an electro-optical modulator (EOM). 75. The method of claim 73, wherein the electro-optical device is an electro-optical deflector (EOD).

[0194] 76. The method of any one of notes 56 to 75, further comprising detecting light from the flow stream with an optical detection system comprising an optical detector and an optical conditioning component configured to reduce an amount of light transmitted from the first laser to the optical detector. 77. The method of claim 76, wherein the optical adjustment component is a bandpass filter. 78. The method of claim 76 or 77, wherein the single photodetector is a photodetector array. 79. The method of any one of claims 76-78, wherein the optical detection system comprises an optical propagation component configured to transmit light from the flow stream to the optical detector.

[0195] 80. Continuously irradiating the flow stream with a first laser; irradiating the flow stream with a second laser at discrete intervals, each discrete interval for irradiation with the second laser being triggered by irradiation of a particle in the flow stream with the first laser; A method comprising: 81. The method of claim 80, wherein a second laser is configured to irradiate the flow stream at a location downstream from the first laser. 82. The method of claim 80 or 81, wherein the wavelength of the light from the first laser is shorter than the wavelength of the light from the second laser. 83. Continuously irradiating the flow stream with a first laser; Irradiating the flow stream with multiple lasers at discrete intervals Including, each discrete interval for illumination by each of the plurality of lasers is triggered by illumination of a particle in the flow stream with a first laser; 83. The method according to any one of appendices 80 to 82. 84. The method of claim 83, wherein multiple lasers are configured to irradiate the flow stream at a location downstream from the first laser.

[0196] 85. The method of claim 84, wherein multiple lasers are configured to irradiate the flow stream at locations spaced 10 μm or less apart from one another. 86. The method of any one of notes 83-85, wherein each of the plurality of lasers outputs light of a different wavelength. 87. Multiple lasers, a second laser configured to irradiate the flow stream at a location downstream from the first laser; a third laser configured to irradiate the flow stream at a location downstream from the second laser; a fourth laser configured to irradiate the flow stream at a location downstream from the third laser; and Equipped with 87. The method according to any one of claims 84 to 86. 88. The wavelength of the light from the second laser is longer than the wavelength of the light from the first laser; the wavelength of the light from the third laser is longer than the wavelength of the light from the second laser; the wavelength of the light from the fourth laser is longer than the wavelength of the light from the third laser; 88. The method according to claim 87. 89. The method of any one of claims 80 to 88, wherein each discrete interval of each laser has a duration between 1 μs and 500 μs.

[0197] 90. The method of any one of claims 80-89, further comprising calculating the timing of irradiation of the flow stream by each of the lasers. 91. Calculating the timing of illumination of the flow stream by each of the lasers; sequentially irradiating particles within the flow stream with each of the lasers; detecting light from the flow stream in response to illumination of the particle with each of the lasers; calculating a time interval between irradiation of the particle by each of the lasers; Including, 91. The method of claim 90. 92. The method of any one of claims 80-91, wherein the method includes turning on a second laser in response to irradiation of the particle by the first laser. 93. The method of any one of claims 80-91, wherein the method includes moving a beam stop positioned in a beam path between the second laser and the flow stream in response to irradiation of the particle by the first laser. 94. The method of any one of claims 80-91, wherein the method includes, in response to irradiation of the particle by the first laser, directing light from a second laser into the flow stream using a beam diverter.

[0198] 95. The method of claim 94, wherein the beam diverter comprises an acousto-optical device. 96. The method of claim 95, wherein the acousto-optical device is an acousto-optical modulator (AOM). 97. The method of claim 95, wherein the acousto-optical device is an acousto-optical deflector (AOD). 98. The method of claim 94, wherein the beam diverter comprises an electro-optical device. 99. The method of claim 98, wherein the electro-optical device is an electro-optical modulator (EOM).

