Multiple laser system with modified beam profile and method of use

Abstract

To provide systems with multiple lasers having modified beam profiles.SOLUTION: Systems include a first laser that produces a first beam of light, a second laser that produces a second beam of light, and a beam shaping component that receives the first beam of light and the second beam of light at substantially the same position from different angles of incidence and is configured to generate, from the first beam of light and the second beam of light, an output beam of light having a predetermined intensity profile along a horizontal axis. Methods for irradiating a sample in a flow stream with the output beam of light are also disclosed. Kits having one or more lasers and a beam shaping component configured to generate, from a first beam of light and a second beam of light, an output beam of light having a predetermined intensity profile along a horizontal axis are also provided.SELECTED DRAWING: Figure 3A

Description

[Background technology]

[0001] Characterizing analytes in biological fluids has become an integral part of the medical diagnosis and assessment of overall health and patient health. 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 conditions.

[0002] Flow cytometry is a technique used to characterize and often sort biomaterials, such as cells in a blood sample or target particles in other types of biological or chemical samples. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer transfers particles (including cells) in the fluid sample into the flow cell as a cell stream while directing the sheath fluid into the flow cell. To characterize the components of the flow stream, the flow stream is illuminated with light. Variations in the material within the flow stream, such as the presence of morphology or fluorescent labeling, can cause variations in the observed light, and these variations allow for characterization and separation.

[0003] To characterize the components within the flowstream, light must collide with and be collected on the flowstream. The light source within the flow cytometer can vary from a wide range of lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flowstream, and the optical response from the irradiated particles is collected and quantified. [Overview of the project]

[0004] Aspects of this disclosure include systems having multiple lasers having modified beam profiles. A system according to a particular embodiment comprises a first laser that generates a first light beam, a second laser that generates a second light beam, and a beam shaping component configured to receive the first and second light beams from different incident angles at substantially the same position and to generate an output light beam having a predetermined intensity profile along the horizontal axis from the first and second light beams. A method for irradiating a sample in a flowstream with the output light beam is also disclosed. Also provided are kits having one or more lasers and beam shaping components configured to generate an output light beam having a predetermined intensity profile along the horizontal axis from the first and second light beams.

[0005] In the embodiment, the output light beam is generated by a beamforming component that receives a first light beam from a first laser and a second light beam from a second laser at substantially the same location from different angles of incidence. The beamforming component is configured to generate an output light beam from the first and second light beams having a predetermined intensity profile along the horizontal axis. The first and second light beams are received by the beamforming component at substantially the same location, such as on the surface of the beamforming component or within the beamforming component (e.g., more than 1 mm within the beamforming component). The first and second light beams are received by the beamforming component at different angles of incidence, including, for example, more than 1 arc minute, more than 5 arc minutes, more than 10 arc minutes, and more than 25 arc minutes.

[0006] To propagate a first light beam from a first laser and a second light beam from a second laser to a beam shaping component, a system according to some embodiments includes a mirror component comprising a first mirror and a second mirror positioned to propagate light from the first mirror to a beam combiner. In some cases, the second mirror is positioned to propagate light orthogonally from the first mirror. In other cases, the second mirror is positioned to propagate light orthogonally from the light beam from the laser. In some cases, the first and second light beams are combined with a dichroic beam combiner. In certain cases, the light from the beam combiner is propagated directly to the beam shaping component (i.e., without further optical changes).

[0007] In some embodiments, the beam shaping component is configured to generate an output optical beam having intensity profiles for a first optical beam and a second optical beam, with a central beam intensity profile where the intensity at the edge along the horizontal axis is 75% to 99.9%. In some cases, the central beam intensity profile of the output optical beam is 90% to 99.9% of the edge intensity of the output optical beam along the horizontal axis. In some cases, the beam shaping component is configured to generate an output optical beam having a top-hat intensity profile along the horizontal axis. In other cases, the beam shaping component is configured to generate an output optical beam having a super-Gaussian intensity profile along the horizontal axis. In these embodiments, the beam shaping component may include a diffractive optical system, a refractive optical system, or a lens array such as a cylindrical lens array. In some embodiments, the beam shaping component is a laser line generator lens, such as a Powell lens. In some cases, the laser line generator lens is positioned to receive two or more different optical beams at substantially the same position at different incident angles and generate an output optical beam having a central beam profile intensity where the intensity at the edge along the horizontal axis is 75% to 99.9%. In one embodiment, the subject system comprises only a single Powell lens and is configured to receive two or more different light beams at substantially the same position with different incident angles and to generate an output light beam having a predetermined beam intensity profile (e.g., a top-hat beam intensity profile) along the horizontal axis.

[0008] In some embodiments, the beam shaping component is configured to generate an output light beam having intensity profiles for a first and second light beam, with a central beam intensity profile where the intensity at the edge along the vertical axis is 75% to 99.9%. In some cases, the central beam intensity profile of the output light beam is 90% to 99.9% of the edge intensity of the output light beam along the vertical axis. In some cases, the beam shaping component is configured to generate an output light beam having a top-hat intensity profile along the vertical axis. In other cases, the beam shaping component is configured to generate an output light beam having a super-Gaussian intensity profile along the vertical axis. In these embodiments, the beam shaping component may include a diffractive optical system, a refractive optical system, or a lens array such as a cylindrical lens array. In some embodiments, the beam shaping component is a Powell lens. In some cases, the Powell lens is positioned to receive two or more different light beams at substantially the same position at different angles of incidence and generate an output light beam having a central beam profile intensity where the intensity at the edge along the vertical axis is 75% to 99.9%. In one embodiment, the beam shaping component is configured to generate an output light beam having a Gaussian distribution along the vertical axis.

[0009] In some embodiments, the subject system includes a flowstream configured to propagate a sample within the flowstream. In some cases, the flowstream includes a core stream and a stacked sheath stream. In these cases, the beam shaping component according to some embodiments is configured to produce an output light beam having a beam profile that is 50% to 99.9% of the spatial width of the core stream along the horizontal axis. In some cases, the beam shaping component is configured to produce an output light beam having an intensity profile that is substantially the same across 50% to 99.9% of the core stream along the horizontal axis. In some cases, the beam shaping component is configured to produce an output light beam having a top-hat intensity profile across 50% to 99.9% of the core stream along the horizontal axis. In other cases, the beam shaping component is configured to produce an output light beam having a super-Gaussian intensity profile across 50% to 99.9% of the core stream along the horizontal axis.

[0010] The subject system includes two or more lasers. In embodiments, the lasers may be independent, continuous, or pulsed lasers. In some cases, one or more lasers are continuous diode lasers, such as ultraviolet diode lasers, visible diode lasers, and near-infrared diode lasers. For example, the diode lasers may be 405 nm diode lasers or 488 nm diode lasers. In other cases, one or more lasers are solid-state lasers, such as pulsed lasers, such as Nd:YAG pulsed lasers or Ti sapphire pulsed lasers. In some embodiments, the system includes multiple lasers. In some embodiments, the beam shaping component is configured to generate output light beams having a predetermined intensity profile along the horizontal axis from multiple lasers. In one embodiment, the beam shaping component is configured to generate multiple output light beams having substantially the same intensity profile along the horizontal axis. In one example, the beam shaping component is configured to generate multiple output light beams having a top-hat intensity profile along the horizontal axis. In another example, the beam shaping component is configured to generate multiple output light beams having a super-Gaussian intensity profile along the horizontal axis. In other embodiments, the beam shaping component is configured to generate a first output laser beam having a first intensity profile along the horizontal axis and a second output laser beam having a second intensity profile along the horizontal axis. In these embodiments, one or more output laser beams may have a top-hat intensity profile along the horizontal axis. In other embodiments, one or more output laser beams may have a super-Gaussian intensity profile along the horizontal axis.

[0011] In some embodiments, the system includes a photosensor, such as a photodetector, for measuring light from a sample in a flowstream. The sensor may be configured to detect forward-scattered light, side-scattered light, transmitted light, emitted light, or a combination thereof. The optical signal may be detected continuously or at periodic intervals. In some embodiments, the sensor is a position-sensing detector, such as a quadrant photodiode or a photodiode array consisting of multiple detectors. Among the multiple detectors, there may be one or more solid-state detectors, such as avalanche photodiodes. In one example, the detector array consists of multiple solid-state detectors, such as an array of avalanche photodiodes.

[0012] Aspects of the present disclosure also include methods for irradiating a sample in a flowstream with an output light beam having a predetermined intensity profile along the horizontal axis, generated from a first light beam and a second light beam. A method according to one embodiment includes irradiating a sample in a flowstream with a first light beam and a second light beam through a beam-shaping component that receives the first light beam and the second light beam at substantially the same position from different angles of incidence. Light from the sample in the flowstream can be detected by forward scattering, side scattering, transmitted light, emission, or a combination thereof. In one embodiment, the method includes a multi-photon count of photons from the light from the sample in the flowstream. In one embodiment, the sample in the flowstream includes cells, and the method includes characterizing one or more cells or extracellular vesicles of cells in the sample. Characterizing extracellular vesicles of cells may include identifying the type of extracellular vesicle within the cell and / or determining the size of the extracellular vesicle within the cell.

[0013] A kit containing one or more components of the subject system is also provided. In one embodiment The kit comprises one or more lasers, a mirror component having a first mirror and a second mirror positioned to propagate light from the first mirror to a beam combiner, and a beam shaping component configured to generate an output laser beam having a predetermined intensity profile along the horizontal axis from the first and second lasers.

[0014] The kit may also include one or more mirrors and a support stage for coupling with the beam shaping components. In one embodiment, the support stage includes a motor, such as a stepper motor. The kit of the subject may also include other beam shaping components, such as focusing lenses, collimators, beam splitters, wavelength separators, or combinations thereof. The kit may also include an optical relay system for propagating light from the sample in the flow stream to the detector, such as a free-space optical relay system or optical fibers (e.g., an optical fiber optical relay bundle). [Brief explanation of the drawing]

[0015] The present invention can be best understood from the following detailed description, when read in conjunction with the accompanying drawings. The drawings include the following figures.

[0016] [Figure 1] The diagram illustrates a comparison between an output light beam having a Gaussian intensity profile and an output light beam having a top-hat intensity profile according to one embodiment. A) shows a plot of the relative intensity of the light beam as a function of the beam spot and distance from the optical axis for an output light beam having a Gaussian intensity profile, and B) shows a plot of the relative intensity of the light beam as a function of the beam spot and distance from the optical axis for an output light beam having a top-hat intensity profile. [Figure 2] A system having multiple lasers and a single beam shaping component according to one embodiment is illustrated. [Figure 3A]Illustrated is an optical beam passing through a beam shaping component to generate an output optical beam having a modified intensity profile along a horizontal axis, according to one embodiment. [Figure 3B] Illustrated is an optical beam passing through a beam shaping component to generate an output optical beam having a modified intensity profile along a horizontal axis, according to one embodiment. [Figure 4A] Illustrated are the intensity profiles of five lasers having different wavelengths propagated through a single beam shaping lens, and the intensity profile of an optical beam when multiple optical beams are received at the same position on a beam shaping component (e.g., a Powell lens). [Figure 4B] Illustrated are the intensity profiles of five lasers having different wavelengths propagated through a single beam shaping lens, and the intensity profile of an optical beam when multiple optical beams are received at the same position on the beam shaping component at different angles of incidence. [Figure 4C] Illustrated is a zoom view of the intensity profile when multiple optical beams are received at the same position on a beam shaping component, and the intensity profiles of five lasers having different wavelengths propagated through a single beam shaping lens. [Figure 4D] Illustrated is the intensity profile of an output beam having a super-Gaussian intensity profile along a horizontal axis, and beam profiles having different input beam sizes, according to an embodiment. [Figure 4E] Illustrated is the intensity profile of an output beam having a super-Gaussian intensity profile along a horizontal axis, and the normalized intensity of beam profiles having different input beam sizes, according to an embodiment. [Figure 5A] Illustrated is the irradiation of a flow stream by three lasers passing through a single beam component, and optical beams propagated along the longitudinal axis of the flow stream at different positions through a single beam shaping component. [Figure 5B]Illustrates the irradiation of a frost stream by three lasers passing through a single beam component, and illustrates a cross-section of the frost stream irradiated by three lasers through a single beam shaping component. [Figure 6] Illustrates a system having three lasers configured to irradiate a frost stream through a single beam shaping component.

Mode for Carrying Out the Invention

[0017] Aspects of the present disclosure include systems having multiple lasers with modified beam profiles. A system according to a particular embodiment includes a first laser that generates a first light beam, a second laser that generates a second light beam, and a beam shaping component configured to receive the first light beam and the second light beam at substantially the same position from different angles of incidence and generate an output light beam having a predetermined intensity profile along a horizontal axis from the first light beam and the second light beam. A method for irradiating a sample in a frost stream with the output light beam is also disclosed. Also provided is a kit having one or more lasers and a beam shaping component configured to generate an output light beam having a predetermined intensity profile along a horizontal axis from the first light beam and the second light beam.

[0018] Before the present invention is described in more detail, it is to be understood that the present invention is not limited to the specific embodiments described, and thus may of course vary. It is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0019] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediate value up to one-tenth of the lower limit unit between the upper and lower limits of that range and any other stated or intermediate value within that stated range is included within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, subject to any specifically excluded restrictions within the stated range, and are also included within the invention. If a stated range includes one or both of the restrictions, the range excluding either or both of those included restrictions is also included within the invention.

[0020] In this specification, the term “approximately” is used to indicate a specific range before a number. In this specification, the term “approximately” is used to provide literal support for the exact number it precedes and for any number that is close to or approximates the number preceded by the term. When determining whether a number is close to or approximates a specifically enumerated number, a close or approximate unenumerated number may, in the context in which it is presented, provide a substantial equivalent of the specifically enumerated number.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may also be used in carrying out or testing the present invention, but representative exemplary methods and materials are described herein.

[0022] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the cited publications. Any citation of any publication is for its disclosure prior to the filing date and shall not be construed as acknowledging that the present invention does not have prior rights to such publication by prior invention. Furthermore, the publication dates provided may differ from the actual publication dates which may need to be independently verified.

[0023] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context otherwise explicitly indicates. It should also be noted that the claims may be drafted to exclude any element. Therefore, this statement is intended to function as an antecedent for the use of exclusive terms such as “simply,” “only,” etc., in relation to the enumeration of elements of the claims or the use of “negative” restrictions.

[0024] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of the invention. Any enumerated method may be performed in the order of the enumerated events, or in any other logically possible order.

[0025] Apparatus and methods are described or will be described for grammatical fluidity with functional description, but claims should not necessarily be construed as being limited by constructing “means” or “step” limitations unless expressly formulated under 35 U.S. SC § 112, and should be granted the meaning of the definitions provided by the claims and the full scope of the equivalent under the judicial theory of equivalents, and if the claims are expressly formulated under 35 U.S. SC § 112, it should be explicitly understood that a full statutory equivalent should be granted under 35 U.S. SC § 112.

[0026] As summarized above, the Disclosure provides a system having multiple lasers having modified beam profiles. In a further description of embodiments of the Disclosure, a system having two or more lasers and a beam shaping component configured to generate from two or more laser output light beams having predetermined intensity profiles along the horizontal axis is first described in more detail. Next, a method for irradiating a sample in a flow stream with the output light beams is described. A kit having one or more lasers and a beam shaping component configured to produce output light beams having predetermined intensity profiles along the horizontal axis is also provided.

