Apparatus and method for quantitative characterization of photodetectors

Abstract

Aspects of the present disclosure include methods for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). Methods according to certain embodiments include illuminating a photodetector positioned in a particle analyzer with a light source (e.g., a continuous wave light source) at a first intensity for a first predetermined time interval, illuminating the photodetector with the light source at a second intensity for a second predetermined time interval, integrating a data signal from the photodetector over a time period including the first and second predetermined time intervals, and determining one or more parameters of the photodetector based on the integrated data signal. The present disclosure also describes systems (e.g., particle analyzers) having a light source and a photodetector for implementing the subject methods. The present disclosure also provides non-transitory computer readable storage media having stored thereon instructions for determining parameters of a photodetector according to the subject methods.

Description

[0001] Cross-reference to related applications

[0002] This application relates to U.S. Provisional Patent Application No. 63 / 012,765, filed April 20, 2020, the disclosure of which is incorporated herein by reference.

[0003] introduction

[0004] Characterization of analytes in biofluids has become an important part of medical diagnosis and assessment of overall patient health and well-being. Detecting analytes in biofluids such as human blood or blood-derived products can provide results that can play a role in determining treatment options for patients with multiple disease conditions.

[0005] Flow cytometry is a technique used to characterize and often classify biological materials, such as cells in a blood sample or preferred particles in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample (such as a blood sample) and a sheath reservoir containing the sheath fluid. The flow cytometer delivers particles (including cells) from the fluid sample as a flow stream into a flow cell, while also directing the sheath fluid into the flow cell. To characterize the components of the flow stream, the flow stream is illuminated. Changes in the material within the flow stream (such as the presence of morphology or fluorescent labels) cause observed changes in light, and these changes allow for characterization and separation.

[0006] To characterize the components in a fluid flow, light must strike the fluid flow and be collected. The light source in a flow cytometer can be different from a broad-spectrum lamp, a light-emitting diode, or a single-wavelength laser. The light source is aligned with the flow, collecting and quantifying the optical response from the illuminated particles. Summary of the Invention

[0007] Various methods disclosed herein include approaches for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). According to some embodiments, the method includes illuminating the photodetector with a light source at a first intensity for a first predetermined time interval, illuminating the photodetector with a light source at a second intensity for a second predetermined time interval, integrating a data signal from the photodetector over a time period including the first and second predetermined time intervals, and determining one or more parameters of the photodetector based on the integrated data signal. In some cases, the light source is a continuous-wave light source. In some cases, the light source is a pulsed light source. In some embodiments, the light source is a light-emitting diode (LED). In some cases, the light source is a narrow-bandwidth light source, such as a source emitting light with wavelengths spanning 20 nm or less.

[0008] In practicing the subject method according to certain embodiments, the intensity of the light source is increased after each predetermined time interval (i.e., the second illumination intensity is greater than the first illumination intensity). The time interval for illuminating the photodetector with each light intensity can vary. In some cases, each time interval is the same. In other cases, each time interval is different. In some embodiments, the method includes illuminating the photodetector with a continuous-wave light source at multiple intensities over multiple predetermined time intervals. In these embodiments, some time intervals may be the same, while some time intervals may be different. In some cases, the method includes increasing the intensity of light from the light source over a time period including at least a first predetermined time interval and a second predetermined time interval. In some cases, the intensity of light from the light source increases linearly over a time period including at least a first predetermined time interval and a second predetermined time interval. In other cases, the intensity of light from the light source increases exponentially over a time period including at least a first predetermined time interval and a second predetermined time interval.

[0009] In embodiments, the method includes integrating a data signal from a photodetector over a time period that includes at least a time interval of illumination at each different light intensity. In some embodiments, the data signal from the photodetector is integrated over a time period that includes a first time interval in which the light source illuminates the photodetector at a first intensity and a second time interval in which the light source illuminates the photodetector at a second intensity. In other embodiments, the data signal from the photodetector is integrated over a time period that includes multiple time intervals, wherein the light source illuminates the photodetector at an increasing light intensity during each of the multiple time intervals.