[0199] 100. The method of claim 98, wherein the electro-optical device is an electro-optical deflector (EOD). 101. The method of any one of claims 80 to 100, further comprising detecting light from the flow stream with an optical detection system comprising an optical detector and an optical conditioning component configured to reduce an amount of light transmitted from the first laser to the optical detector. 102. The method of claim 101, wherein the optical adjustment component is a bandpass filter. 103. The method of any one of claims 101 to 102, wherein the photodetector comprises a photodetector array. 104. The method of any one of claims 101-103, wherein the optical detection system comprises an optical propagation component configured to transmit light from the flow stream to the optical detector.

[0200] 105. The method of claim 104, wherein the light propagating component comprises an optical fiber. 106. The method of any one of notes 101-105, wherein the photodetector is a single photodetector configured to detect light from each of the lasers of the light source. 107. The method of claim 105 or 106, wherein the light propagating component comprises a single optical fiber.

[0201] 108. A non-transitory computer-readable storage medium having instructions stored thereon, the instructions comprising: an algorithm for continuously irradiating the flow stream with a first laser; an algorithm for irradiating the flow stream with a second laser at discrete intervals, each discrete interval for irradiation by the second laser being triggered by irradiation of a particle in the flow stream with the first laser; an algorithm for detecting light from particles in the flow stream illuminated by each of the lasers with a single photodetector coupled to a single optical fiber; an algorithm for calculating the timing of illumination of the flow stream by each of the lasers; A non-transitory computer readable storage medium comprising: 109. The non-transitory computer-readable storage medium of claim 108, wherein the instructions include an algorithm for irradiating the flow stream with a second laser at a location downstream from the first laser. 110. The non-transitory computer-readable storage medium of claim 108, wherein the wavelength of the light from the first laser is shorter than the wavelength of the light from the second laser. 111. The command, an algorithm for continuously irradiating the flow stream with a first laser; Algorithms for irradiating a flow stream with multiple lasers at discrete intervals Including, each discrete interval for illumination by each of the plurality of lasers is triggered by illumination of a particle in the flow stream with a first laser; 109. The non-transitory computer-readable storage medium of claim 108. 112. The non-transitory computer-readable storage medium of claim 111, wherein the multiple lasers are configured to irradiate the flow stream at a location downstream from the first laser.

[0202] 113. The non-transitory computer-readable storage medium of claim 112, wherein the multiple lasers are configured to irradiate the flow stream at locations spaced 10 μm or less apart from one another. 114. The non-transitory computer-readable storage medium of claim 112, wherein each of the multiple lasers outputs light of a different wavelength. 115. The command, an algorithm for illuminating the flow stream with a second laser at a location downstream from the first laser; an algorithm for irradiating the flow stream with a third laser at a location downstream from the second laser; an algorithm for irradiating the flow stream with a fourth laser at a location downstream from the third laser; Including, 15. A non-transitory computer-readable storage medium according to any one of appendices 112 to 114. 116. The wavelength of light from the second laser is longer than the wavelength of light from the first laser; the wavelength of the light from the third laser is longer than the wavelength of the light from the second laser; the wavelength of the light from the fourth laser is longer than the wavelength of the light from the third laser; 116. The non-transitory computer-readable storage medium of claim 115. 117. The non-transitory computer-readable storage medium of any one of claims 108 to 116, wherein each discrete interval of each laser comprises a duration between 1 μs and 500 μs.

[0203] 118. The command, an algorithm for sequentially irradiating particles in the flow stream with each of the lasers; an algorithm for detecting light from the flow stream in response to illumination of a particle with each of the lasers; an algorithm for calculating the time interval between irradiation of a particle by each of the lasers; Including, 18. A non-transitory computer-readable storage medium according to any one of appendices 108 to 117. 119. The non-transitory computer-readable storage medium of any one of claims 108 to 118, wherein the instructions include an algorithm for turning on a second laser in response to irradiation of a particle by the first laser. 120. A non-transitory computer-readable storage medium according to any one of claims 108 to 118, wherein the instructions include an algorithm for moving a beam stop positioned in a beam path between a second laser and the flow stream in response to irradiation of a particle by the first laser. 121. The non-transitory computer-readable storage medium of any one of claims 108 to 118, wherein the instructions include an algorithm for directing light from a second laser into the flow stream using a beam diverter in response to irradiation of a particle by the first laser. 122. The non-transitory computer-readable storage medium of claim 121, wherein the beam diverter comprises an acousto-optic device.