[0027] A system for producing an output laser beam with a predetermined intensity profile, and multiple laser beams. Aspects of this disclosure include systems for generating laser beams having modified intensity profiles. A system according to a particular embodiment includes a first laser for generating a first optical beam, a second laser for generating a second optical beam, and a beamforming component configured to receive the first and second optical beams from different incident angles at substantially the same position and to generate an output optical beam from the first and second optical beams having a predetermined intensity profile along the horizontal axis. The term “beamforming” is used herein, in its conventional sense, to mean that the optical beam profiles from each laser along one or more of the horizontal and vertical axes are modified as desired. As will be described in more detail below, the beamforming component is configured in embodiments to generate an optical beam having a predetermined intensity profile along one or more of the horizontal and vertical axes. The beamforming component is configured to generate an output optical beam having a beam profile along one or more of the horizontal and vertical axes, with a central intensity of 75% to 99.9% of the edge intensity. In some embodiments, the beam shaping component is configured to produce an output optical beam having a beam profile with substantially constant intensity from each edge to the center, including, for example, when the intensity across the horizontal axis of the beam profile varies by 10% or less, e.g., 9% or less, e.g., 8% or less, e.g., 7% or less, e.g., 6% or less, e.g., 5% or less, e.g., 4% or less, e.g., 3% or less, e.g., 2% or less, e.g., 1% or less, e.g., 0.5% or less, e.g., 0.05% or less, e.g., 0.01% or less, and when the intensity across the horizontal axis of the beam profile varies by 0.001% or less.In other embodiments, the beam shaping component is configured to generate an output optical beam having a beam profile with substantially constant intensity from each edge to the center, including, for example, when the intensity along the vertical axis of the beam profile varies by 10% or less, e.g., 9% or less, e.g., 8% or less, e.g., 7% or less, e.g., 6% or less, e.g., 5% or less, e.g., 4% or less, e.g., 3% or less, e.g., 2% or less, e.g., 1% or less, e.g., 0.5% or less, e.g., 0.05% or less, e.g., 0.01% or less, and when the intensity along the vertical axis of the beam profile varies by 0.001% or less.

[0028] The intensity of the output light beam can be measured using any convenient protocol, including, but not limited to, other types of photodetectors, such as scanning slit profilers, charge-coupled devices (CCDs, e.g., enhanced charge-coupled devices, ICCDs), positioning sensors, power sensors (e.g., thermoelectric sensors), optical power sensors, energy meters, digital laser photodetectors, and laser diode detectors. In some cases, to determine the intensity profile of the output light beam, the relative intensity of each output laser light beam is plotted as a function of the distance from the optical axis (along the orthogonal horizontal axis) of the output light beam to determine the intensity profile at the point of illumination. In one embodiment, the deviation of relative intensity at a given distance from the optical axis is calculated to determine whether the beam profile of the output light beam exhibits substantially constant intensity from each edge to the center along the horizontal axis. In other embodiments, the deviation of relative intensity is calculated over the entire horizontal axis of the beam profile of the output light beam to determine whether the output light beam exhibits substantially constant intensity from the edge to the center.

[0029] In one embodiment, the beamforming component is configured to produce an output light beam having a top-hat intensity profile along the horizontal axis. The term “top-hat” is used herein in its conventional sense to refer to an irradiation beam (e.g., light) having substantially uniform flow rate (energy density) along one or more axes perpendicular to the optical axis of the irradiation beam. In the embodiment, the output light beam having a top-hat intensity profile exhibits little to no deviation in relative intensity from each edge to the center along the horizontal axis, and the light beam having a top-hat intensity profile in question has an intensity at the center that is 95% to 99.9% of the intensity at the edges along the horizontal axis, and an intensity of 96% to 99.5% including, for example, 98% to 99% of the intensity at the edges along the horizontal axis.

[0030] In other embodiments, the beam shaping component is configured to produce an output optical beam having a super-Gaussian intensity profile along the horizontal axis. The term "super-Gaussian" is used herein, in its conventional sense, to refer to an irradiated beam having a slightly higher energy density at the center of the beam profile along one or more axes orthogonal to the optical axis of the irradiated beam. In embodiments, an output optical beam having a super-Gaussian intensity profile exhibits a greater flow rate at the edges of the beam along the horizontal axis than the corresponding Gaussian intensity profile. For example, an optical beam having a super-Gaussian intensity profile has an intensity at the edges along the horizontal axis that is 70% to 90% of the intensity at the center of the beam, and an intensity at the edges that is, for example, 80% to 90% of the intensity at the center of the beam along the horizontal axis, and is 75% to 85%.

[0031] Figures 1A) and 1B) illustrate a comparison of an output light beam with a Gaussian intensity profile and an output light beam with a top-hat intensity profile according to one embodiment. Figure 1A) shows a plot of the relative intensity of the light beam as a function of the beam spot and distance from the optical axis for an output light beam with a Gaussian intensity profile. Figure 1B) shows a plot of the relative intensity of the light beam as a function of the beam spot and distance from the optical axis for an output light beam with a top-hat intensity profile. As described above, a Gaussian-shaped beam exhibits greater relative intensity near the optical axis, with a decrease in intensity at the edges of the output light beam. On the other hand, a top-hat-shaped beam exhibits a nearly uniform flow rate, where the energy of the light beam at the optical axis is equal to the energy at the edges of the light beam.

[0032] In the subject system, the beam shaping component can be any convenient beam shaper that modifies the beam profile of a light beam over one or more of the horizontal and vertical axes. As described herein, the horizontal and vertical axes refer to axes perpendicular to the optical axis (i.e., beam path) of the light beam and, in embodiments, form the XY plane of the beam profile. As will be described in more detail below, the horizontal axis of the output light beam is perpendicular to the longitudinal axis of the flow stream (e.g., the flow stream in a flow cytometer), and the vertical axis of the output light beam is parallel to the longitudinal axis of the flow stream. In some embodiments, the beam shaping component modifies the beam profile of the light beam propagated over the horizontal axis. In other embodiments, the beam shaping component modifies the beam profile of the light beam propagated over the vertical axis. In yet another embodiment, the beam shaping component modifies the beam profile of the light beam propagated over both the horizontal and vertical axes. In these embodiments, the beam shaping component may include a diffractive optical system, a refractive optical system, or a lens array such as a cylindrical lens array. In some embodiments, the beam shaping component is an aspherical cylindrical lens having a right-angled cylindrical axis, such as a laser line generator lens (e.g., a Powell lens). Examples of laser line generator lenses, but not limited to these, include those described in U.S. Patents No. 4,826,299, No. 5,283,694, No. 7,400,457, and No. 7,329,860, the disclosures of which are incorporated herein by reference.

[0033] The beam shaping components to be used (e.g., cylindrical lenses, laser line generator lenses, Powell lenses) may be formed from any suitable material, but are not limited to, glass (e.g., N-SF10, N-SF11, N-SF57, N-BK7, N-LAK21, or N-LAF35 glass), silica (e.g., condensed silica), quartz, crystal (e.g., CaF2 crystal), zinc selenide (ZnSe), F2, germanium (Ge) titanate (e.g., S-TIH11), and borosilicate (e.g., BK7). In some embodiments, the beam shaping component has a transmittance window in the following wavelength ranges: 150 nm to 5 μm, 180 nm to 8 μm, 185 nm to 2.1 μm, 200 nm to 6 μm, 200 nm to 11 μm, 250 nm to 1.6 μm, 350 nm to 2 μm, 600 nm to 16 μm, 1.2 μm to 8 μm, 2 μm to 16 μm, or several other wavelength ranges. The refractive index of the beam shaping component in question may vary in the range of 1 to 3, e.g., 1.1 to 2.9, e.g., 1.2 to 2.8, e.g., 1.3 to 2.7, e.g., 1.4 to 2.6, e.g., 1.5 to 2.7, e.g., 1.6 to 2.6, e.g., 1.7 to 2.5, e.g., 1.8 to 2.4, and 1.9 to 2.3.

[0034] In some cases, the beam shaping component is positioned to receive two or more different light beams at substantially the same position at different incidence angles and to produce an output light beam with a beam profile intensity at the center that is 75% to 99.9% of the intensity at the edge along the horizontal axis. Depending on the laser in the subject system, the Powell lens may have a diameter that varies in range, including 2 mm to 15 mm, e.g., 2.5 mm to 14.5 mm, e.g., 3 mm to 14 mm, e.g., 3.5 mm to 13.5 mm, e.g., 4 mm to 13 mm, e.g., 4.5 mm to 12.5 mm, e.g., 5 mm to 12 mm, e.g., 5.5 mm to 11.5 mm, e.g., 6 mm to 11 mm, and 7 mm to 10 mm. The fan angle of the Powell lens can also vary, including Powell lenses having fan angles of 0.1° to 90°, e.g., 0.5° to 85°, e.g., 1° to 80°, e.g., 5° to 75°, e.g., 10° to 70°, e.g., 15° to 65°, and 20° to 60°. In one embodiment, the system of the subject comprises only a single beam-shaping optical component (e.g., a single Powell lens) and is configured to receive two or more different light beams at substantially the same position with different incident angles and to produce an output light beam having a predetermined beam intensity profile (e.g., a top-hat beam intensity profile) along the horizontal axis.

[0035] In the embodiment, the generated output light beam retains the power intensity of each laser received by the beam shaping component, and as a result, the power from each laser is reduced by 10% or less, including, for example, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, and 0.0001% or less. The generated output power of the light beam according to the embodiment can be determined by any convenient protocol, including, but not limited to, measuring the incident and output power with a power sensor (e.g., thermocouple power sensor), optical power sensor, energy meter, digital laser photodetector, laser diode detector, CCD or CMOS photodetector, among other types of photodetectors. To determine the change in power of the generated output light beam, the incident light on the beam-shaping component may be measured with one or more of the aforementioned power meters, such as a handheld optical power meter or a thermocouple power meter, and compared with the laser light power propagated through the beam-shaping component (i.e., the power of the generated output light beam).

[0036] In some embodiments, the beam shaping component is configured to produce an output light beam having a beam profile, wherein each light beam has a central intensity that is 50% to 99.9% of the intensity at the edge along the horizontal axis, for example 55% to 99%, for example 60% to 95%, for example 65% to 90%, for example 70% to 85%, and a central intensity that is 75% to 80% of the intensity at the edge along the horizontal axis. In one embodiment, the beam shaping component is configured to produce an output light beam having a beam profile, wherein each light beam has a central intensity that is 90% to 99.9% of the intensity at the edge along the horizontal axis. In the embodiment, the line length of 80% of the output light beam exhibits substantial uniformity, and the normalized intensity of the laser light along the line length of 80% of each output laser beam along the horizontal axis includes, for example, 4.5% or less, for example 4% or less, for example 3.5% or less, for example 3% or less, for example 2.5% or less, for example 2% or less, for example 1.5% or less, for example 1% or less, for example 0.5% or less, for example 0.1% or less, for example 0.01% or less, for example 0.001% or less, and 0.0001% or less, with deviations of 5% or less. The 80% line length in each output light beam along the horizontal axis exhibits deviations in contained power of 5% or less, including, for example, 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, and 0.0001% or less.

[0037] In some embodiments, the beam shaping component is configured to produce an output light beam having a beam profile, wherein each light beam has a central intensity that is 50% to 99.9% of the intensity at the edge along the vertical axis, for example 55% to 99%, for example 60% to 95%, for example 65% to 90%, for example 70% to 85%, and a central intensity that is 75% to 80% of the intensity at the edge along the horizontal axis. In one embodiment, the beam shaping component is configured to produce an output light beam having a beam profile, wherein each light beam has a central intensity that is 90% to 99.9% of the intensity at the edge along the vertical axis. In the embodiment, the line length of 80% of the output light beam exhibits substantial uniformity, and the normalized intensity of the laser light along the line length of 80% of each output laser beam along the vertical axis includes, for example, 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, and 0.0001% or less, with deviations of 5% or less. The 80% line length in each output light beam along the vertical axis exhibits deviations in contained power of 5% or less, including, for example, 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, and 0.0001% or less.

[0038] As described above, the beam shaping components of the subject are configured to produce an output light beam having a beam profile that exhibits substantially constant intensity from each edge to the center along one or more of the horizontal and vertical axes. In some embodiments, incident light beams from two or more lasers exhibit a beam profile that is not substantially constant from the edge to the center. In some cases, incident light beams from two or more lasers exhibit independently distinct beam profiles, and the intensity at the center of the beam is greater than the intensity at the edges of the beam, for example, by 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, 99% or more, 2 times or more, 3 times or more, and 5 times or more. In other embodiments, incident light beams from two or more lasers exhibit independently distinct beam profiles, and the intensity at the edges is greater than the intensity at the center, for example, by 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, 99% or more, 2 times or more, 3 times or more, and 5 times or more. In some embodiments, the incident light beam exhibits a Gaussian beam profile.

[0039] In the embodiment, the system includes two or more lasers, each outputting a light beam that is received at substantially the same location by a beam-forming component. "Substantially the same location" means that each light beam propagates through a common location within the beam-forming component, in one case the light beams are received by the beam-forming component at a distance of, for example, 0.5 mm or less, 0.1 mm or less, 0.05 mm or less, 0.01 mm or less, 0.005 mm or less, 0.001 mm or less, and within 0.0001 mm of each other, and within 1 mm of each other. In other cases, each light beam from the two or more lasers overlaps in the beam-forming component, including overlaps of, for example, 0.001 μm or more, 0.005 μm or more, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, and 100 μm or more.

[0040] In some embodiments, a beamforming component receives light beams from two or more lasers at the same location on the surface of the beamforming component. The “surface” of the beamforming component means the space in which the beamforming component is in contact with the external environment and can extend from about 0.0001 mm to about 0.1 mm on the beamforming component, for example, 0.0005 mm to about 0.09 mm, for example, 0.001 mm to about 0.08 mm, for example, about 0.005 mm to about 0.07 mm, for example, about 0.01 mm to about 0.05 mm, etc. In some cases, the system of the subject includes two lasers, and the beamforming component receives a first light beam from the first laser and a second light beam from the second laser at the same location on the surface of the beamforming component. In other cases, the system of the subject includes three lasers, and the beamforming component receives a first light beam from the first laser, a second light beam from the second laser, and a third light beam from the third laser at the same location on the surface of the beamforming component. In yet another example, the subject system includes four lasers, and the beamforming component receives a first light beam from the first laser, a second light beam from the second laser, a third light beam from the third laser, and a fourth light beam from the fourth laser at the same location on the surface of the beamforming component. It receives the fourth light beam. In yet another example, the subject system includes five lasers, and the beamforming component receives the first light beam from the first laser, the second light beam from the second laser, the third light beam from the third laser, the fourth light beam from the fourth laser, and the fifth light beam from the fifth laser at the same location on the surface of the beamforming component.

[0041] In other embodiments, the beamforming component receives light beams from two or more lasers at the same location within the beamforming component. For example, the beamforming component may be positioned to receive light beams from two or more lasers at locations of 0.1 mm or more within the beamforming component, including 0.2 mm or more, e.g., 0.3 mm or more, e.g., 0.4 mm or more, e.g., 0.5 mm or more, e.g., 0.6 mm or more, e.g., 0.7 mm or more, e.g., 0.8 mm or more, e.g., 0.9 mm or more, e.g., 1 mm or more, e.g., 1.5 mm or more, e.g., 2 mm or more, and 2.5 mm or more. Depending on the size of the beamforming component, the beamforming component may be positioned to receive light beams from two or more lasers at positions within the beamforming component that include 15 mm or less, e.g., 14 mm or less, e.g., 13 mm or less, e.g., 12 mm or less, e.g., 11 mm or less, e.g., 10 mm or less, e.g., 9 mm or less, e.g., 8 mm or less, e.g., 7 mm or less, e.g., 6 mm or less, e.g., 5 mm or less, e.g., 4 mm or less, e.g., 3 mm or less, e.g., 2 mm or less, and 1 mm or less. In some cases, the system of the subject includes two lasers, and the beamforming component receives a first light beam from the first laser and a second light beam from the second laser at the same position within the beamforming component that is 0.1 mm or more. In other cases, the system of the subject includes three lasers, and the beamforming component receives a first light beam from the first laser, a second light beam from the second laser, and a third light beam from the third laser at the same position within the beamforming component that is 0.1 mm or more. In yet another example, the subject system includes four lasers, and the beamforming component receives a first light beam from the first laser, a second light beam from the second laser, a third light beam from the third laser, and a fourth light beam from the fourth laser at the same location, which is more than 0.1 mm within the beamforming component.In yet another example, the subject system includes five lasers, and the beamforming component receives a first light beam from the first laser, a second light beam from the second laser, a third light beam from the third laser, a fourth light beam from the fourth laser, and a fifth light beam from the fifth laser at the same location, which is more than 0.1 mm within the beamforming component.