[0010] In some embodiments, integrating the data signal from the photodetector includes calculating the signal amplitude over the time period. In some cases, calculating the signal amplitude includes calculating one or more of the following: median signal amplitude, average signal amplitude, standard deviation of the signal amplitude, and variance and coefficient of variation of the signal amplitude. In some cases, the method further includes comparing the calculated signal amplitude with the light intensity of the light source. Based on one or more of the calculated signal amplitude and the comparison between the calculated signal amplitude and the light intensity of the light source, parameters of the photodetector are calculated. For example, the method may include determining parameters for the photodetector, such as a minimum detection threshold, a maximum detection threshold, detector sensitivity, detector dynamic range, detector signal-to-noise ratio, or number of photoelectrons per unit output. Detector parameters may be determined over the operating voltage range of the photodetector, such as over the entire operating voltage range of the photodetector. In some embodiments, the photodetector is located within a particle analyzer, such as where the photodetector is part of the photodetector's light detection module. In some cases, the particle analyzer is incorporated into a flow cytometer, where the photodetector is positioned to detect light from particles in the flowing stream.

[0011] In some embodiments, the method includes determining one or more parameters of a photodetector (e.g., a photodetector in a particle analyzer) by irradiating particles in a flowing stream, wherein the particles comprise one or more fluorophores. In some cases, the particles are beads (e.g., polystyrene beads). In some cases, the method for determining the parameters of the photodetector includes: irradiating a flowing stream having particles comprising one or more fluorophores at a first intensity for a first predetermined time interval, and irradiating the flowing stream having particles comprising one or more fluorophores at a second intensity for a second predetermined time interval; a photodetector having a light source detecting light from the flowing stream; generating a data signal from the photodetector at the first irradiation intensity; generating a data signal from the photodetector at the second irradiation intensity; and determining one or more parameters of the photodetector based on the data signals generated at the first and second intensities. In some cases, the method includes determining an average fluorescence intensity from the particles at the first and second irradiation intensities. In some cases, the method includes determining a change in the average fluorescence intensity at the first and second irradiation intensities. In some cases, the method includes determining statistical photoelectrons (SPEs) at the first and second irradiation intensities. In some cases, the method also includes calculating the detector efficiency (Q) of the photodetector for each fluorophore on the particle based on statistical photoelectrons and the average fluorescence intensity of the identified fluorophores. det In some embodiments, the method includes determining the detector efficiency of each detector channel of the photodetector. In some embodiments, the method further includes determining the background signal of each photodetector. In some embodiments, the method further includes determining the electronic noise from each photodetector. In some embodiments, the method further includes determining the detection limit of the photodetector.

[0012] Various embodiments of this disclosure also include a system having a light source and a photodetector configured to detect light from the light source at a first intensity for a first predetermined time interval and at a second intensity for a second predetermined time interval; and a processor having a memory operatively coupled to the processor, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to: integrate a data signal from the photodetector over a time interval including the first and second predetermined time intervals; and determine one or more parameters of the photodetector based on the integrated data signal. In some embodiments, the system is a particle analyzer. In some cases, the photodetector is part of the light detection module of the particle analyzer. In some cases, the particle analyzer is incorporated into a flow cytometer.

[0013] In some embodiments, the light source of the subject system is a continuous-wave light source. In other embodiments, the light source is a pulsed light source. In some embodiments, the light source is a light-emitting diode (LED). In some cases, the light source is a narrow-bandwidth light source, such as an LED emitting light with wavelengths spanning 20 nm or less. The light source is configured to illuminate a photodetector at two or more different intensities for predetermined time intervals. In some embodiments, the light source is configured to illuminate the photodetector at a first intensity for a first predetermined time interval and at a second intensity for a second predetermined time interval. In other embodiments, the light source is configured to illuminate the photodetector at multiple intensities within multiple predetermined time intervals. In embodiments, each time interval may be the same duration or different durations. In some embodiments, the light source is configured to increase the illumination intensity after each predetermined time interval (i.e., increase the light intensity for each consecutive illumination interval). In some embodiments, the light source is configured to increase the intensity over a time period including at least the first and second predetermined time intervals. In some cases, the intensity of the light source is configured to increase gradually. In other cases, the intensity of the light source is configured to increase exponentially.

[0014] A preferred system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to integrate a data signal from a photodetector. In some embodiments, the memory includes instructions for calculating the signal amplitude from the photodetector in each time interval. In other embodiments, the memory includes instructions for calculating the median signal amplitude. In some embodiments, the memory includes instructions for comparing the calculated signal amplitude with an illumination intensity during each predetermined time interval.