[0204] 123. The non-transitory computer-readable storage medium of claim 122, wherein the acousto-optical device is an acousto-optical modulator (AOM). 124. The non-transitory computer-readable storage medium of claim 122, wherein the acousto-optic device is an acousto-optic deflector (AOD). 125. The non-transitory computer-readable storage medium of claim 121, wherein the beam diverter comprises an electro-optical device. 126. The non-transitory computer-readable storage medium of claim 125, wherein the electro-optical device is an electro-optical modulator (EOM). 127. The non-transitory computer-readable storage medium of claim 125, wherein the electro-optical device is an electro-optical deflector (EOD). 128. The non-transitory computer-readable storage medium of any one of appendixes 108 to 127, wherein the instructions further include an algorithm for detecting light from the flow stream with an optical detection system comprising a photodetector and an optical conditioning component configured to reduce an amount of light transmitted from the first laser to the photodetector.

[0205] 129. A light source including a first laser and a second laser; An optical fiber; Photodetector and A kit comprising: 130. The kit of claim 129, further comprising an optical tuning component configured to reduce the passage of wavelengths of light of the first laser. 131. The kit of claim 130, wherein the optical adjustment component is a bandpass filter. 132. The kit of claim 130, wherein the optical adjustment component is a dichroic filter. 133. The kit of any one of claims 129 to 132, further comprising a plurality of lasers. 134. The kit of claim 133, wherein the plurality of lasers comprises a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0206] 135. A light source including a first laser and a second laser; an optical conditioning component configured to reduce passage of a wavelength of light of the first laser; A kit comprising: 136. The kit of claim 135, further comprising a plurality of lasers. 137. The kit of claim 136, wherein the plurality of lasers comprises a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser. 138. A kit according to any one of claims 135 to 137, wherein the optical adjustment component is a bandpass filter. 139. The kit of any one of notes 135 to 137, wherein the optical adjustment component is a dichroic filter.

[0207] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of this invention, certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0208] Thus, the above merely illustrates the principles of the invention. It is understood that those skilled in the art can devise various arrangements that embody the principles of the invention and are within the spirit and scope of the invention, although not expressly described or shown herein. Furthermore, all examples and conditional language described herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts that the inventors contribute to furthering the art, and should not be construed as being limited to such specifically described examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, regardless of structure, i.e., any elements developed to perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly set forth in the claims.

[0209] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly defined as being invoked for a limitation in a claim only if the precise phrase "means for" or the precise phrase "step for" appears at the beginning of such limitation in the claim, and if such precise phrase is not used in the limitation in the claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0210] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Patent Application No. 62 / 978,751, filed February 19, 2020, the disclosure of which is incorporated herein by reference.

Claims

1. a first laser configured for continuous illumination of the flow stream; a second laser configured for irradiating the flow stream at discrete intervals, each discrete interval of irradiation by said second laser being triggered by irradiation of a particle in the flow stream by said first laser; A light source comprising: a single optical fiber configured to collect light from particles in the flow stream illuminated by each of the lasers; a single photodetector configured to detect light transmitted by the single optical fiber; An optical detection system comprising: a processor comprising a memory operatively coupled to the processor, the memory including instructions stored therein that, when executed by the processor, cause the processor to calculate a timing for irradiating the flow stream with the second laser; A system comprising:

2. 10. The system of claim 1, wherein the second laser is configured to irradiate the flow stream at a location downstream from the first laser, and wherein a wavelength of light from the first laser is shorter than a wavelength of light from the second laser.