[0042] Light from each laser strikes the beamforming component at an incident angle that varies in the range of 0.1° to 60°, including, for example, 5° to 55°, for example, 10° to 50°, and 15° to 45°. In the embodiment, the beamforming component receives light beams from each laser at different incident angles, and the incident angles on the beamforming component for each light beam include, for example, 2 arc minutes or more, for example, 3 arc minutes or more, for example, 5 arc minutes or more, for example, 10 arc minutes or more, for example, 15 arc minutes or more, for example, 20 arc minutes or more, for example, 25 arc minutes or more, for example, 50 arc minutes or more, for example, 75 arc minutes or more, for example, 100 arc minutes or more, for example, 150 arc minutes or more, and locations where the incident angles on the beamforming component by each laser differ by 250 arc minutes or more, and by 1 arc minute or more. Depending on the type of beamforming component, the incidence angles on the beamforming component relative to the light beam are, in some embodiments, different from each other, up to 500 arc minutes, including, for example, 450 arc minutes or less, 400 arc minutes or less, 350 arc minutes or less, 300 arc minutes or less, 250 arc minutes or less, 200 arc minutes or less, 150 arc minutes or less, 100 arc minutes or less, 50 arc minutes or less, and 25 arc minutes or less.

[0043] As will be described in more detail below, each output light beam propagated through the beam shaping component can be projected to a different position in the flow stream. For example, each output light beam, The projections can be projected onto the flowstream at different positions along the longitudinal axis of the flowstream (for example, at intervals of 1 mm or more and 2 mm or more along the longitudinal axis, as detailed below). Depending on the refractive index and incident angle of the beamforming component, the refraction angle of each output light beam can vary in the range of 0.1° to 60°, including, for example, 5° to 55°, for example, 10° to 50°, and 15° to 45°. In embodiments, the refraction angles of each output light beam can differ from each other by 1 arc minute or more, including, for example, 2 arc minutes or more, for example, 3 arc minutes or more, for example, 5 arc minutes or more, for example, 10 arc minutes or more, for example, 15 arc minutes or more, for example, 20 arc minutes or more, for example, 25 arc minutes or more, for example, 50 arc minutes or more, for example, 75 arc minutes or more, for example, 100 arc minutes or more, for example, 150 arc minutes or more, and cases where the incident angle on the beamforming component by each laser differs by 250 arc minutes or more. In some cases, the refraction angles of each output light beam include, for example, 400 arc minutes or less, 300 arc minutes or less, 250 arc minutes or less, 200 arc minutes or less, 150 arc minutes or less, 100 arc minutes or less, 75 arc minutes or less, 50 arc minutes or less, 25 arc minutes or less, 20 arc minutes or less, 15 arc minutes or less, 10 arc minutes or less, 5 arc minutes or less, 3 arc minutes or less, 2 arc minutes or less, and 1 arc minute or less, and differ from each other by 500 arc minutes or less.

[0044] In some embodiments, the beamforming component may be spatially adjustable. The beamforming component may be adjusted to change its horizontal position, its vertical position, its orientation angle, or a combination thereof. In some embodiments, the beamforming component is configured to be spatially adjustable and to change its horizontal position (e.g., in the XY plane). For example, the horizontal position of the beamforming component may be moved by 0.0001 mm or more, including moving the horizontal position of the beamforming component by 0.0001 mm or more, including moving it by 0.0005 mm or more, 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 10 mm or more, and moving the horizontal position of the beamforming component by 25 mm or more. Depending on the size of the beam forming component, the horizontal position of the beam forming component may be configured in some embodiments to move by 50 mm or less, including, for example, 40 mm or less, 30 mm or less, 20 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, and 1 mm or less.

[0045] In other embodiments, the beam forming component is spatially adjustable and configured to change the vertical position of the beam forming component. For example, the vertical position of the beam forming component can be moved by, for example, 0.0005 mm or more, for example 0.001 mm or more, for example 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 2 mm or more, for example 3 mm or more, for example 4 mm or more, for example 5 mm or more, for example 10 mm or more, and by 0.0001 mm or more, including moving the vertical position of the beam forming component by 25 mm or more. Depending on the size of the beam forming component, the vertical position of the beam forming component may be configured in some embodiments to move by 50 mm or less, including, for example, 40 mm or less, 30 mm or less, 20 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, and 1 mm or less.

[0046] In other embodiments, the beam forming component is spatially adjustable and configured to change the orientation angle of the beam forming component. For example, the beam forming component can be, for example, 0.0005° or more, for example 0.001° or more, for example 0.005° or more, for example 0 The beam forming component may be rotated by 0.0001° or more (for example, in the XY plane, XZ plane, or YZ plane), including rotating it by 0.0001° or more, e.g., 0.05° or more, e.g., 0.1° or more, e.g., 0.2° or more, e.g., 0.3° or more, e.g., 0.4° or more, e.g., 0.5° or more, e.g., 1° or more, e.g., 2° or more, e.g., 3° ​​or more, e.g., 4° or more, e.g., 5° or more, e.g., 10° or more, e.g., 15° or more, e.g., 20° or more, e.g., 25° or more, e.g., 30° or more, and 45° or more, e.g., 35° or more, e.g., 20° or more, e.g., 25° or more, e.g., 30° or more, and 45° or more, e.g., 0.0001° or more (for example, in the XY plane, XZ plane, or YZ plane). In some embodiments, the beam forming component may be configured to rotate by 60° or less, e.g., 55° or less, e.g., 50° or less, e.g., 55° or less, e.g., 45° or less, e.g., 35° or less, e.g., 30° or less, e.g., 20° or less, e.g., 15° or less, e.g., 10° or less, e.g., 5° or less, and 1° or less.

[0047] In one embodiment, the beamforming component is configured to rotate by 0.001 arc minutes or more (for example, in the XY plane, XZ plane, or YZ plane), including, for example, 0.005 arc minutes or more, for example, 0.01 arc minutes or more, for example, 0.05 arc minutes or more, for example, 0.1 arc minutes or more, for example, 0.5 arc minutes or more, for example, 1 arc minute or more, for example, 2 arc minutes or more, for example, 3 arc minutes or more, for example, 4 arc minutes or more, for example, 5 arc minutes or more, for example, 6 arc minutes or more, for example, 7 arc minutes or more, for example, 8 arc minutes or more, for example, 9 arc minutes or more, and 10 arc minutes or more.

[0048] In one example, the beam shaping component is configured to fine-tune the laser beam position on the flowstream and is configured to rotate by 5 arc minutes or less (for example, in the XY plane, XZ plane, or YZ plane), including, for example, 4.5 arc minutes or less, 4 arc minutes or less, 3.5 arc minutes or less, 3 arc minutes or less, 2.5 arc minutes or less, 2 arc minutes or less, 1.5 arc minutes or less, 1 arc minute or less, 0.5 arc minutes or less, 0.1 arc minutes or less, 0.05 arc minutes or less, 0.01 arc minutes or less, 0.005 arc minutes or less, and 0.001 arc minutes or less.

[0049] The beam forming component is configured to be spatially adjustable, either continuously or in separate increments, including displacing the beam forming component along the horizontal axis in increments of, for example, 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, and in increments of 5 mm or more. In some embodiments, the beam forming component may be configured to be displaced in increments of 25 mm or less, including increments of, for example, 20 mm or less, 15 mm or less, 10 mm or less, 5 mm or less, 4.5 mm or less, 4 mm or less, 3.5 mm or less, 3 mm or less, 2.5 mm or less, 2 mm or less, 1.5 mm or less, 1 mm or less, and 1 mm or less.

[0050] In another example, the beam forming component is configured to be displaced along the vertical axis in increments of, for example, 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, and 5 mm or more, and is displaced along the vertical axis in separate increments. In some embodiments, the beam forming component is configured to be displaced in increments of 25 mm or less, including increments of, for example, 20 mm or less, 15 mm or less, 10 mm or less, 5 mm or less, 4.5 mm or less, 4 mm or less, 3.5 mm or less, 3 mm or less, 2.5 mm or less, 2 mm or less, 1.5 mm or less, 1 mm or less, and 1 mm or less.

[0051] In yet another example, the orientation angle of the beam-forming component is adjustable in separate increments, for example, 0.1° or more, including, for example, 0.2° or more, for example, 0.3° or more, for example, 0.4° or more, for example, 0.5° or more, for example, 1° or more, for example, 2° or more, for example, 3° or more, for example, 4° or more, and 5° or more. In another embodiment, the beam-forming component is adjustable in separate increments, for example, 10° or less, including, for example, 9° or less, for example, 8° or less, for example, 7° or less, for example, 6° or less, for example, 5° or less, for example, 4° or less, for example, 3° or less, for example, 2° or less, for example, 1° or less, for example, 0.5° or less, and 0.1° or less.

[0052] In yet another example, the beamforming component is configured for rotation in separate increments, for example, 0.0001 arc minutes or more, including, for example, 0.0005 arc minutes or more, for example, 0.001 arc minutes or more, for example, 0.005 arc minutes or more, for example, 0.01 arc minutes or more, for example, 0.05 arc minutes or more, for example, 0.1 arc minutes or more, for example, 0.5 arc minutes or more, for example, 1 arc minute or more, for example, 2 arc minutes or more, for example, 3 arc minutes or more, for example, 4 arc minutes or more, and 5 arc minutes or more. In another embodiment, the beamforming component is configured for rotation in separate increments, for example, 15 arc minutes or less, including, for example, 12.5 arc minutes or less, for example, 10 arc minutes or less, for example, 9 arc minutes or less, for example, 8 arc minutes or less, for example, 7 arc minutes or less, for example, 6 arc minutes or less, for example, 5 arc minutes or less, for example, 4 arc minutes or less, for example, 3 arc minutes or less, for example, 2 arc minutes or less, and 1 arc minute or less.

[0053] In some embodiments, the beam-forming component is movable (e.g., manually, mechanically, or by a motor-driven displacement device). In other embodiments, the beam-forming component is coupled to a movable support stage. In some cases, the beam-forming component is configured to be moved manually. In other cases, the beam-forming component is configured to be moved mechanically, such as by being directly coupled to a suitable actuator, such as a mechanical lead screw assembly or a mechanically operated geared converter, or by a mechanical lead screw assembly or a mechanically operated geared converter being coupled to a support stage. In yet another case, the beam-forming component is configured to move with a motor-driven displacement device, such as by being coupled to a motor-driven displacement stage, a motor-driven lead screw assembly, or a motor-operated geared actuator using, among other types of motors, a stepping motor, a servo motor, a brushless electric motor, a brushed DC motor, a microstepping motor, or a high-resolution stepping motor.

[0054] In some embodiments, the system is configured to be spatially adjustable to produce an output light beam having a spatial width of 50% to 99.9% of the width of the flowstream along the horizontal axis, including cases where the output light beam has a spatial width of 55% to 99%, 60% to 95%, 65% to 90%, 70% to 85%, and 75% to 95% of the width of the flowstream along the horizontal axis. In some embodiments, as will be described in more detail below, the flowstream has a core stream and a stacked sheath stream, and the system is configured to be spatially adjustable to produce an output light beam having a spatial width of 50% to 99.9% of the width of the corestream along the horizontal axis, including cases where the output light beam has a spatial width of 55% to 99%, 60% to 95%, 65% to 90%, 70% to 85%, and 75% to 95% of the width of the corestream along the horizontal axis. In these embodiments, the beam shaping component is configured to generate an output light beam having a substantially uniform intensity profile across 50% to 99.9% of the spatial width of the flowstream along the horizontal axis. In one embodiment, the beam shaping component is configured to generate an output light beam having a substantially uniform intensity profile across 50% to 99.9% of the spatial width of the corestream along the horizontal axis. In some cases, the beam shaping component is configured to be adjustable to generate an output light beam having a top-hat intensity profile traversing the corestream along the horizontal axis. In other cases, the beam shaping component is configured to be adjustable to generate an output light beam having a super-Gaussian intensity profile across the corestream along the horizontal axis.

[0055] In some embodiments, the subject system is configured to dynamically adjust the output beam to have a width that is between 50% and 99.9% of the spatial width of the flowstream. In these embodiments, the laser, one or more optical adjustment components, or beam shaping components may be adjusted to produce an output beam having a desired spatial width. In one example, the spatial width of the output beam is increased by 5% or more, including, for example, 10% or more, 25% or more, 50% or more, 75% or more, and 90% or more, in order to achieve a desired incident width on the flowstream. In another example, the spatial width of the output beam is decreased by 5% or more, including, for example, 10% or more, 25% or more, 50% or more, 75% or more, and 90% or more, in order to achieve a desired incident width on the flowstream. In some embodiments, the output optical beam is adjusted to match the spatial width of the flowstream. For example, the output optical beam may be adjusted to match the spatial width of the core stream of the flowstream.

[0056] Light from each laser can be propagated directly to a beam shaping component or through one or more optical tuning components. The term “optical tuning” is used herein, in its conventional sense, to refer to any device that can modify spatial width irradiation, or several other characteristics of irradiation from laser light, such as irradiation direction, wavelength, beam width, beam intensity, focus, and pulse width. An optical tuning protocol may be any convenient device that modifies one or more characteristics of a laser, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimating protocols, and combinations thereof. In some embodiments, the system in question includes one or more focusing lenses. In one example, the focusing lens may be a non-magnifying lens. In another example, the focusing lens is a magnifying lens. In other embodiments, the system in question includes one or more mirrors. In yet another embodiment, the system in question includes optical fibers. In some embodiments, the light beams from each laser are combined by a beam combiner, such as a dichroic mirror beam combiner. In these embodiments, the beam combiner combines the light beams from each laser and propagates the light to a beam shaping component.

[0057] In some embodiments, light from each laser is propagated to a beamforming component via a mirror component. In these embodiments, the mirror component may include a first mirror and a second mirror positioned to propagate light from a first mirror to the beamforming component. In embodiments, the second mirror is positioned to propagate light from the first mirror at an angle that varies with respect to the first mirror, for example, including 1° to 90°, 5° to 85°, 10° to 80°, 15° to 75°, 20° to 70°, 25° to 65°, and 30° to 60°. In some cases, the second mirror is positioned to propagate light orthogonally from the first mirror. In other embodiments, the second mirror is positioned to propagate light from the first mirror at angles that vary with respect to the laser, including, for example, 1° to 90°, 5° to 85°, 10° to 80°, 15° to 75°, 20° to 70°, 25° to 65°, and 30° to 60°. In some cases, the second mirror is positioned to propagate light orthogonally with respect to the laser. In some embodiments, the second mirror is also a beam combiner configured to combine light beams from two or more lasers. In these embodiments, the second mirror may be a dichroic mirror that selectively passes wavelengths of light as desired.

[0058] In some embodiments, the subject system does not include prisms for combining light from two or more lasers and propagating the combined light beam to a beam-shaping component. In these embodiments, the optical tuning component for combining two or more light beams from multiple lasers is prism-less and may use mirror components to propagate and combine light from each laser to the beam-shaping component, as described above.