[0015] In one embodiment, the system includes a memory operatively coupled to a processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine parameters of the photodetector based on one or more of a calculated signal amplitude and a comparison between the calculated signal amplitude and the light intensity of a light source. For example, the memory may include instructions for determining a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, or the number of photoelectrons per unit output. The system can be configured to determine detector parameters over the operating voltage range of the photodetector, such as over the entire operating voltage range of the photodetector.

[0016] Various embodiments of this disclosure also include a non-transitory computer-readable storage medium for determining one or more parameters of a photodetector. In embodiments, the non-transitory computer-readable storage medium includes an algorithm for illuminating the photodetector with a light source at a first intensity for a first predetermined time interval; an algorithm for illuminating the photodetector with a light source at a second intensity for a second predetermined time interval; an algorithm for integrating a data signal from the photodetector over a time period including the first and second predetermined time intervals; and an algorithm for determining one or more parameters of the photodetector based on the integrated data signal. In some cases, the non-transitory computer-readable storage medium includes an algorithm for illuminating the photodetector with multiple light intensities over multiple time intervals. In these cases, the non-transitory computer-readable storage medium includes an algorithm for integrating a data signal from the photodetector over a time period including multiple illumination time intervals.

[0017] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating signal amplitude. In some examples, the non-transitory computer-readable storage medium includes an algorithm for calculating one or more of the following: median signal amplitude, average signal amplitude, standard deviation of the signal amplitude, and variance and coefficient of variation of the signal amplitude. In some cases, the non-transitory computer-readable storage medium includes an algorithm for comparing the calculated signal amplitude with the light intensity of a light source. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining parameters of a photodetector based on one or more of the calculated signal amplitude and a comparison between the calculated signal amplitude and the light intensity of a light source. For example, the non-transitory computer-readable storage medium may include an algorithm for determining a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, or the number of photoelectrons per unit output. The non-transitory computer-readable storage medium may include an algorithm for determining detector parameters over the operating voltage range of the photodetector, such as wherein the parameters of the photodetector are determined over the entire operating voltage range of the photodetector.

[0018] In some embodiments, the present disclosure also includes multispectral particles (e.g., beads) having one or more fluorophores for implementing one or more subject methods. According to some embodiments, the multispectral particles contain one or more fluorophores, such as two or more, three or more, five or more, and including ten or more fluorophores. In some cases, preferred particles comprise single-peak multifluorophore beads that provide a bright photodetector signal across all light source wavelengths (e.g., across all LEDs or lasers in the system) and across the detection wavelengths of the photodetector. Attached Figure Description

[0019] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures:

[0020] Figure 1A and Figure 1B The measurement of light intensity variations from a light source over multiple discrete time intervals is described using a photodetector according to certain embodiments.

[0021] Figure 1A A 50-step light intensity ramp spanning 2601 ms was depicted.

[0022] Figure 1B Depicting Figure 1A The first 5 steps of the 50-step light intensity ramp (spanning 250ms).

[0023] Figure 2 Measurements of continuously varying light intensity according to certain embodiments are described.

[0024] Figure 3A A flowchart illustrating a method for determining one or more parameters of a photodetector according to certain embodiments is depicted.

[0025] Figure 3B A graph depicting the initial detector gain for setting the photodetector according to certain embodiments is shown.

[0026] Figure 4A A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization, according to certain embodiments, is depicted.

[0027] Figure 4B A flow cytometer according to certain embodiments is described.

[0028] Figure 5 A functional block diagram of an example particle analyzer system according to certain embodiments is depicted.

[0029] Figure 6A A schematic diagram of a particle classifier system according to certain embodiments is depicted.

[0030] Figure 6B A schematic diagram of a particle classifier system according to certain embodiments is depicted.

[0031] Figure 7 A block diagram of a computing system according to certain embodiments is depicted. Detailed Implementation

[0032] Various methods disclosed herein include methods for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). A method according to some embodiments includes: illuminating a photodetector positioned in a particle analyzer with a light source (e.g., a continuous wave light source) at a first intensity for a first predetermined time interval; illuminating the photodetector with a light source at a second intensity for a second predetermined time interval; integrating a data signal from the photodetector over a time interval including the first and second predetermined time intervals; and determining one or more parameters of the photodetector based on the integrated data signal. This disclosure also describes a system (e.g., a particle analyzer) having a light source and a photodetector for implementing the subject method. This disclosure also provides a non-transitory computer-readable storage medium storing instructions for determining parameters of a photodetector according to the subject method.

[0033] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as such modifications are naturally possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention; the scope of the invention will be limited only by the appended claims.