3. a first laser configured for continuous illumination of the flow stream; a plurality of lasers configured for irradiating the flow stream at discrete intervals; each discrete interval of illumination of the flow stream by each of the plurality of lasers is independently triggered by illumination of a particle in the flow stream with the first laser; 3. A system according to claim 1 or 2.

4. The memory includes instructions stored in the memory that, when executed by the processor, cause the processor to control timing of illumination of the flow stream by each of the lasers; irradiating particles in the flow stream with each of the lasers; detecting light from the flow stream in response to illumination of the particle with each of the lasers; calculating a time interval between illumination of said particle by each of said lasers; Let it be calculated by A system according to any one of claims 1 to 3.

5. The memory of the processor includes instructions stored in the memory that, when executed by the processor, cause the processor to: turning on the second laser in response to irradiation of the particle by the first laser; moving a beam stop out of a beam path between the second laser and the flow stream in response to irradiation of a particle by the first laser; or redirecting light from the second laser into the flow stream with a beam diverter in response to irradiation of the particle by the first laser. To carry out A system according to any one of claims 1 to 4.

6. The system of claim 5 , wherein the beam diverter is selected from an acousto-optical device and an electro-optical device.

7. continuously irradiating the flow stream with a first laser; irradiating the flow stream at discrete intervals with a second laser, each discrete interval for irradiation by the second laser being triggered by irradiation of a particle in the flow stream with the first laser; detecting light from particles in the flow stream illuminated by each of the lasers with a single photodetector coupled to a single optical fiber; calculating a timing of illumination of the flow stream by the second laser; A method comprising:

8. continuously irradiating the flow stream with a first laser; illuminating the flow stream with a plurality of lasers at discrete intervals; each discrete interval for illumination by each of the plurality of lasers is triggered by illumination of a particle in the flow stream with the first laser; The method of claim 7.

9. The method of claim 8 , wherein the plurality of lasers are configured to irradiate the flow stream at a location downstream from the first laser, and each of the plurality of lasers outputs a different wavelength of light.

10. calculating the timing of illumination of the flow stream by each of the lasers; sequentially irradiating particles within the flow stream with each of the lasers; detecting light from the flow stream in response to illumination of the particle with each of the lasers; calculating a time interval between illumination of said particle by each of said lasers; Including, 10. The method according to claim 8 or 9.

11. turning on the second laser in response to irradiation of the particle by the first laser; moving a beam stop positioned in a beam path between the second laser and the flow stream in response to irradiation of a particle by the first laser; or directing light from the second laser into the flow stream using a beam diverter in response to irradiation of the particle by the first laser. Including, The method according to any one of claims 7 to 10.

12. The method of claim 11 , wherein the beam diverter is selected from an acousto-optical device and an electro-optical device.

13. A non-transitory computer-readable storage medium having instructions stored thereon, the instructions comprising: an algorithm for continuously irradiating the flow stream with a first laser; an algorithm for irradiating the flow stream with a second laser at discrete intervals, each discrete interval for irradiation by the second laser being triggered by irradiation of a particle in the flow stream with the first laser; an algorithm for detecting light from particles in the flow stream illuminated by each of the lasers with a single photodetector coupled to a single optical fiber; an algorithm for calculating the timing of illumination of the flow stream by the second laser; and Including, A non-transitory computer-readable storage medium.

14. The instruction: an algorithm for sequentially irradiating particles within the flow stream with each of the lasers; an algorithm for detecting light from the flow stream in response to illumination of the particle with each of the lasers; an algorithm for calculating a time interval between irradiation of said particle by each of said lasers; Including, 14. The non-transitory computer-readable storage medium of claim 13.

15. The instruction: an algorithm for turning on the second laser in response to irradiation of a particle by the first laser; an algorithm for moving a beam stop positioned in a beam path between the second laser and the flow stream in response to irradiation of a particle by the first laser; or an algorithm for directing light from the second laser into the flow stream using a beam diverter in response to irradiation of a particle by the first laser. Including, 15. A non-transitory computer-readable storage medium according to claim 13 or 14.

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