[0059] In some embodiments, the optical adjustment component (e.g., one or more of the first and second mirrors of a mirror component) is movable. In some cases, the optical adjustment component is movable in two dimensions, for example, in the XY plane. In other cases, the optical adjustment component is movable in three dimensions. In one embodiment, the optical adjustment component is one or more mirrors of a mirror component configured to move to adjust the irradiation position on a beam shaping component. In some embodiments, one or more mirrors may be configured to move in the XY plane. In other embodiments, one or more mirrors may be configured to change their angle, for example, by tilting with respect to the laser or beam shaping component. For example, the system may be configured to change the irradiation position on the beam shaping component by changing the angle of the mirrors with respect to the laser by 5° or more, including, for example, 10° or more, for example 15° or more, for example 20° or more, for example 30° or more, for example 45° or more, for example 60° or more, and 75° or more.

[0060] If the optical adjustment components (e.g., one or more mirrors) are configured to move, the optical adjustment components may be configured to move continuously or at distinct intervals. In some embodiments, the movement of the optical adjustment components is continuous. In other embodiments, the optical adjustment components are movable at distinct intervals, including, for example, increments of 0.01 microns or more, 0.05 microns or more, 0.1 microns or more, 0.5 microns or more, 1 micron or more, 10 microns or more, 100 microns or more, 500 microns or more, 1 mm or more, 5 mm or more, 10 mm or more, and 25 mm or more.

[0061] Any displacement protocol may be used to move the optical adjustment component structure, such as by coupling it to a movable support stage or directly to a motor-driven translation stage, a lead screw translation assembly, or a geared conversion device using a stepping motor, servo motor, brushless electric motor, brushed DC motor, microstep drive motor, or high-resolution stepping motor, among other types of motors.

[0062] In embodiments, the beam shaping component receives light from two or more lasers, including, for example, three or more lasers, four or more lasers, five or more lasers, six or more lasers, and ten or more lasers. The lasers in question may include pulsed lasers or continuous-wave lasers. The type and number of lasers may vary and may be gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other cases, the system includes irradiating an acousto-optical device with a dye laser such as a stilbene, coumarin, or rhodamine laser. In further cases, the system includes metal vapor lasers such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper or gold lasers, and combinations thereof. In even further cases, the system includes ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, slim YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, and combinations thereof. In even further cases, the system includes semiconductor diode lasers, optically pumped semiconductor lasers (OPSLs), or implementations of any of the lasers described above at 2x or 3x frequency.

[0063] Depending on the desired wavelength of light produced by the output laser beam (for example, for use when irradiating a sample in a flow stream), each laser may have a specific wavelength varying between 200 nm and 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. The lasers may include any combination of several types of lasers. For example, in some embodiments, the system of the subject includes an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers. In some embodiments, the system of the subject includes an array of continuous-wave diode lasers.

[0064] Each laser may be configured to irradiate continuously or at separate intervals. In some cases, the system includes lasers configured to irradiate continuously, such as continuous-wave lasers. In other cases, the system in question includes lasers configured to irradiate at separate intervals, including 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 several other intervals. When each laser is configured to irradiate at separate intervals, the system may include one or more additional components to provide intermittent irradiation at each laser. For example, the system in question in these embodiments may include one or more laser beam choppers, manual or computer-controlled beam stop units for blocking and exposing beam shaping components at each laser.

[0065] In some embodiments, the system includes a flow cell configured to propagate a sample in a flow stream. Any convenient flow cell for propagating a fluid sample into a sample inspection area may be used, and in some embodiments, the flow cell includes a cylindrical flow cell, a frustoconical flow cell, or a distal conical portion terminating at a flat surface having an orifice that is transverse to the longitudinal axis.

[0066] In the embodiment, the sample flowstream is emitted from an orifice at the distal edge of the flow cell. Depending on the desired characteristics of the flowstream, the flow cell orifice may be any suitable shape, and the cross-sectional shape of the subject is not limited to these, but includes, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic base joined to the top of a plane. In one embodiment, the flow cell of the subject has a circular orifice. The nozzle orifice size can vary in some embodiments from 1 μm to 20000 μm, including, for example, 2 μm to 17500 μm, 5 μm to 15000 μm, 10 μm to 12500 μm, 15 μm to 10000 μm, 25 μm to 7500 μm, 50 μm to 5000 μm, 75 μm to 1000 μm, 100 μm to 750 μm, and 150 μm to 500 μm. In some embodiments, the nozzle orifice is 100 μm.

[0067] In some embodiments, the flow cell includes a sample injection port configured to supply the sample to the flow cell. In embodiments, the sample injection system is configured to provide a suitable flow of the sample into the internal chamber of the flow cell. Depending on the desired characteristics of the flow stream, the rate of the sample transmitted to the flow cell chamber by the sample injection port is, for example, 2 μL / min or more, for example, 3 μL / min or more, for example, 5 μL / min or more, for example, 10 μL / min The above includes, for example, 15 μL / min or more, for example, 25 μL / min or more, for example, 50 μL / min or more, and 100 μL / min or more, and may also be 1 μL / min or more. In some cases, the rate of the sample transmitted to the flow cell chamber by the sample injection port is 1 μL / second or more, including, for example, 2 μL / second or more, for example, 3 μL / second or more, for example, 5 μL / second or more, for example, 10 μL / second or more, for example, 15 μL / second or more, for example, 25 μL / second or more, for example, 50 μL / second or more, and 100 μL / second or more.

[0068] The sample injection port may be an orifice positioned in the wall of the internal chamber, or a conduit positioned at the proximal end of the internal chamber. If the sample injection port is an orifice positioned in the wall of the internal chamber, the sample injection port orifice may be any preferred shape, and the cross-sectional shape of the subject is not limited, but includes, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic base coupled to a plane top. In one embodiment, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in some cases it may have an opening in the range of 0.1 mm to 5.0 mm, including 1.25 mm to 1.75 mm, for example 0.2 to 3.0 mm, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, and 1.5 mm.

[0069] In some cases, the sample injection port is a conduit positioned at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a conduit positioned to have an orifice of the sample injection port along the flow cell orifice. If the sample injection port is a conduit positioned along the flow cell orifice, the cross-sectional shape of the sample injection tube may be any preferred shape, and the cross-sectional shapes in question are not limited to these, but include, for example, straight cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as, for example, a parabolic base joined to the top of a plane. The orifice of the conduit may vary depending on the shape, and in some cases, it may have an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and for example, 1.5 mm, including 1.25 mm to 1.75 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 in the range of 1° to 10°, including bevel angles of, for example, 2° to 9°, 3° to 8°, 4° to 7°, and 5°.

[0070] In some embodiments, the flow cell also includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to provide a flow of sheath fluid into the internal chamber of the flow cell, for example, together with the sample, to create a layered 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 transmitted to the flow cell chamber can be 25 μL / s or more, including, for example, 50 μL / s or more, 75 μL / s or more, 100 μL / s or more, 250 μL / s or more, 500 μL / s or more, 750 μL / s or more, 1000 μL / s or more, and 2500 μL / s or more.

[0071] In some embodiments, the sheath fluid injection port is an orifice positioned in the wall of the internal chamber. The sheath fluid injection port orifice may have any preferred shape, and the cross-sectional shape of the subject is not limited to these, but includes, for example, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons; curved cross-sectional shapes such as circles and ellipses; and irregular shapes such as a parabolic base coupled to a plane top. The size of the sample injection port orifice may vary depending on the shape, and in some cases, for example, 0.2 to 3.0 It has an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.5 mm to 2.5 mm, for example, 0.75 mm to 2.25 mm, for example, 1 mm to 2 mm, and 1.25 mm to 1.75 mm, such as 1.5 mm.

[0072] In some embodiments, the system further includes a pump that fluid-communicates with the flow cell to propagate a flow stream through the flow cell. Any convenient fluid pump protocol may be used to control the flow of the flow stream through the flow cell. In some cases, the system includes a peristaltic pump with a pulse damper, for example. The pump in the subject system is configured to transmit fluid through the flow cell at a rate suitable for multi-photon counting light from the sample in the flow stream. In some cases, the rate of sample flow in the flow cell is 1 nL / min or more, including, for example, 2 nL / min or more, for example, 3 nL / min or more, for example, 5 nL / min or more, for example, 10 nL / min or more, for example, 25 nL / min or more, for example, 50 nL / min or more, for example, 75 nL / min or more, for example, 100 nL / min or more, for example, 250 nL / min or more, for example, 500 nL / min or more, for example, 750 nL / min or more, and 1000 nL / min or more. For example, the system may include a pump configured to flow a sample through a flow cell at a rate in the range of 1 nL / min to 500 nL / min, including, for example, 1 nL / min to 250 nL / min, for example, 1 nL / min to 100 nL / min, for example, 2 nL / min to 90 nL / min, for example, 3 nL / min to 80 nL / min, for example, 4 nL / min to 70 nL / min, for example, 5 nL / min to 60 nL / min, and 10 nL / min to 50 nL / min. In one embodiment, the flow rate of the flow stream is 5 nL / min to 6 nL / min.

[0073] Figure 2 illustrates a system having multiple lasers and a single beam shaping component according to one embodiment. System 200 includes lasers 201a, 201b, 201c, 201d, and 201e, each having a different irradiation wavelength. Light from each of the lasers 201a, 201b, 201c, 201d, and 201e is irradiated through optical adjustment components 202a, 202b, 202c, 202d, 202e (e.g., prisms) and 203a, 203b, 203c, 203d, 203e (e.g., beam expanders, de-sensing lenses), each of which propagates the light from the lasers to mirror components including first mirrors 204a1, 204b1, 204c1, 204d1, and 204e1, which also serve as dichroic beam combiners, reflecting the light from the lasers to second mirrors 204a2, 204b2, 204c2, 204d2, and 204e2. The light from the beam combiners is received by a beam shaping component 206, which generates an output light beam with an intensity profile modified along the horizontal axis. The beam-shaped output light beam is focused onto the flow cell 208 by a focusing lens 207.

[0074] Figure 3A illustrates, according to one embodiment, a light beam passing through a beamforming component to generate an output light beam having a horizontally modified intensity profile. Light beams 301a, 301b, and 301c are received by the beamforming lens 300 at the same location on surface 302 at different angles of incidence. The beamforming lens 300 generates output light beams 301a1, 301b1, and 301c1 from the light beams 301a, 301b, and 301c, which have a horizontally modified intensity profile. Figure 3B illustrates, according to another embodiment, a light beam passing through a beamforming component to generate an output light beam having a horizontally modified intensity profile. Light beams 302a, 302b, and 302c are received by the beamforming lens 300a at the same location within the beamforming lens 304 at different angles of incidence. The beam shaping lens 300a generates output light beams 302a1, 302b1, and 302c1 from the light beams 302a, 302b, and 302c, which have an intensity profile modified along the horizontal axis.

[0075] Figures 4A to 4C show the propagation through a single beam-forming lens according to one embodiment. The intensity profiles of five lasers with different wavelengths are illustrated. Figure 4A illustrates the intensity profiles of the 355nm, 405nm, 488nm, 561nm, and 640nm lasers after propagation through a Powell lens. Each light beam from the laser is received by the Powell lens at the same position and angle of incidence. Figure 4B illustrates the intensity profiles of the 355nm, 405nm, 488nm, 561nm, and 640nm lasers after propagation through the Powell lens at the same position but with different angles of incidence. The light beam from the 488nm laser has an angle of incidence of 0 degrees, the light beam from the 355nm laser has an angle of incidence of -2 degrees, the light beam from the 405nm laser has an angle of incidence of -1 degree, the light beam from the 561nm laser has an angle of incidence of 1 degree, and the light beam from the 640nm laser has an angle of incidence of 2 degrees. Figure 4C shows zoomed-in intensity profiles of 355nm, 405nm, 488nm, 561nm, and 640nm lasers after propagation through a Powell lens at the same location and different incidence angles. The 80% line length of the output light beam exhibits substantial uniformity, for example, when the normalized intensity of the laser light along the 80% line length of each output laser beam along the vertical axis deviates by less than 1%. As shown in Figures 4A to 4C, propagating multiple light beams through the same location at different incidence angles results in a top-hat intensity profile, providing effective beam shaping.

[0076] Figures 4D and 4E illustrate the intensity profiles of an output beam with a super-Gaussian profile compared to an output beam with a Gaussian profile and a top-hat profile according to one embodiment. Figure 4D shows a comparison of beam profiles of an output beam with a super-Gaussian profile and an output beam with a superimposed Gaussian profile, where the beam diameter is reduced by 10% and 20%. Figure 4D also illustrates an output beam with a top-hat profile generated by a Powell lens from a Gaussian input beam. Figure 4E illustrates the output beam of Figure 4D with standard beam intensity to show a comparison of the intensity profiles of each type of beam along the horizontal axis.

[0077] In some embodiments, the flowstream is irradiated with an output light beam having a predetermined intensity profile along the horizontal axis. In some embodiments, the beam shaping component is configured to generate one or more output beams of light having spatial widths, for example, over 50% or more of the flowstream, for example 55% or more, for example 60% or more, for example 65% or more, for example 70% or more, for example 75% or more, for example 80% or more, for example 85% or more, for example 90% or more, for example 95% or more, for example 97% or more, for example 99% or more, and over 100% or more of the width of the flowstream (i.e., the horizontal axis). In one embodiment, one or more of the output light beams have a spatial width that extends beyond the width of the flow stream (i.e., extends more than 100% across the flow stream), including, for example, a spatial width that extends beyond the width of the flow stream by 5% or more of the width of the flow stream, for example, 10% or more, for example 15% or more, for example 20% or more, for example 25% or more, for example 30% or more, for example 35% or more, for example 40% or more, for example 45% or more, and a spatial width that extends beyond the width of the flow stream by 50% or more of the width of the flow stream. Depending on the width of the flowstream, the spatial width of each output light beam can be independently 0.00001 mm or more, including, for example, 0.00005 mm or more, for example 0.0001 mm or more, for example 0.0005 mm or more, for example 0.001 mm or more, for example 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 5 mm or more, for example 10 mm or more, and 25 mm or more. In some embodiments, the beam shaping component includes generating an output light beam having an intensity profile that is substantially the same over, for example 55% to 95%, for example 60% to 90%, for example 65% to 85%, and over 70% to 80% of the flowstream along the horizontal axis, and is configured to generate an output light beam having an intensity profile that is substantially the same over 50% to 99.9% of the flowstream along the horizontal axis.

[0078] As described above, the refraction angle of each output light beam may vary depending on the refractive index and incident angle of the beam shaping component. In embodiments, each output light beam may propagate from the beam shaping component to different locations along the longitudinal axis of the flowstream. For example, each output light beam may propagate along the flowstream at locations along the longitudinal axis that differ by 0.0001 mm or more, including, for example, 0.0005 mm or more, for example, 0.001 mm or more, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2 mm or more, for example, 3 mm or more, for example, 4 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 15 mm or more, and 25 mm or more. In one embodiment, each output light beam can propagate along a flowstream at positions along different longitudinal axes of 50 mm or less, including, for example, 25 mm or less, 15 mm or less, 10 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, 1 mm or less, 0.5 mm or less, 0.1 mm or less, and 0.001 mm or less.

[0079] Figure 5A illustrates the illumination of a flowstream by three lasers through a single beamforming component according to one embodiment. The optical beams 501a, 501b, and 501c propagate through the beamforming component 500 at substantially the same location at different angles of incidence. The respective output optical beams 502a, 502b, and 502c from the incident optical beams 501a, 501b, and 501c are focused by focusing the lens 503 onto the flowstream 504 at different locations along the longitudinal axis. Figure 5B illustrates a cross-section of the flowstream illuminated by the three lasers through a single beamforming component according to one embodiment. The respective beam spots from the output optical beams 502a, 502b, and 502c propagate onto the flowstream 504 at different locations along the longitudinal axis. As shown in Figure 5B, the beam spots of each output optical beam extend beyond the width of the flowstream.