[0034] Where numerical ranges are provided, it should be understood that, unless the context clearly indicates otherwise, every intermediate value between the upper and lower limits of the range up to one-tenth of the lower limit unit, and any other stated or intermediate value within the range, is covered by this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also within this invention, subject to any particular exclusions. When the range includes one or both limits, the range excluding one or both of those included limits is also included in this invention.

[0035] The numerical ranges given in this document are preceded by the term "approximately". The term "approximately" is used in this document to provide textual support for the exact number preceding it and for numbers that are close to or approximate to the number preceding the term. In determining whether a number is close to or approximate to a specifically listed number, the unlisted number that is close to or approximate to may be a number that is substantially equivalent to the specifically listed number in the context in which it is presented.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, representative exemplary methods and materials are described hereafter.

[0037] All publications and patents referenced in this specification 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 referenced publications. Reference to any publication is for its publication prior to the filing date and should not be construed as an admission that the invention is not entitled to prior rights to such publication due to prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0038] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a prior basis for using exclusive terms such as “solely,” “only,” etc., or for using the limiting word “no,” in relation to the description of the claim elements.

[0039] Those skilled in the art will understand upon reading this disclosure that each of the various embodiments described and illustrated herein has discrete components and features, which can be readily separated from or combined with features of any of the 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.

[0040] Although the apparatus and method have been or will be described using a functional interpretation for the sake of grammatical fluency, it should be clearly understood that, unless expressly set forth in 35 U.SC § 112, these claims should not be construed as necessarily being limited in any way to the construction of “apparatus” or “step,” but should conform to the meaning and full scope of equivalents provided by the claims under the doctrine of judicial equivalence, and in the case of claims expressly set forth in 35 U.SC § 112, should conform to all legal equivalents in 35 U.SC § 112.

[0041] As summarized above, this disclosure provides methods and systems for determining one or more parameters of a photodetector (e.g., a photodetector in a particle analyzer). In further describing embodiments of this disclosure, a method comprising irradiating the photodetector with a first intensity during a first predetermined time interval and with a second intensity during a second predetermined time interval, and integrating data signals from the photodetector over a time period including the first and second predetermined time intervals, is first described in more detail. Next, a system (e.g., a particle analyzer) having a light source and a photodetector for practicing the subject method is described. This disclosure also provides a non-transitory computer-readable storage medium having instructions stored thereon for determining parameters of the photodetector according to the subject method.

[0042] Methods for determining the parameters of a photodetector

[0043] Various methods disclosed herein include approaches for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). In implementing the methods of this subject matter, the photodetector is illuminated with a light source of a first intensity for a first predetermined time interval, and then illuminated with a light source of a second intensity for a second predetermined time interval. In some embodiments, the light source is a continuous light source. The term "continuous light source" is used herein in its conventional sense to refer to a light source that provides an uninterrupted luminous flux and maintains illumination of the photodetector with little or no undesired variation in light intensity. In some embodiments, the continuous light source emits non-pulsed or non-flickering illumination. In some embodiments, the continuous light source provides substantially constant emitted light intensity. For example, a continuous light source may provide a luminous intensity that varies by 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, such as 0.00001% or less, and includes a luminous intensity variation of 0.000001% or less during the illumination time interval. The intensity of the light output can be measured using any convenient protocol, including but not limited to scanning slit profilers, charge-coupled devices (CCDs) such as enhanced charge-coupled devices (ICCDs), positioning sensors, power sensors (e.g., thermopile power sensors), optical power sensors, energy meters, digital laser photometers, laser diode detectors, and other types of photodetectors.

[0044] In some cases, the light source is a pulsed light source. The term "pulsed light source" is used herein in its conventional sense to refer to a light source that provides luminous flux at predetermined time intervals, such as by stroboscopic illumination. The pulse duration can vary depending on the type of light source and can be 0.001 ns or more, such as 0.005 ns or more, such as 0.01 ns or more, such as 0.05 ns or more, such as 0.1 ns or more, such as 0.5 ns or more, such as 1 ns or more, such as 2 ns or more, such as 3 ns or more, such as 5 ns or more, such as 10 ns or more, such as 25 ns or more, such as 50 ns or more, such as 100 ns or more, such as 500 ns or more, such as 1000 ns or more, and includes pulse durations of 5 μs or more. For example, the pulse duration of a pulsed light source can range from 0.00001 μs to 1000 μs, such as from 0.00005 μs to 900 μs, such as from 0.0001 μs to 800 μs, such as from 0.0005 μs to 700 μs, such as from 0.001 μs to 600 μs, such as from 0.005 μs to 500 μs, such as from 0.01 μs to 400 μs, such as from 0.05 μs to 300 μs, such as from 0.1 μs to 200 μs, and includes pulse durations ranging from 1 μs to 100 μs.

[0045] The light source can be any convenient light source and can include both laser and non-laser light sources. In some embodiments, the light source is a non-laser light source, such as a narrowband light source emitting a specific wavelength or a narrow wavelength range. In some cases, the narrowband light source emits light with a narrow wavelength range, such as 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less, and includes light sources emitting light of a specific wavelength (i.e., monochromatic light). Any convenient narrowband light source protocol can be used, such as narrow-wavelength LEDs.

[0046] In other embodiments, the light source is a broadband light source, such as a broadband light source coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof. In some cases, the broadband light source emits light with a wide range of wavelengths, such as spanning 50 nm or more, such as 100 nm or more, such as 150 nm or more, such as 200 nm or more, such as 250 nm or more, such as 300 nm or more, such as 350 nm or more, such as 400 nm or more, and including spanning 500 nm or more. For example, a suitable broadband light source emits light with wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with wavelengths from 400 nm to 1000 nm. Any convenient broadband light source protocol can be used, such as halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with a continuous spectrum, superluminescent diodes, semiconductor light-emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, and other broadband light sources or any combination thereof. In some embodiments, the light source used to illuminate the flow of particles passing through an opening in the particle sorting module during image capture includes an infrared LED array.

[0047] In some embodiments, the light source is a laser, such as a continuous-wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser can be a helium-neon laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) stimulated excimer laser, a krypton-fluorine (KrF) stimulated excimer laser, a xenon-chlorine (XeCl) stimulated excimer laser, or a xenon-fluorine (XeF) stimulated excimer laser, or a combination thereof. In other cases, the system includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In other cases, preferred lasers include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof. In other cases, the light source is a solid-state laser, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers, and combinations thereof.

[0048] In some embodiments, the light source is a narrow bandwidth light source. In some cases, the light source is a light source that outputs a specific wavelength ranging from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm, and including 400 nm to 800 nm. In some embodiments, the continuous wave light source emits light having wavelengths of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm, or 850 nm.

[0049] The photodetector can be illuminated by a light source from any suitable distance from the flow, such as at a distance of 0.001 mm or longer, such as 0.005 mm or longer, such as 0.01 mm or longer, such as 0.05 mm or longer, such as 0.1 mm or longer, such as 0.5 mm or longer, such as 1 mm or longer, such as 5 mm or longer, such as 10 mm or longer, such as 25 mm or longer, and including at a distance of 100 mm or longer. Furthermore, the illumination of the photodetector can be at any suitable angle, such as angles from 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including angles from 30° to 60°, for example, 90°.

[0050] In embodiments, the method includes illuminating a photodetector with a light source for two or more discrete time intervals. The term "discrete time interval" is used herein in its conventional sense to refer to illuminating the photodetector with a light source for a predetermined duration, followed by a period of time during which the intensity of the light source changes (e.g., increases), and then by subsequent discrete time intervals or illuminating sessions. In some embodiments, the method includes illuminating the photodetector at discrete time intervals of 0.1 ms or more, such as 0.5 ms or more, such as 1.0 ms or more, such as 5 ms or more, such as 10 ms or more, such as 20 ms or more, such as 30 ms or more, such as 40 ms or more, such as 50 ms or more, such as 60 ms or more, such as 70 ms or more, such as 80 ms or more, such as 90 ms or more, including 100 ms or more. In some embodiments, each predetermined time interval for illuminating the photodetector is the same duration. For example, each predetermined time interval according to the subject method may be 50 ms. In other embodiments, each predetermined time interval is different. In some embodiments, the method includes illuminating a photodetector with a continuous-wave light source at multiple intensities, each intensity occurring over multiple discrete time intervals, such as three or more discrete time intervals, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more, such as fifty or more, such as seventy-five or more, and including one hundred or more discrete time intervals.

[0051] In some embodiments, each of the plurality of time intervals has the same duration. In other embodiments, each of the plurality of time intervals has a different duration. In still other embodiments, some time intervals may have the same duration, while some time intervals may have different durations.