[0080] Figure 6 illustrates a system having three lasers configured to irradiate a frost stream through a single beam shaping component. Lasers 601a, 601b, and 601c generate optical beams 602a, 602b, and 602c that propagate through optical adjustment components 603, 604, and 605 (e.g., focusing components, position control components, and polarization control components), and 606 to beam shaping component 607. Each optical beam 602a, 602b, and 602c is received by beam shaping component 607 at the same position with different angles of incidence. The output optical beam from beam shaping component 607 is focused by focusing lens 608 onto flow cell 609 at different positions along the longitudinal axis of frost stream 610.

[0081] The system of interest also includes one or more photodetectors for detecting optical signals from a sample irradiated with the frost stream. The optical detection protocol in the system of interest may be any convenient optical detection protocol including, but not limited to, 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, and combinations thereof. In one embodiment, the system of interest includes a photodiode array having two or more photodiodes, e.g., two or more, e.g., three or more, e.g., five or more, and ten or more photodiodes, each photodiode being, e.g., 0.05 cm 2 ~9 cm 2 e.g., 0.1 cm 2 ~8 cm 2 e.g., 0.5 cm [[ID=;13]] 2 ~7 cm 2 and 1 cm 2 ~5 cm 2 including, 0.01 cm 2 ~10 cm 2Each region may have an active detection surface area within that range.

[0082] The photodetector may be positioned at any suitable distance from the flowstream, as long as a usable optical signal is detectable. For example, the detector in the subject system may be positioned at least 1 mm from the flowstream, including, for example, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, 50 mm or more, 100 mm or more, 150 mm or more, 250 mm or more, and 500 mm or more from the flowstream. The detector may also be positioned at any angle from the flowstream. For example, the detector may be angled with respect to the vertical axis of the flowstream between 10° and 90°, including, for example, 15° to 85°, 20° to 80°, 25° to 75°, and 30° to 60°. In some cases, one or more detectors are positioned between 30° and 60° from the vertical axis of the flowstream.

[0083] In some embodiments, the system is configured to detect forward-scattered light, side-scattered light, emitted light, transmitted light, or a combination thereof. In some embodiments, an optical signal from an irradiated flowstream may be detected by one or more detectors configured as forward-scatter detectors. In these embodiments, the forward-scatter detectors are positioned on the opposite side of the flowstream from the light source and are positioned to collect and detect forward-propagated (e.g., scattered) light. In other embodiments, the system of the subject is configured to detect an optical signal from light propagated upstream by total internal reflection. In some embodiments, the system of the subject is configured with a flow cell nozzle described in U.S. Patent Publication No. 2014 / 0320861, filed April 23, 2014, the disclosure of which is incorporated herein by reference.

[0084] Aspects of the present invention further include a flow cytometry system having a plurality of lasers, as described above, and a beam shaping component configured to generate an output light beam having a predetermined beam profile intensity along the horizontal axis. Suitable flow cytometry systems are not limited to those mentioned above, but include: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan, 49(pt 1):17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30(5):502-11; Alison, et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier. This includes those described in Syst.24(3):203-255, the disclosures of which are incorporated herein by reference. In some cases, the flow cytometry systems covered include the BD Biosciences FACSCanto® flow cytometer, BD Biosciences FACSVantage®, BD Biosciences FACSort®, BD Biosciences FACSCount®, BD Biosciences FACScan®, and BD Biosciences FACSCalibur® systems, BD Biosciences Influx® cell sorters, BD Biosciences Jazz® cell sorters, and BD Biosciences Aria® cell sorters.

[0085] In some embodiments, the subject system is, for example, U.S. 3,960,449 No. 4,347,935, No. 4,667,830, No. 4,704,891, No. 4,770,992, No. 5,030,002, No. 5,040,890, No. 5,047,321, No. 5,245,318, No. 5,317 , No. 5,464,581, No. 5,483,469, No. 5,602,039, No. 5,620,842, No. 5,627,040, No. 5,643,796, No. 5,700,692, No. 6,372,506, No. 6, Flow cytometry systems, such as those described in Patent Nos. 809,804, 6,813,017, 6,821,740, 7,129,505, 7,201,875, 7,544,326, 8,140,300, 8,233,146, 8,753,573, 8,975,595, 9,092,034, 9,095,494, and 9,097,640, are incorporated herein by reference in their entirety.

[0086] In some embodiments, the subject system is a flow cytometry system having an excitation module that generates multiple frequency-shifted optical beams using radio frequency multiplexing. In these embodiments, the laser light generator may include multiple lasers and one or more acousto-optical components (e.g., acoustic deflectors, acoustic frequency shifters) for generating multiple frequency-shifted comb beams. One or more of the frequency-shifted comb beams and local oscillator beams may be configured to be received by beam-shaping components such as those described herein to generate one or more beams of frequency-shifted light having substantially constant intensity profiles. In some cases, the subject system is a flow cytometry system having a laser excitation module as described in U.S. Patent 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.

[0087] Method for irradiating a sample in a flowstream Aspects of the present disclosure also include a method for irradiating a sample in a flowstream with two or more lasers having a predetermined intensity profile along the horizontal axis. In embodiments, the method includes irradiating a sample in a flowstream with a first and a second optical beam through an optical shaping component configured to receive a first and a second optical beam from substantially the same position from different incident angles and to generate an output optical beam from the first and second optical beams having a predetermined intensity profile along the horizontal axis. As described above, the beam shaping component is configured to modify the optical beam profile from each laser along one or more of the horizontal and vertical axes. In practice of the method according to one embodiment, the generated output optical beam has a beam profile having a central intensity that is 75% to 99.9% of the edge intensity along the horizontal axis. In some embodiments, the method includes irradiating a beam shaping component to generate an output light beam having a beam profile with substantially constant intensity from each edge to the center, including, for example, when the intensity along the horizontal axis of the beam profile varies by 10% or less, such as, for example, 9% or less, for example, 8% or less, for example, 7% or less, for example, 6% or less, for example, 5% or less, for example, 4% or less, for example, 3% or less, for example, 2% or less, for example, 1% or less, for example, 0.5% or less, for example, 0.1% or less, for example, 0.05% or less, for example, 0.01% or less, and when the intensity along the horizontal axis of the beam profile varies by 0.001% or less. In some embodiments, the method includes irradiating a beam shaping component to generate an output light beam having a top-hat intensity profile along the horizontal axis.

[0088] When implementing the subject method, irradiating the beam shaping components with each laser means, for example, that each output light beam is less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% The power of each output light beam is not reduced, for example, when reduced by 10% or less, including 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, and 0.0001% or less. In the embodiment, the line length of 80% of the output light beam exhibits substantial uniformity, and for example, the normalized intensity of the laser light along the line length of 80% of each output laser beam deviates by 5% or less, including 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, and 0.0001% or less. The 80% line length in each output light beam exhibits deviations in contained power of 5% or less, including, for example, 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, 0.001% or less, and 0.0001% or less.

[0089] The beamforming component is irradiated by each laser at substantially the same position at different incident angles. As described above, each light beam propagates through common positions within the beamforming component, and in some cases the light beams are received by the beamforming component at positions of 1 mm or less from each other, including, for example, 0.5 mm or less, 0.1 mm or less, 0.05 mm or less, 0.01 mm or less, 0.005 mm or less, 0.001 mm or less, and 0.0001 mm or less from each other. In other cases, each light beam from two or more lasers overlaps in the beamforming component, including, for example, overlaps of 0.001 μm or more, 0.005 μm or more, 0.01 μm or more, 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 5 μm or more, 10 μm or more, and 100 μm or more.

[0090] In some cases, the method involves irradiating the beamforming component with each laser at a position within the beamforming component at a distance of approximately 0.0001 mm to approximately 0.1 mm from the surface of the beamforming component, for example, 0.0005 mm to approximately 0.09 mm, for example, approximately 0.001 mm to approximately 0.08 mm, for example, 0.005 mm to approximately 0.07 mm, for example, 0.01 mm to approximately 0.05 mm, etc. In other cases, the method involves irradiating the beamforming component with each laser at a position within the beamforming component at a distance of 0.1 mm or more, for example, 0.2 mm or more, for example, 0.3 mm or more, for example, 0.4 mm or more, for example, 0.5 mm or more, for example, 0.6 mm or more, for example, 0.7 mm or more, for example, 0.8 mm or more, for example, 0.9 mm or more, for example, 1 mm or more, for example, 1.5 mm or more, for example, 2 mm or more, and 2.5 mm or more. Depending on the size of the beamforming component, the method includes irradiating the beamforming component with each laser at positions within the beamforming component, including 15 mm or less, e.g., 14 mm or less, e.g., 13 mm or less, e.g., 12 mm or less, e.g., 11 mm or less, e.g., 10 mm or less, e.g., 9 mm or less, e.g., 8 mm or less, e.g., 7 mm or less, e.g., 6 mm or less, e.g., 5 mm or less, e.g., 4 mm or less, e.g., 3 mm or less, e.g., 2 mm or less, and 1 mm or less.

[0091] Each laser irradiates the beamforming component at different angles of incidence, such as angles in the range of 0.1° to 60°, including, for example, 5° to 55°, 10° to 50°, and 15° to 45°. In embodiments, the method includes irradiating the beamforming component at different angles of incidence, where the angles of incidence on the beamforming component for each light beam are, for example, 2 arc minutes or more, 3 arc minutes or more, 5 arc minutes or more, 10 arc minutes or more, 15 arc minutes or more, 20 arc minutes or more, 25 arc minutes or more, 50 arc minutes or more, 75 arc minutes or more, 100 arc minutes or more, 150 arc minutes or more, and This includes cases where the incident angles on the beamforming components by each laser differ by 250 arc minutes or more, and by 1 arc minute or more. In some embodiments, the method includes irradiating the beamforming components at different angles of 500 arc minutes or less, including, for example, 450 arc minutes or less, for example 400 arc minutes or less, for example 350 arc minutes or less, for example 300 arc minutes or less, for example 250 arc minutes or less, for example 200 arc minutes or less, for example 150 arc minutes or less, for example 100 arc minutes or less, for example 50 arc minutes or less, and 25 arc minutes or less.

[0092] Beam shaping components can be irradiated directly by each laser or through one or more optical tuning components. As described above, optical tuning may include changing the spatial width or several other characteristics of the irradiation from the laser light, such as the irradiation direction, wavelength, beam width, beam intensity, focus, and pulse width. Optical tuning protocols may include, but are not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimating protocols, and combinations thereof, to adjust one or more characteristics of each laser. In some embodiments, the light beams from each laser are combined, for example, by irradiating 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 components.

[0093] In some embodiments, light from each laser is propagated to a beamforming component through a mirror component. In these embodiments, the mirror component may include a first mirror and a second mirror positioned to propagate light from a first mirror to the beamforming component. In embodiments, the second mirror is positioned to propagate light from the first mirror at an angle that varies with respect to the first mirror, for example, including 1° to 90°, 5° to 85°, 10° to 80°, 15° to 75°, 20° to 70°, 25° to 65°, and 30° to 60°. In some cases, the second mirror is positioned to propagate light orthogonally from the first mirror. In other embodiments, the second mirror is positioned to propagate light from the first mirror at angles that vary with respect to the laser, including, for example, 1° to 90°, 5° to 85°, 10° to 80°, 15° to 75°, 20° to 70°, 25° to 65°, and 30° to 60°. In some cases, the second mirror is positioned to propagate light orthogonally with respect to the laser. In some embodiments, the second mirror is also a beam combiner configured to combine one or more light beams. In some cases, the second mirror is a dichroic beam combiner that selectively passes a desired wavelength of light.

[0094] The beamforming component may be irradiated by a laser at any suitable distance, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, and at a distance of 100 mm or more from the beamforming component, such as at a distance of 0.001 mm or more. When practicing the method of the subject according to one embodiment, the beamforming component may be irradiated by each laser continuously or at separate intervals. In some cases, the method includes irradiating the flowstream continuously through the beamforming component. In other cases, the method includes irradiating the flowstream through the beamforming component at separate intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at several other intervals. If a flowstream is irradiated through beam-shaping components at separate intervals, the frequency of irradiation may depend on the concentration of components in the flowstream (e.g., cells, beads, non-cell particles) and the flow rate of the flowstream.

[0095] In some embodiments, the method includes spatially adjusting the beamforming component. Depending on the embodiment, spatially adjusting the beamforming component may include changing the horizontal position of the beamforming component, changing the vertical position of the beamforming component, changing the orientation angle (e.g., rotation angle) of the beamforming component, or a combination thereof. In some embodiments, the beamforming component is spatially adjusted by changing the horizontal position of the beamforming component (e.g., in the XY plane). For example, the horizontal position of the beamforming component may be moved by 0.0001 mm or more, e.g., 0.0005 mm or more, e.g., 0.01 mm or more, e.g., 0.05 mm or more, e.g., 0.1 mm or more, e.g., 0.5 mm or more, e.g., 1 mm or more, e.g., 2 mm or more, e.g., 3 mm or more, e.g., 4 mm or more, e.g., 5 mm or more, e.g., 10 mm or more, and moving the horizontal position of the beamforming component by 25 mm or more, and by 0.0001 mm or more. In some embodiments, the method includes moving the horizontal position of the beam forming component by 50 mm or less, including, for example, 40 mm or less, 30 mm or less, 20 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, and 1 mm or less.

[0096] In other embodiments, the beam forming component is spatially adjusted by changing the vertical position of the beam forming component (for example, along the longitudinal axis). For example, the vertical position of the beam forming component can be moved by 0.0001 mm or more, including moving the vertical position of the beam forming component by 0.0001 mm or more, including moving it by 0.0005 mm or more, 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 10 mm or more, and 25 mm or more, including moving the vertical position of the beam forming component by 0.0001 mm or more. In some embodiments, the method includes moving the vertical position of the beam forming component by 50 mm or less, including, for example, 40 mm or less, 30 mm or less, 20 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, and 1 mm or less.

[0097] In other embodiments, the beamforming component is spatially adjusted by changing the orientation angle of the beamforming component. For example, the beamforming component may be rotated by, for example, 0.0005° or more, for example, 0.001° or more, for example, 0.005° or more, for example, 0.01° or more, for example, 0.05° or more, for example, 0.1° or more, for example, 0.2° or more, for example, 0.3° or more, for example, 0.4° or more, for example, 0.5° or more, for example, 1° or more, for example, 2° or more, for example, 3° or more, for example, 4° or more, for example, 5° or more, for example, 10° or more, for example, 15° or more, for example, 20° or more, for example, 25° or more, for example, 30° or more, and 45° or more, and may be rotated by 0.0001° or more (for example, in the XY plane, XZ plane, or YZ plane). In some embodiments, the beam forming component rotates by 60° or less, including, for example, 55° or less, 50° or less, 45° or less, 40° or less, 35° or less, 30° or less, 20° or less, 15° or less, 10° or less, 5° or less, and 1° or less.

[0098] In one embodiment, the beamforming component rotates in 0.001 arc minutes or more (for example, in the XY plane, XZ plane, or YZ plane), including, for example, 0.005 arc minutes or more, for example 0.01 arc minutes or more, for example 0.05 arc minutes or more, for example 0.1 arc minutes or more, for example 0.5 arc minutes or more, for example 1 arc minute or more, for example 2 arc minutes or more, for example 3 arc minutes or more, for example 4 arc minutes or more, for example 5 arc minutes or more, for example 6 arc minutes or more, for example 7 arc minutes or more, for example 8 arc minutes or more, for example 9 arc minutes or more, and 10 arc minutes or more. In some cases, the method includes spatially adjusting the position of a beamforming component to fine-tune the laser beam position on the flowstream, and the method includes rotating the beamforming component (for example, in the XY plane, XZ plane, or YZ plane) by 5 arc minutes or less, including, for example, 4.5 arc minutes or less, for example, 4 arc minutes or less, for example, 3.5 arc minutes or less, for example, 3 arc minutes or less, for example, 2.5 arc minutes or less, for example, 2 arc minutes or less, for example, 1.5 arc minutes or less, for example, 1 arc minute or less, for example, 0.5 arc minutes or less, for example, 0.1 arc minutes or less, for example, 0.05 arc minutes or less, for example, 0.01 arc minutes or less, for example, 0.005 arc minutes or less, and 0.001 arc minutes or less.