[0052] In some embodiments, the illumination intensity of the light source is substantially constant over the duration of each predetermined time interval, such as where the illumination intensity varies by 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, such as 0.00001% or less, and includes where the illumination intensity of the light source varies by 0.000001% or less over the duration of the predetermined time interval.

[0053] In some embodiments, the intensity of the light source is varied after each discrete illumination interval. In some embodiments, the illumination intensity of the light source is increased. In other embodiments, the intensity of the light source is decreased. The intensity of the light used to illuminate the photodetector may vary by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and including 100% or more, in each subsequent time interval. In some cases, the light intensity is varied by 1.5 times or more, such as 2 times or more, such as 3 times or more, such as 4 times or more, and including 5 times or more. In some embodiments, the method includes increasing the light intensity for each subsequent time interval, such as by increasing the light intensity for each subsequent time interval by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and including 100% or more. In other embodiments, the method includes increasing the light intensity for each subsequent time interval by 1.5 times or more, such as 2 times or more, such as 3 times or more, such as 4 times or more, and including 5 times or more.

[0054] In some cases, the method includes keeping the photodetector illuminated by the light source while changing the intensity (e.g., while increasing the light intensity). In some cases, the method includes increasing the intensity of the light from the light source over a time period including at least a first predetermined time interval and a second predetermined time interval. In some cases, the intensity of the light from the light source increases linearly over a time period including at least a first predetermined time interval and a second predetermined time interval. In other cases, the intensity of the light from the light source increases exponentially over a time period including at least a first predetermined time interval and a second predetermined time interval.

[0055] In other cases, the method includes stopping the illumination of the photodetector by the light source for the duration during which the intensity of the light source is changed (e.g., by turning off the light source or by blocking the light source, such as with a chopper, beam stop, etc.). Any convenient protocol can be used to provide intermittent illumination, such as an electronic switch for turning the light source on and off, such as a computer-controlled switch triggered based on a data signal (e.g., a received or input data signal), as described in more detail below. In some embodiments, the time interval for changing the intensity of the light source can be 0.001 ms or more, such as 0.005 ms or more, such as 0.01 ms or more, such as 0.05 ms or more, such as 0.1 ms or more, such as 0.5 ms or more, such as 1 ms or more, such as 2 ms or more, such as 3 ms or more, such as 4 ms or more, such as 5 ms or more, such as 6 ms or more, such as 7 ms or more, such as 8 ms or more, such as 9 ms or more, and includes 10 ms or more. For example, the time interval between each predetermined time interval for illuminating the photodetector with a light source can be from 0.001ms to 25ms, such as from 0.005ms to 20ms, such as from 0.01ms to 15ms, such as from 0.05ms to 10ms, and includes from 0.1ms to 5ms.

[0056] According to some embodiments, the method of this disclosure further includes detecting light using a photodetector. The photodetector used to implement the methods of this subject matter can be any convenient light detection protocol, including but not limited to light sensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-coupled devices (ICCDs), light-emitting diodes, photon counters, radiation thermometers, thermoelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or avalanche photodiodes (APDs), silicon photomultiplier tubes and combinations thereof, and other photodetectors. In some embodiments, the photodetector is a photomultiplier tube, such as one with an active detection surface area of ​​0.01 cm² per region. 2 -10cm 2 Such as 0.05cm 2 -9cm 2 Such as 0.1cm 2 -8cm 2 Such as 0.5cm 2 -7cm 2 And including 1cm 2 -5cm 2 The photomultiplier tube. In other embodiments, the photodetector is an avalanche photodiode, such as an avalanche photodiode, with an effective detection surface area of ​​0.01 cm² per region. 2Up to 10cm 2 Within a range, such as within 0.05cm 2 Up to 9cm 2 Within a range, such as within 0.1cm 2 Up to 8cm 2 Within a range, such as within 0.5cm 2 Up to 7cm 2 Within the range, and including 1cm 2 up to 5cm 2 Within the range. In some cases, light is detected by a photodetector array, such as a photodetector array having 2 or more photodetectors, such as 3 or more photodetectors, such as 5 or more photodetectors, such as 10 or more photodetectors, such as 25 or more photodetectors, such as 50 or more photodetectors, such as 75 or more photodetectors, such as 100 or more photodetectors, such as 500 or more photodetectors, and including a photodetector array having 1000 or more photodetectors. In some cases, light is detected by an array of avalanche photodiodes, such as a photodetector array having two or more avalanche photodiodes, such as three or more avalanche photodiodes, such as five or more avalanche photodiodes, such as ten or more avalanche photodiodes, such as 25 or more avalanche photodiodes, such as 50 or more avalanche photodiodes, such as 75 or more avalanche photodiodes, such as 100 or more avalanche photodiodes, such as 500 or more avalanche photodiodes, and including a photodetector array having 1000 or more avalanche photodiodes.

[0057] In embodiments of this disclosure, light can be measured by a photodetector at one or more wavelengths, such as at two or more wavelengths, such as at five or more different wavelengths, such as at ten or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and includes measuring light from particles in the flowing stream at 400 or more different wavelengths.

[0058] In embodiments, light can be measured continuously or at discrete intervals. In some cases, a preferred detector is configured to measure light continuously. In other cases, a preferred detector is configured to measure light at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0059] During each discrete time interval, one or more measurements of light from the light source can be performed, such as two or more, three or more, five or more, and including ten or more. In some embodiments, the light from the light source is measured two or more times by a photodetector, and in some cases the data are averaged.

[0060] Figure 1A and 1B The measurement of light intensity variations from a light source over multiple discrete time intervals is depicted using a photodetector according to certain embodiments. For each 50 ms time interval, the photodetector is illuminated with a constant light intensity from the light source, followed by a 2 dB increase in light intensity over a 1 ms ramp time interval. Figure 1A A 50-step light intensity ramp spanning 2601 ms was depicted. Figure 1B Depicting Figure 1A The first 5 steps of the 50-step light intensity ramp (spanning 250ms). Figure 2 Measurements of continuously varying light intensity are described according to certain embodiments. A photodetector is illuminated with light intensity increasing continuously over time, and the data signal is integrated over two or more predetermined time intervals to determine the parameters of the photodetector.

[0061] Figure 3 illustrates a flowchart for determining one or more parameters of a photodetector according to certain embodiments. In step 301, the photodetector is illuminated with a continuous-wave light source at a first light intensity for a first predetermined discrete time interval. In step 302, the photodetector is illuminated with a light source of a second intensity for a second predetermined discrete time interval. In step 303, the data signal from the photodetector is integrated over a time period that includes at least the first and second time intervals. In step 304, the signal amplitude from the photodetector is calculated based on the integrated data signal. In step 305, the calculated signal amplitude is used to determine one or more parameters, such as wherein the calculated signal amplitude is compared with the light intensity during each illumination interval. For example, by comparing the calculated signal amplitude with the light intensity during each illumination interval, a minimum detection threshold 305a, a maximum detection threshold 305b, a detector sensitivity 305c, a detector dynamic range 305d, a detector signal-to-noise ratio 305e, or the number of photoelectrons output per unit 305f can be determined.

[0062] In some embodiments, the method includes integrating data signals from a photodetector. In some embodiments, integrating the data signals from the photodetector includes integrating the data signals over 10% or more of the duration of each discrete illumination interval, such as 15% or more, such as 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and includes integrating the data signals over 99% of the duration of each discrete illumination interval. In some embodiments, according to the main method, the data signals from the photodetector are integrated over the entire duration of each discrete illumination time interval.

[0063] In some embodiments, the method includes integrating a data signal from a photodetector for a period of time, which includes at least each discrete time interval of illumination at each different light intensity. For example, in the case where a continuous wave light source illuminates the photodetector over 50 or more discrete intervals, the method includes integrating the data signal from the photodetector over a time period comprising at least 50 discrete time intervals. In some embodiments, the method includes integrating the data signal from the photodetector over a time period including the duration prior to illuminating the photodetector, according to the subject method, to measure a noise component of the photodetector signal. In these embodiments, the method includes integrating the data signal from the photodetector for 0.001 ms or more, such as 0.005 ms or more, such as 0.01 ms or more, such as 0.05 ms or more, such as 0.1 ms or more, such as 0.5 ms or more, such as 1 ms or more, such as 2 ms or more, such as 3 ms or more, such as 4 ms or more, such as 5 ms or more, such as 10 ms or more, such as 25 ms or more, such as 50 ms or more, such as 100 ms or more, and includes integrating for 250 ms or more before illuminating the photodetector. In other embodiments, the method includes integrating the data signal from the photodetector after the final discrete illumination time interval, such as 0.005 ms or more, such as 0.01 ms or more, such as 0.05 ms or more, such as 0.1 ms or more, such as 0.5 ms or more, such as 1 ms or more, such as 2 ms or more, such as 3 ms or more, such as 4 ms or more, such as 5 ms or more, such as 10 ms or more, such as 25 ms or more, such as 50 ms or more, such as 100 ms or more, and including 250 ms or more.