[0099] In some embodiments, the method includes evaluating the output light beams generated from the beam shaping components. In some cases, evaluating the output light beams generated from the beam shaping components includes evaluating the beam profile along the horizontal axis to determine, for example, the intensity of each output light beam, the power content of 80% of the linewidth, the edge power, the normalized intensity deviation across the horizontal axis of each output light beam, the shape of the beam profile, the spatial width of the beam profile, and the power distribution of each light beam along the horizontal axis.

[0100] In some embodiments, the method includes evaluating the illumination of a flowstream by the output light beam generated from the beamforming component. In some cases, evaluating the illumination of a flowstream by the output light beam generated from the beamforming component includes determining the spatial position of each laser beam spot. In other cases, evaluating the illumination of a flowstream by the output light beam generated from the beamforming component includes determining the spatial width of each laser beam spot traversing the flowstream (e.g., the proportion of the flowstream's spatial width illuminated by the spatial width of each generated output light beam).

[0101] In one embodiment, the position of the beamforming component is adjusted in response to the evaluated irradiation of the flowstream. For example, in some cases where the irradiation position by each generated output light beam on the flowstream is determined to produce a suboptimal optical signal, the beamforming component may be displaced to a position that produces the maximum optical signal amplitude. In these cases, the method may include mapping the spatial positioning (e.g., horizontal position, vertical position, orientation angle) of the beamforming component that produces the maximum optical signal amplitude and matching the spatial position of the beamforming component that produces the maximum optical signal amplitude.

[0102] The position of the illumination on the flowstream by the output light beam generated from the beam-shaping components can be adjusted by any convenient protocol, such as by directly moving one or more of the laser and beam-shaping components (manually, mechanically, or using a motor-driven displacement device), by moving a support stage coupled to the laser or beam-shaping components, and by changing the position, configuration, or orientation angle of one or more optical adjustment protocols (as described above). In some embodiments, the position of the illumination on the flowstream by the output light beam generated from the beam-shaping components is adjusted by manually adjusting one or more of the spatial positions of the beam-shaping components and the laser (e.g., by hand). In one example, the horizontal or vertical position or orientation angle of the beam-shaping components may be adjusted manually. In another example, the horizontal or vertical position of the laser is adjusted manually. In yet another example, the horizontal or vertical position or orientation angle of the beam-shaping components is adjusted manually, and the horizontal or vertical position of the laser is adjusted manually.

[0103] Using any convenient mechanical actuator, the spatial position of beam shaping components or lasers can be mechanically adjusted, for example, by a mechanically operated geared conversion device coupled to a mechanical lead screw assembly or support stage. In another example, the horizontal or vertical position or orientation angle of the beam-forming component is mechanically adjusted. In yet another example, the horizontal or vertical position of the laser is mechanically adjusted.

[0104] In yet another embodiment, the position of irradiation on the flowstream by the output beam generated from the beam shaping component is adjusted by adjusting the spatial position of one or more of the beam shaping component and the laser with a motor-driven displacement device. Any convenient motor-driven actuator may be used, for example, a motor-driven displacement stage, a motor-driven lead screw assembly, or other types of motors, including a stepping motor, a servo motor, a brushless electric motor, a brushed DC motor, a microstepped drive motor, or a motor-driven geared actuator using a high-resolution stepping motor.

[0105] In one embodiment, the position of irradiation on the flowstream by the output light beam generated from the beamforming component is adjusted by changing the position or orientation of one or more optical adjustment components. For example, the orientation of the optical adjustment protocol (e.g., one or more mirrors among the mirror components described above) may be changed to position each laser light beam on different parts of the beamforming component, for example, by increasing the angle of the optical adjustment protocol by 5° or more, including, for example, 10° or more, for example 15° or more, for example 20° or more, for example 30° or more, for example 45° or more, for example 60° or more, and 75° or more.

[0106] In one embodiment, the method includes dynamically adjusting the output beam, for example, by adjusting the laser, one or more optical adjustment components, or beam shaping components to have a width that is 50% to 99.9% of the spatial width of the flowstream. In one example, the spatial width of the output beam is increased by 5% or more, including, for example, 10% or more, 25% or more, 50% or more, 75% or more, and 90% or more, in order to achieve a desired incident width on the flowstream. In another embodiment, the spatial width of the output beam is decreased by 5% or more, including, for example, 10% or more, 25% or more, 50% or more, 75% or more, and 90% or more, in order to achieve a desired incident width on the flowstream. In one embodiment, the output light beam is adjusted to match the spatial width of the flowstream. For example, the output light beam may be adjusted to match the spatial width of the core stream of the flowstream.

[0107] In practicing a method according to one embodiment, a flowstream is illuminated by an output light beam generated from a beamforming component, and light from the flowstream is collected and detected. The light from the flowstream may be forward scattered light, side scattered light, transmitted light, emitted light (e.g., fluorescence or phosphorescence), or a combination thereof. In some embodiments, the method includes collecting and detecting forward scattered light from the flowstream. In other embodiments, the method includes collecting and detecting side scattered light from the flowstream. In yet another embodiment, the method includes collecting and detecting light transmitted through the flowstream. In yet another embodiment, the method includes collecting and detecting synchrotron radiation (e.g., fluorescence or phosphorescence) from the flowstream.

[0108] The flowstream can be irradiated at any suitable vertical position along the flowstream, as long as the optical signal from the flowstream is sufficiently detected. In one embodiment, the flowstream is a flow cytometer flowstream and is configured to irradiate at a position directly adjacent to the flow cell orifice. In other embodiments, the flowstream is irradiated at positions downstream of the flow cell nozzle orifice, such as 0.001 mm from the flow cell nozzle orifice, including, for example, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 5 mm or more, and 10 mm or more downstream from the flow cell nozzle orifice. The flowstream can be irradiated at one or more vertical positions, including, for example, two or more, three or more, four or more, five or more, and ten or more vertical positions.

[0109] The laser in question may include pulsed lasers or continuous-wave lasers. For example, the laser may be a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, xenon-fluorine (XeF) excimer laser, or a combination thereof. In other cases, the method includes aligning a dye laser, such as a stilbene, coumarin, or rhodamine laser, with a flow stream. In yet another case, the method includes aligning a metal vapor laser, such as a helium-cadmium (HeCd) laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser, copper laser or gold laser, or a combination thereof, with a flow stream. In other cases, the method involves aligning a solid-state laser, such as a ruby ​​laser, Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO4 laser, Nd:YCa4O(BO3)3 laser, Nd:YCOB laser, titanium-sapphire laser, slim YAG laser, ytterbium YAG laser, Yb2O3 laser, or cerium-doped laser, or combinations thereof, with a flowstream.

[0110] As summarized above, the method involves irradiating a flowstream with two or more lasers through a beam-shaping component that generates an output light beam having a predetermined intensity profile along the horizontal axis. In some embodiments, the method involves irradiating with three or more lasers, including, for example, four or more lasers, for example, five or more lasers, and ten or more lasers. Any combination of laser types can be aligned with the flowstream. For example, in some embodiments, the method involves irradiating the flowstream through a beam-shaping component with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0111] Each laser irradiation may occur simultaneously, sequentially, or in combination thereof. If each laser irradiation is sequential, each laser may be configured to irradiate independently for a duration of 0.001 microseconds or more, including, for example, 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 30 microseconds or more, and 60 microseconds or more. For example, the method may include irradiating with each laser for a duration in the range of 0.001 microseconds to 100 microseconds, including, for example, 0.01 microseconds to 75 microseconds, 0.1 microseconds to 50 microseconds, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In embodiments, the irradiation durations of each laser may be the same or different.

[0112] The interval between each laser pulse can also be individually separated and varied by delays of 0.001 microseconds or more, including, for example, 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 15 microseconds or more, 30 microseconds or more, and 60 microseconds or more, as needed. For example, the interval between each laser pulse may be in the range of 0.001 microseconds to 60 microseconds, including, for example, 0.01 microseconds to 50 microseconds, 0.1 microseconds to 35 microseconds, 1 microseconds to 25 microseconds, and 5 microseconds to 10 microseconds. In one embodiment, the interval between each laser pulse is 10 microseconds. It's a cross-second.

[0113] The flowstream may be irradiated continuously or at separate intervals. In some cases, the method involves irradiating the flowstream continuously. In other cases, the flowstream is irradiated at separate intervals including 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 intervals.

[0114] The optical signal from the flowstream can be detected by any convenient position-sensing detection protocol, including, but not limited to, other photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhanced charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In one embodiment, the optical signal is detected by a quadrant photodiode. When the optical signal is detected by a quadrant photodiode, the active detection surface area of ​​each region of the quadrant photodiode is, for example, 0.05 cm². 2 ~9cm 2 For example, 0.1 cm 2 ~8cm 2 For example, 0.5 cm 2 ~7cm 2 , and 1cm 2 ~5cm 2 Including, for example, 0.01 cm 2 ~10cm 2 These can vary. In some cases, the photodetector is a photodiode array having two or more photodiodes, for example, two or more photodiodes, for example, three or more, for example, five or more, and ten or more photodiodes.

[0115] In some embodiments, the detector is positioned in space away from the flowstream, and light from the flowstream is propagated to the detector through an optical relay system, such as an optical fiber or a free-space optical relay system. For example, the optical relay system may be an optical fiber relay bundle, and the light is transmitted to the detector through the optical fiber relay bundle. Any optical fiber relay system may be used to propagate light to the detector. In some embodiments, a suitable optical fiber relay system for propagating light to the detector includes, but is not limited to, optical fiber relay systems described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference. In other embodiments, the optical relay system is a free-space optical relay system. The term “free-space optical relay” is used herein in its conventional sense to refer to light propagation that directs light through free space to a detector using a configuration of one or more optical components. In some embodiments, the free-space optical relay system includes a housing having a proximal edge and a distal edge, the proximal edge being coupled to the detector. A free-space relay system may include any combination of different beam-shaping components, such as one or more of lenses, mirrors, slits, pinholes, wavelength separators, or combinations thereof. For example, in some embodiments, the free-space optical relay system in question includes one or more focusing lenses. In other embodiments, the free-space optical relay system in question includes one or more mirrors. In yet another embodiment, the free-space optical relay system includes a collimating lens. In some embodiments, a suitable free-space optical relay system for propagating light to a detector is, but is not limited to, optical relay systems described, for example, U.S. Patents 7,643,142, 7,728,974, and 8,223,445, the disclosures of which are incorporated herein by reference.

[0116] The method also includes detecting light from a sample within a flowstream. The detected light may be side-scattered light, forward-scattered light, emitted light, or a combination thereof. Preferred photodetection protocols include, but are not limited to, optical sensors or photodetectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhanced charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof. In one embodiment, light from a flowstream irradiated in the sample inspection area of ​​a particle sorting module is measured by a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In one embodiment, the light is measured by a charge-coupled device (CCD).

[0117] The optical signal from the flowstream may be measured at one or more wavelengths, including measuring the light from the flowstream at, for example, two or more wavelengths, for example, five or more different wavelengths, for example, ten or more different wavelengths, for example, twenty-five or more different wavelengths, for example, fifty or more different wavelengths, for example, one hundred or more different wavelengths, for example, two hundred or more different wavelengths, for example, three hundred or more different wavelengths, and four hundred or more different wavelengths. In some embodiments, the method includes measuring the light over a range of wavelengths (e.g., 200 nm to 1000 nm). For example, the method may include collecting the spectrum of light over one or more wavelengths within the 200 nm to 1000 nm range. In yet other embodiments, the method includes measuring the light from the flowstream at one or more specific wavelengths. For example, the light can be measured at one or more of the following wavelengths: 450nm, 518nm, 519nm, 561nm, 578nm, 605nm, 607nm, 625nm, 650nm, 660nm, 667nm, 670nm, 668nm, 695nm, 710nm, 723nm, 780nm, 785nm, 647nm, 617nm, and any combination thereof. In one embodiment, the method includes measuring the wavelength of light corresponding to the fluorescence peak wavelength of a certain fluorophore.

[0118] Light from a flowstream can be measured continuously or at separate intervals. In some cases, the method involves measuring the light continuously. In other cases, the light is measured at separate intervals, including 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.

[0119] Light measurements may be performed once or more during the method of the subject, including, for example, two or more times, three or more times, five or more times, and ten or more times. In some embodiments, light propagation is measured two or more times, and in some cases, the data is averaged.

[0120] The flow rate of the flowstream according to the embodiment may vary depending on the intensity of the laser light, and may be 1 nL / min or more, including, for example, 2 nL / min or more, for example, 3 nL / min or more, for example, 5 nL / min or more, for example, 10 nL / min or more, for example, 25 nL / min or more, for example, 50 nL / min or more, for example, 75 nL / min or more, for example, 100 nL / min or more, for example, 250 nL / min or more, for example, 500 nL / min or more, for example, 750 nL / min or more, and 1000 nL / min or more. In one embodiment, the flow rate of the flow stream in the method of the subject is in the range of 1 nL / min to 500 nL / min, including, for example, 1 nL / min to 250 nL / min, for example, 1 nL / min to 100 nL / min, for example, 2 nL / min to 90 nL / min, for example, 3 nL / min to 80 nL / min, for example, 4 nL / min to 70 nL / min, for example, 5 nL / min to 60 nL / min, and 10 nL / min to 50 nL / min. In one embodiment, the flow rate of the flow stream is 5 nL / min to 6 nL / min.

[0121] In embodiments, the method may include irradiating different positions along the longitudinal axis of the flowstream with one or more of the output light beams. For example, the method may include irradiating at output light beam positions along the longitudinal axis of the flowstream that are 0.0001 mm or more apart, including, for example, 0.0005 mm or more, for example, 0.001 mm or more, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2 mm or more, for example, 3 mm or more, for example, 4 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 15 mm or more, and 25 mm or more. In one embodiment, each output light beam can propagate along a flowstream at positions along different longitudinal axes of 50 mm or less, including, for example, 25 mm or less, 15 mm or less, 10 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, 1 mm or less, 0.5 mm or less, 0.1 mm or less, and 0.001 mm or less.

[0122] In some cases, the sample is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, plant, fungus, or, in some cases, a subset of animal tissues, cells, or constituent parts that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, sheep's marrow blood, urine, vaginal fluid, and semen. Thus, “biological sample” refers to both a natural organism or a subset of its tissues, as well as homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory, gastrointestinal, cardiovascular, and urinary tract, tears, saliva, milk, blood cells, tumors, and organs. A biological sample may be any type of biological tissue, including both healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In some embodiments, the biological sample is blood or a derivative thereof, such as plasma, or other biological fluid samples, such as tears, urine, or semen. In some cases, the sample is a blood sample containing whole blood, such as blood obtained by venipuncture or from a fingertip (the blood may or may not be combined with any reagents before the assay, such as preservatives or anticoagulants).

[0123] In some embodiments, the source of the sample is “mammal” or “mammalian animal,” and these terms are broadly used to describe organisms within mammals, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is human. The method may be applied to samples obtained from human subjects of both sexes and to any stage of development (i.e., neonatal, infant, adolescent, adult), and in some embodiments, the human subject is a child, adolescent, or adult. It should be understood that the present invention may be applied to samples from human subjects, but is not limited thereto, and may also be carried out to samples from other animal subjects (i.e., in “non-human subjects”) such as birds, mice, rats, dogs, cats, livestock, and horses.

[0124] In some embodiments, the biological sample contains cells. Cells that may be present in the sample include eukaryotic cells (e.g., mammalian cells) and / or prokaryotic cells (e.g., bacterial or archaeal cells). The sample may be obtained from an in vitro source (e.g., a suspension of cells derived from cultured and grown laboratory cells) or an in vivo source (e.g., mammalian subjects, human subjects, etc.). In some embodiments, the cell sample is obtained from an in vitro source. The in vitro source includes, but is not limited to, prokaryotic (e.g., bacterial, archaeological) cell cultures, environmental samples containing prokaryotic and / or eukaryotic (e.g., mammalian, antimicrobial, fungal, etc.) cells, eukaryotic cell cultures (e.g., cultures of established cell lines, cultures of known or purchased cell lines, cultures of immortalized cell lines, cultures of primary cells, cultures of experimental yeast, etc.), tissue cultures, etc.

[0125] If the biological sample contains cells, the methods of this disclosure may include cell characterizing components, such as cell fragments, fragmented cell membranes, organelles, dead cells, or lysed cells. In some embodiments, the methods include characterizing extracellular vesicles of cells. Characterizing extracellular vesicles of cells may include identifying the type of extracellular vesicle within the cell or determining the size of the extracellular vesicle within the cell.

[0126] In some embodiments, light (e.g., forward scattered light, side scattered light, synchrotron radiation, etc.) is detected directly from the sample in the flowstream. In other embodiments, light from the sample in the flowstream is propagated to a detector having one or more beam optical adjustment components. For example, the beam path, direction, focus, or collimation of light from the sample in the flowstream may be modified by the optical adjustment components. In some cases, the dimensions of the light collected from the sample in the flowstream are adjusted by increasing the dimensions by, for example, 10% or more, 25% or more, 50% or more, and 75% or more, and by increasing the dimensions by 5% or more, or by focusing the light to reduce the dimensions of the light by, for example, 10% or more, 25% or more, 50% or more, and 75% or more. In other cases, optical adjustment includes collimating the light. The term “collimate” is used in its conventional sense to refer to optically adjusting the collinearity of light propagation, or reducing divergence due to light from a common propagation axis. In some cases, collimation involves narrowing the spatial cross-section of a light beam. In some embodiments, the optical tuning component is a wavelength separator. The term “wavelength separator” is used herein in its conventional sense to refer to an optical protocol for separating polychromatic light into its constituent wavelengths. Wavelength separation according to some embodiments may involve selectively passing or blocking specific wavelengths or wavelength ranges of polychromatic light. Wavelength separation protocols in question include, but are not limited to, colored glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, monochromators, and combinations thereof, among other wavelength separation protocols. In some embodiments, the wavelength separator is an optical filter. For example, an optical filter may be a bandpass filter having a minimum bandwidth in the range of 2 nm to 100 nm, including bandpass filters having minimum bandwidths in the ranges of, for example, 3 nm to 95 nm, 5 nm to 95 nm, 10 nm to 90 nm, 12 nm to 85 nm, 15 nm to 80 nm, and 20 nm to 50 nm.

[0127] The method in one embodiment also includes data acquisition, analysis, and recording by a computer or the like, with multiple data channels recording data from the sample as the sample passes through the detection area of ​​the system. In these embodiments, the analysis may include classifying and counting cells or components of cells (extracellular vesicles) such that each component exists as a set of digitized parameter values. The system of the subject may be configured to trigger on selected parameters to distinguish the particles of interest from the background and noise. A “trigger” refers to a pre-set threshold for detecting the parameter and may be used as a means to detect the passage of the components of interest through the detection area. Detection of an event exceeding the threshold of the selected parameter triggers data acquisition of the sample components. With respect to components in the medium being chemically analyzed, no data is acquired that would result in a response below the threshold.

[0128] Computer control system Aspects of the present disclosure further include a computer-controlled system for practicing the subject method, the system further including one or more computers for full or partial automation of the system for practicing the method described herein. In some embodiments, the system includes a computer having a computer-readable storage medium in which a computer program is stored, the computer program including irradiating a sample in a flow stream with a first light beam and a second light beam through an optical tuning component configured, once loaded into the computer, to receive a first light beam and a second light beam from different angles of incidence at substantially the same position and to generate an output light beam from the first light beam and the second light beam having a predetermined intensity profile along the horizontal axis.

[0129] In some embodiments, the computer-readable storage medium is raw from the beam forming components. The algorithm includes an algorithm for evaluating the generated output light beam. In some cases, the algorithm for evaluating the generated output light beam includes, for example, an algorithm for evaluating the beam profile along the horizontal axis to determine the intensity of each output light beam, the power content of 80% of the linewidth, the edge power, the normalized intensity deviation across the horizontal axis of each output light beam, the shape of the beam profile, the spatial width of the beam profile, and the power distribution of each light beam along the horizontal axis.

[0130] In other embodiments, the computer-readable storage medium includes an algorithm for evaluating the illumination of the flowstream by the output light beams generated from the beam shaping components. In some embodiments, the computer-readable storage medium includes an algorithm for determining the spatial position of each laser beam spot. In other embodiments, the computer-readable storage medium includes an algorithm for determining the spatial width of each laser beam spot across the flowstream (e.g., the ratio of the spatial width of the flowstream illuminated by the spatial width of each generated output light beam).

[0131] In one example, the system includes a computer having a computer-readable storage medium in which a computer program is stored, and the computer program, when loaded onto the computer, further includes instructions having one or more algorithms for spatially adjusting a beam-shaping component, changing the orientation angle of a beam-shaping component, spatially adjusting one or more lasers, deflecting the orientation angle of one or more lasers, spatially adjusting one or more optical adjustment components (e.g., one or more mirrors of a mirror component as described above), and changing the orientation angle of one or more optical adjustment components.

[0132] The subject system 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, and as a result, the functional elements of the system, i.e., those elements of the system that perform specific tasks (such as managing input / output of information, processing information, etc.), may be operated by the execution of software applications on and across one or more computer platforms represented by the system.

[0133] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses memory having instructions stored thereon to perform steps of the method of the subject. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, 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 will become available. The processor runs the operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor coordinating and executing the functions of various computer programs that can be written in various 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 works with the processor to coordinate and execute the functions of 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, according to all known techniques. The processor may be any preferred analog or digital system. In some embodiments, the processor includes analog electronics that allow the user to manually align the light source with the flow stream based on a first optical signal and a second optical signal. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0134] 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 resident hard disks or tapes, optical media such as read-and-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including compact disk drives, tape drives, removable hard disk drives, or disk drives. Such types of memory storage devices typically read from and / or write to program storage media such as compact disks, magnetic tapes, removable hard disks, or magnetic disks (not shown), respectively. Any of these program storage media, or others currently in use or to be developed in the future, can be considered computer program products. As is understood, these program storage media typically store computer software programs and / or data. Computer software programs, also called computer control logic, are typically stored in program storage devices used in conjunction with system memory and / or memory storage devices.

[0135] In some embodiments, a computer program product is described that includes a computer-usable medium in which control logic (a computer software program including program code) is stored. When the control logic is executed by a processor, it causes the computer, the processor, to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state device. Implementations of hardware state devices for performing the functions described herein will be apparent to those skilled in the art.

[0136] Memory may be any suitable device from which the processor can store and retrieve data, for example, magnetic, optical, or solid-state storage devices (including magnetic or optical disks, or tapes or RAM, or any other suitable device, whether fixed or portable). The processor may include a general-purpose digital microprocessor preferably programmed from a computer-readable medium that carries the required program code. The programming may be provided to the processor remotely via a communication channel, or may be pre-stored in a computer program product such as memory or some other portable or fixed computer-readable storage medium using one of those devices in relation to memory. For example, a magnetic disk or optical disk may carry programming and be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of a computer program product, and algorithms for use in practicing the above methods. 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 magnetic disks, hard disk storage media, and magnetic tapes; optical storage media such as CD-ROMs; electrical storage media such as RAM and ROMs; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0137] The processor may also have access to a communication channel for communicating with a user at a remote location. A remote location is a location where the user does not have direct contact with the system, and communicates via a wide area network ("WAN"), telephone network, satellite network, or mobile phone (i.e., This means relaying input information from an external device, such as a computer connected to any other suitable communication channel (including a smartphone), to the input manager.

[0138] In some embodiments, the systems according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but is 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).

[0139] In one embodiment, the communication interface is configured to include one or more communication ports, such as a physical port or interface, such as a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the subject system and other external devices, such as computer edge terminals (e.g., in a doctor's office or hospital environment) configured for similar complementary data communication.

[0140] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the subject system of the subject to communicate with other devices, such as computer terminals and / or networks, communicable mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with them.

[0141] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet Protocol (IP) via a mobile phone 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 Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0142] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface using a common standard such as 802.11 or Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device such as a desktop computer or appliance. In some embodiments, the server device has a display such as a liquid crystal display (LCD) and input devices such as buttons, a keyboard, a mouse, or a touchscreen.

[0143] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically with a network or server device and the system of the subject, for example, data stored in any data storage unit, using one or more of the communication protocols and / or mechanisms described above.

[0144] An output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of picture elements. A graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input. Functional elements of a computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. An output manager may also provide information generated by a processing module to a user at a remote location, for example, via the internet, telephone, or satellite network, according to known techniques. The presentation of data by the output manager may be implemented according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may also include an internet URL address so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. 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 computer platform. However, they may also be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cable or other communication system, including wireless systems such as networks. They may be in the same location or physically separated.Depending on the type and / or design of the selected computer platform, various operating systems may be employed on any of the computer platforms. Suitable operating systems include Windows 10, Windows NT®, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux®, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, Ubuntu, Zorin OS, and others.

[0145] kit Aspects of the present invention further include a kit comprising one or more lasers, a mirror component having a first mirror and a second mirror positioned to propagate light from the first mirror to a beam combiner, and a beam shaping component configured to generate an output light beam having a predetermined intensity profile along the horizontal axis from the first and second light beams, as described herein.

[0146] The various assay components of the kit may be located in separate containers, or some or all of them may be pre-assembled. For example, in some cases, one or more components of the kit, such as each beam shaping component, mirror, or laser, may be located in a sealed pouch, such as a sterile foil pouch or envelope.

[0147] In addition to the components described above, the subject kit may (in some embodiments) further include instructions for practicing the subject method. These instructions may be present in the subject kit in various forms, and one or more of them may be present in the kit. One possible form in which these instructions may be present is as information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, the kit packaging, accompanying documentation, etc. Yet another form of these instructions may be a computer-readable medium on which the information is recorded, such as a diskette, compact disc (CD), portable flash drive, etc. Yet another possible form of these instructions may be a website address that can be used over the internet to access the information at a deleted site.

[0148] Utility The subject system, method, and computer system are used in fluid culture media such as biological samples. The disclosure finds applications in various fields where it is desirable to analyze and sort particle components within a sample. The disclosure also finds applications in flow cytometry, where it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more precise particle charging, and enhanced particle deflection during cell sorting. In embodiments, the disclosure reduces the need for user input or manual adjustment during sample analysis with a flow cytometer. In some embodiments, the system of the subject provides a fully automated protocol, resulting in little to no adjustment to the flow cytometer in use, even with any human input.

[0149] This disclosure also finds applications in which cells prepared from biological samples may be desired for use in research, laboratory testing, or therapy. In some embodiments, the methods and devices of the subject can facilitate the acquisition of individual cells prepared from a biological fluid or tissue sample of interest. For example, the methods and systems of the subject facilitate the acquisition of cells from a fluid or tissue sample used as a study or diagnostic specimen for diseases such as cancer. Similarly, the methods and systems of the subject facilitate the acquisition of cells from a fluid or tissue sample used in therapy. Compared to conventional flow cytometry systems, the methods and devices of the disclosure enable the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, fluids) with improved efficiency and lower cost.

[0150] Embodiments of the subject matter described herein may be useful on their own or in combination with one or more other embodiments or forms. Without limiting the description, some non-limiting embodiments of the present disclosure numbered 1 to 104 are provided below. As will be apparent to those skilled in the art when reading the present disclosure, each individually numbered embodiment may be used in any of the individually numbered embodiments or in combination with them. This is intended to provide support for all such combinations of embodiments, and is not limited to the combinations of embodiments expressly provided below.

[0151] 1. A first laser that generates the first light beam, A second laser that generates a second light beam, A beam shaping component configured to receive a first and second light beam from different incident angles at substantially the same position, and to generate an output light beam having a predetermined intensity profile along the horizontal axis from the first and second light beams. A system equipped with these features. 2. The system according to embodiment 1, wherein the beam forming component receives a first light beam and a second light beam at the same position on the surface of the beam forming component. 3. The system according to embodiment 1, wherein the beam shaping component receives the first light beam and the second light beam at the same location within the beam shaping component. 4. The system according to embodiment 3, wherein the beam forming component receives the first light beam and the second light beam at a position of 1 mm or more within the beam forming component. 5. The system according to any one of embodiments 1 to 4, wherein the first laser and the second laser are in optical communication with a mirror component configured to combine the first light beam and the second light beam, respectively.

[0152] 6. The system according to embodiment 5, wherein the mirror component comprises a first mirror and a second mirror positioned to propagate light from the first mirror to a beam shaping component. 7. The system according to embodiment 6, wherein the second mirror is a beam combiner. 8. The system according to embodiment 7, wherein the beam combiner is a dichroic beam combiner. 9. The system according to any one of embodiments 1 to 8, wherein the intensity at the center of the output light beam is 75% to 99.9% of the intensity at the edge of the output light beam along the horizontal axis. 10. The system according to embodiment 9, wherein the intensity at the center of the output light beam is 90% to 99.9% of the intensity at the edge of the output light beam along the horizontal axis.

[0153] 11. The system according to any one of embodiments 1 to 10, wherein the beam shaping component is configured to generate an output light beam having a top-hat intensity profile along the horizontal axis. 12. The system according to any one of embodiments 1 to 10, wherein the beam shaping component is configured to generate an output light beam having an ultra-Gaussian intensity profile along the horizontal axis. 13. The beam shaping component is a system according to any one of embodiments 1 to 12, comprising a diffractive optical element. 14. The beam shaping component is a system according to any one of embodiments 1 to 13, comprising a refractive optical element. 15. The beam forming component is a system according to any one of embodiments 1 to 14, comprising a Powell lens.

[0154] 16. The beam forming component is a system according to any one of embodiments 1 to 15, comprising a cylindrical lens array. 17. The system according to any one of embodiments 1 to 16, wherein the intensity at the center of the output light beam is 75% to 99.9% of the intensity at the edge of the output light beam along the vertical axis. 18. The system according to embodiment 16, wherein the intensity at the center of the output light beam is 90% to 99.9% of the intensity at the edge of the output light beam along the vertical axis. 19. The system according to embodiment 17 or 18, wherein the beam shaping component is configured to generate an output light beam having a top-hat intensity profile along the vertical axis. 20. The system according to embodiment 17 or 18, wherein the beam shaping component is configured to generate an output light beam having an ultra-Gaussian intensity profile along the vertical axis.

[0155] 21. The system according to embodiment 17 or 18, wherein the intensity at the center of the output light beam is 75% to 99.9% of the intensity at the edges of the output light beam along the horizontal and vertical axes. 22. The system according to embodiment 21, wherein the beam shaping component is configured to generate an output beam of an optical beam having a top-hat intensity profile along the horizontal and vertical axes. 23. The system according to embodiment 21, wherein the beam shaping component is configured to generate an output beam of an optical beam having an ultra-Gaussian intensity profile along the horizontal and vertical axes. 24. The system according to any one of embodiments 1 to 23, wherein the output light beam includes a Gaussian distribution along the vertical axis of the output laser beam. 25. The system according to any one of embodiments 1 to 24, further comprising a beam combiner configured to combine a first light beam from a first laser and a second light beam from a second laser.

[0156] 26. A system according to any one of embodiments 1 to 25, wherein the first laser and the second laser have different wavelengths. 27. The system according to any one of embodiments 1 to 26, wherein the angle of incidence of the first light beam onto the beam shaping component differs from the angle of incidence of the second light beam onto the beam shaping component by 0.5 degrees or more. 28. The system according to embodiment 27, wherein the angle of incidence of the first light beam onto the beam shaping component differs from the angle of incidence of the second light beam onto the beam shaping component by 2 degrees or more. 29. Further equipped with multiple lasers, the beam shaping component receives multiple lasers along the horizontal axis. A system according to any one of embodiments 1 to 28, configured to generate an output light beam having a predetermined intensity profile. 30. The system according to embodiment 29, wherein the beam shaping component is configured to generate multiple output light beams having substantially the same intensity profile along the horizontal axis.

[0157] 31. The system according to embodiment 30, wherein the beam shaping component is configured to generate multiple output light beams having a top-hat intensity profile along the horizontal axis. 32. The system according to embodiment 30, wherein the beam shaping component is configured to generate multiple output light beams having an ultra-Gaussian intensity profile along the horizontal axis. 33. The system according to embodiment 29, wherein the beam shaping component is configured to generate a first output laser beam having a first intensity profile along the horizontal axis and a second output laser beam having a second intensity profile along the horizontal axis. 34. The system according to embodiment 33, wherein the first output laser beam includes a top-hat intensity profile along the horizontal axis. 35. The system according to embodiment 33, wherein the first output laser beam includes an ultra-Gaussian intensity profile along the horizontal axis.

[0158] 36. The system according to any one of embodiments 33 to 35, wherein the second output laser beam includes a top-hat intensity profile along the horizontal axis. 37. The system according to any one of embodiments 33 to 35, wherein the second output laser beam includes an ultra-Gaussian intensity profile along the horizontal axis. 38. The system according to any one of embodiments 1 to 37, further comprising a flow cell configured to propagate a sample within a flow stream. 39. The system according to embodiment 38, wherein the output light beam is positioned to illuminate a spatial width that is 50% to 99.9% of the flow stream along the horizontal axis. 40. The system according to embodiment 38, wherein the output light beam is positioned along the horizontal axis to illuminate 90% to 99.9% of the spatial width of the flowstream.

[0159] 41. The system according to any one of embodiments 38 to 40, wherein the beam shaping component is configured to generate an output optical beam having a beam profile that is 50% to 99.9% of the width of the flowstream along the horizontal axis. 42. The system according to any one of embodiments 38 to 41, wherein the beam shaping component is configured to produce an output optical beam having an intensity profile that is substantially the same over 50% to 99.9% of the flowstream along the horizontal axis. 43. The system according to any one of embodiments 38 to 41, wherein the beam shaping component is configured to produce an output optical beam having an intensity profile that is substantially the same over 90% to 99.9% of the flowstream along the horizontal axis. 44. The system according to embodiment 38, wherein the flow stream includes a core stream and a stacked sheath stream. 45. The system according to embodiment 44, wherein the beam shaping component is configured to generate an output optical beam having a beam profile that is 50% to 99.9% of the width of the core stream along the horizontal axis.

[0160] 46. ​​The system according to embodiment 45, wherein the beam shaping component is configured to generate an output optical beam having a beam profile that is 90% to 99.9% of the width of the core stream along the horizontal axis. 47. The system according to any one of embodiments 44 to 46, wherein the beam shaping component is configured to produce an output optical beam having an intensity profile that is substantially the same over 50% to 99.9% of the core stream along the horizontal axis. 48. The beam forming components are located along the horizontal axis, covering 90% to 99.9% of the core stream. A system according to any one of embodiments 44 to 46, configured to produce an output light beam having substantially the same intensity profile over time. 49. The system according to any one of embodiments 38 to 48, further comprising a detector for detecting light from a sample in a flowstream. 50. The system according to any one of embodiments 38 to 49, wherein the generated output light beam is configured to illuminate different positions along the longitudinal axis of the flowstream.

[0161] 51. The system according to embodiment 50, wherein the output beams are separated from each other by at least 1 mm along the longitudinal axis of the flowstream. 52. The system according to any one of embodiments 1 to 51, wherein the beam forming component consists of a single beam forming lens. 53. A method comprising irradiating a sample in a flowstream with a first light beam and a second light beam through a beam shaping component configured to receive a first light beam and a second light beam from substantially the same position from different incident angles and to generate an output light beam having a predetermined intensity profile along the horizontal axis from the first light beam and the second light beam. 54. The method according to embodiment 53, wherein the beam shaping component receives a first light beam and a second light beam at the same location on the surface of the beam shaping component. 55. The method according to embodiment 53, wherein the beam shaping component receives the first light beam and the second light beam at the same location within the beam shaping component.

[0162] 56. The method according to embodiment 55, wherein the beam forming component receives the first light beam and the second light beam at a position of 1 mm or more within the beam forming component. 57. The method according to any one of embodiments 53 to 56, wherein the first laser and the second laser are each in optical communication with a mirror component configured to combine the first light beam and the second light beam. 58. The method according to embodiment 57, wherein the mirror component comprises a first mirror and a second mirror positioned to propagate light from the first mirror to a beam shaping component. 59. The method according to embodiment 58, wherein the second mirror is a beam combiner. 60. The method according to embodiment 59, wherein the beam combiner is a dichroic beam combiner.

[0163] 61. The method according to any one of embodiments 53 to 60, wherein the intensity at the center of the output light beam is 75% to 99.9% of the intensity at the edge of the output light beam along the horizontal axis. 62. The method according to embodiment 61, wherein the intensity at the center of the output light beam is 90% to 99.9% of the intensity at the edge of the output light beam along the horizontal axis. 63. The method according to any one of embodiments 53 to 62, wherein the beam shaping component is configured to produce an output light beam having a top-hat intensity profile along the horizontal axis. 64. The method according to any one of embodiments 53 to 62, wherein the beam shaping component is configured to generate an output light beam having an ultra-Gaussian intensity profile along the horizontal axis. 65. The beam shaping component having a diffractive optical element, according to any one of embodiments 53 to 64.

[0164] 66. The beam shaping component having a refractive optical element, according to any one of embodiments 53 to 65. 67. The beam forming component having a Powell lens, according to any one of embodiments 53 to 66. 68. The beam forming component has a cylindrical lens array, any of embodiments 53 to 67. One method. 69. The method according to any one of embodiments 53 to 68, wherein the intensity at the center of the output light beam is 75% to 99.9% of the intensity at the edge of the output light beam along the vertical axis. 70. The method according to embodiment 69, wherein the intensity at the center of the output light beam is 90% to 99.9% of the intensity at the edge of the output light beam along the vertical axis.

[0165] 71. The method according to embodiment 69 or 70, wherein the beam shaping component is configured to produce an output light beam having a top-hat intensity profile along the vertical axis. 72. The method according to embodiment 69 or 70, wherein the beam shaping component is configured to generate an output light beam having an ultra-Gaussian intensity profile along the vertical axis. 73. The method according to any one of embodiments 69 to 72, wherein the intensity at the center of the output light beam is 75% to 99.9% of the intensity at the edges of the output light beam along the horizontal and vertical axes. 74. The method according to embodiment 73, wherein the beam shaping component is configured to generate an output light beam having a top-hat intensity profile along the horizontal and vertical axes. 75. The method according to embodiment 73, wherein the beam shaping component is configured to generate an output light beam having an ultra-Gaussian intensity profile along the horizontal and vertical axes.

[0166] 76. The method according to any one of embodiments 53 to 75, wherein the output light beam includes a Gaussian distribution along the vertical axis of the output laser beam. 77. The method according to any one of embodiments 53 to 76, further comprising combining a first light beam from a first laser and a second light beam from a second laser with a beam combiner. 78. The method according to embodiment 77, wherein the first laser and the second laser have different wavelengths. 79. The method according to any one of embodiments 53 to 78, wherein the angle of incidence of the first light beam onto the beam shaping component differs from the angle of incidence of the second light beam onto the beam shaping component by 0.5 degrees or more. 80. The method according to embodiment 79, wherein the angle of incidence of the first light beam onto the beam shaping component differs from the angle of incidence of the second light beam onto the beam shaping component by 2 degrees or more.

[0167] 81. The method according to any one of embodiments 53 to 80, further comprising irradiating a flowstream passing through a beam shaping component with a plurality of lasers. 82. The method according to embodiment 81, wherein multiple output light beams have substantially the same intensity profile along the horizontal axis. 83. The method according to embodiment 82, wherein multiple output light beams have a top-hat intensity profile along the horizontal axis. 84. The method according to 82, wherein multiple output light beams have supergaussian intensity profiles along the horizontal axis. 85. The method according to embodiment 81, comprising generating a first output laser beam having a first intensity profile along the horizontal axis and generating a second output laser beam having a second intensity profile along the horizontal axis.

[0168] 86. The method according to embodiment 85, wherein the first output laser beam includes a top-hat intensity profile along the horizontal axis. 87. The method according to embodiment 86, wherein the first output laser beam includes an ultra-Gaussian intensity profile along the horizontal axis. 88. The method according to any one of embodiments 85 to 87, wherein the second output laser beam includes a top-hat intensity profile along the horizontal axis. 89. The second output laser beam has an ultra-Gaussian intensity profile along the horizontal axis. The method according to any one of embodiments 85 to 87. 90. The method according to any one of embodiments 53 to 89, comprising irradiating a spatial width of 50% to 99.9% of the flow stream along the horizontal axis.

[0169] 91. The method according to embodiment 90, comprising irradiating a spatial width of 90% to 99.9% of the flowstream along the horizontal axis. 92. The method according to any one of embodiments 53 to 91, wherein the flow stream includes a core stream and a stacked sheath stream. 93. The method according to embodiment 92, comprising generating an output optical beam having a beam profile that is 50% to 99.9% of the width of the core stream along the horizontal axis. 94. The method according to embodiment 93, comprising generating an output optical beam having a beam profile that is 90% to 99.9% of the width of the core stream along the horizontal axis. 95. The method of any one of embodiments 92 to 94, comprising generating an output light beam having an intensity profile that is substantially the same over 50% to 99.9% of the core stream along the horizontal axis.

[0170] 96. The method of embodiment 95, comprising generating an output light beam having an intensity profile that is substantially the same over 90% to 99.9% of the core stream along the horizontal axis. 97. The method according to any one of embodiments 53 to 96, wherein the output light beam is irradiated at different positions along the longitudinal axis of the flowstream. 98. The method according to embodiment 97, wherein the positions are separated by 1 mm or more. 99. The method according to any one of embodiments 53 to 98, further comprising detecting light from a sample in a flowstream. 100.1 or more lasers, A mirror component comprising a first mirror and a second mirror positioned to propagate light from the first mirror to a beam-shaping component, A beam shaping component configured to generate an output light beam having a predetermined intensity profile along the horizontal axis from a first light beam and a second light beam, and A kit that includes the following:

[0171] 101. The beam forming component is a kit according to embodiment 100, having a Powell lens. 102. The beam forming component is a kit according to embodiment 100 or 101, having a cylindrical lens array. 103. The second mirror is a beam combiner, as described in any one of embodiments 100 to 102 of the kit. 104. The beam combiner is a dichroic beam combiner, as described in the kit according to embodiment 103.

[0172] Although the above invention has been described in some detail as an example and illustration for the sake of clear understanding, it will be readily apparent to those skilled in the art that certain modifications and alterations may be made in light of the teachings of the invention without departing from the spirit or scope of the appended claims.

[0173] Therefore, what has been stated above is merely illustrative of the principles of the present invention. Those skilled in the art will understand that various arrangements embodying the principles of the present invention and falling within its spirit and scope can be devised, although these are not expressly described or shown herein. Furthermore, all examples and conditional language enumerated herein are primarily intended to help the reader understand the principles of the present invention and the concepts to which the inventors contribute to further advance the art. It should be interpreted that this is not limited to such specifically enumerated examples and conditions. Furthermore, all descriptions herein enumerating the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. In addition, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any elements developed to perform the same function, regardless of their structure. Furthermore, nothing disclosed herein is intended to be for the public only, whether such disclosure is expressly enumerated in the claims or not.

[0174] Accordingly, 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 are embodied by the appended claims. In the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) is expressly defined as being invoked for limitation in the claims only when the exact phrase “means for” or the exact phrase “steps for” is enumerated at the beginning of such limitation in the claims, and if such exact phrase is not used in limitation in the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) is not invoked. Cross-reference of related applications

[0175] In accordance with 35 U.S. SC § 119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 62 / 662,115, filed on 24 April 2018, the disclosure of which is incorporated herein by reference.

Claims

1. Multiple lasers that generate multiple light beams, The beam shaping component comprises a single beam shaping lens and is configured to receive each of the plurality of light beams from different incident angles at the same position on its surface or at the same position within itself, and to generate an output light beam having a predetermined intensity profile along the horizontal axis from the plurality of light beams. A flow cell configured to propagate a sample within a flow stream It is equipped with, Light from each laser is directly propagated to the beam shaping component. The beam shaping component directs the output light beam towards the flow cell. system.

2. The system according to claim 1, further comprising a focusing lens that receives the output light beam from the beam shaping component and directs the output light beam toward the flow cell.

3. Two or more of the aforementioned plurality of lasers are optically in communication with a mirror component configured to combine two or more light beams generated by the two or more lasers. The mirror component comprises a first mirror and a second mirror positioned to propagate light from the first mirror to the beam shaping component. The system according to claim 1 or 2.

4. The system according to claim 1 or 2, wherein the intensity at the center of the output light beam is 90% to 99.9% of the intensity at the edge of the output light beam along the horizontal axis.

5. The beam forming component is The system is configured to generate an output light beam having a top-hat intensity profile along the horizontal axis, or a super-Gaussian intensity profile along the horizontal axis. The system according to claim 1 or 2.

6. The system according to claim 1 or 2, wherein the output light beam includes a Gaussian distribution along the vertical axis of the output laser beam.

7. The system according to claim 1 or 2, wherein the incident angles of each light beam to the beam shaping component differ by 0.5 degrees or more.

8. The output light beam is configured to illuminate a spatial width that is 90% to 99.9% of the flow stream along the horizontal axis. The system according to claim 1 or 2.

9. The system according to claim 8, wherein the beam shaping component is configured to generate a plurality of output light beams, and the generated output light beams are configured to irradiate different positions along the longitudinal axis of the flow stream.

10. The system according to claim 1 or 2, wherein the single beam-forming lens is a Powell lens.

11. A method comprising irradiating a sample in a flowstream with multiple light beams through a beam-forming lens configured to receive each of multiple light beams generated by multiple lasers from different incident angles at the same location on its surface or at the same location within itself, and to generate an output light beam having a predetermined intensity profile along the horizontal axis from the multiple light beams, wherein the light from each laser is propagated directly to the beam-forming lens.

12. The method according to claim 11, further comprising focusing the output light beam onto the sample using a focusing lens that receives the output light beam from the beam shaping lens and directs the output light beam toward the sample.

13. The method according to claim 11, comprising generating an output light beam having a top-hat intensity profile along the horizontal axis, or an ultra-Gaussian intensity profile along the horizontal axis, through the beam shaping lens.

14. The method according to claim 11, comprising generating an output light beam that irradiates a spatial width that is 90% to 99.9% of the flow stream along the horizontal axis.

15. A kit used to carry out the method described in any one of claims 11 to 14, One or more lasers, A mirror component having a first mirror and a second mirror positioned to directly propagate light from the first mirror to a beam-forming lens, A beam shaping lens configured to generate an output light beam having a predetermined intensity profile along the horizontal axis from multiple light beams, and A kit that includes the following:

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