[0064] In some embodiments, integrating the data signal from the photodetector includes calculating the signal amplitude over the time period. In some cases, calculating the signal amplitude includes calculating the median signal amplitude. In some cases, the method further includes comparing the calculated signal amplitude with the light intensity of the light source. In other cases, the method includes calculating the average signal amplitude. In some cases, the method further includes calculating the standard deviation of the signal amplitude. In other cases, the method includes calculating the variance and coefficient of variation of the signal amplitude (e.g., CV = standard deviation / mean). Based on one or more of the calculated signal amplitude and the comparison between the calculated signal amplitude and the light intensity of the light source, parameters of the photodetector are calculated. For example, the method may include determining parameters for the photodetector, such as a minimum detection threshold, a maximum detection threshold, detector sensitivity (i.e., the ratio of detector output to detector input), detector dynamic range (the range of the detector signal from the minimum to the maximum detection threshold), detector signal-to-noise ratio, or the number of photoelectrons per unit output.

[0065] Each detector parameter can be determined within the operating voltage range of the photodetector. In some embodiments, the parameter is determined based on a calculated signal amplitude at 10% or more of the photodetector's operating voltage, such as 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, or 90% or more, and the parameter is determined at 99% or more of the photodetector's operating voltage. In some cases, each parameter can be determined across the entire operating voltage range of the photodetector.

[0066] In some cases, the parameters of the photodetector may be adjusted based on either a calculated signal amplitude or a comparison between the calculated signal amplitude and the illumination intensity during each discrete time interval. The term "adjustment" is used herein in its conventional sense to refer to changing one or more functional parameters of the photodetector. For example, adjusting the photodetector may include increasing or decreasing the photodetector voltage gain. In some embodiments, adjusting one or more parameters of the photodetector based on a calculated signal amplitude or a comparison between the calculated signal amplitude and the illumination intensity during preferred discrete time intervals can be fully automated, requiring minimal or no human intervention or manual input from the user.

[0067] In some embodiments, the method includes determining one or more parameters of a photodetector (e.g., a photodetector in a particle analyzer) by irradiating particles in a flowing stream, wherein the particles comprise one or more fluorophores. In some cases, the particles are beads (e.g., polystyrene beads), as described in more detail below. In some cases, the subject matter method described below provides parameters for determining photodetectors that include a specified relative fluorescence unit (e.g., ABD unit) for each photodetector, robust coefficient of variation (rCV) for one or more photodetectors, maximum and minimum linearity for each photodetector, relative change of rCV relative to a baseline, relative change of detector gain relative to a baseline, and imaging specifications of the photodetector, such as RF power or axial optical loss.

[0068] In some cases, a method for determining parameters of a photodetector includes: irradiating a flow of particles comprising one or more fluorophores at a first intensity for a first predetermined time interval, and irradiating the flow of particles comprising one or more fluorophores at a second intensity for a second predetermined time interval; a photodetector having a light source detecting light from the flow; generating a data signal from the photodetector at the first irradiation intensity; generating a data signal from the photodetector at the second irradiation intensity; and determining one or more parameters of the photodetector based on the data signals generated at the first and second intensities.

[0069] In some embodiments, the method includes determining the average fluorescence intensity (M) from the particles under a first irradiation intensity and a second irradiation intensity. In some cases, the method includes determining the variance of the average fluorescence intensity (V(M)) under the first irradiation intensity and the second irradiation intensity. In some cases, the method includes determining the %rCV (robust coefficient of variation) of the photodetector. In some embodiments, a linear fit of the variance is calculated according to the following formula:

[0070]

[0071] Q led Given by 1 / c1, and is the statistical photoelectrons per unit average fluorescence intensity (M) (i.e., SPE / MFI). In some embodiments, the variance is plotted according to the following formula to determine a linear fit of the variance:

[0072] y = c1x + c0

[0073] In embodiments, the average fluorescence intensity and variance can be determined for multiple different irradiation intensities, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, and including 15 or more different irradiation intensities.

[0074] In some embodiments, the method includes determining statistical photoelectrons (SPEs) at one or more illumination intensities, such as at least a first illumination intensity and a second illumination intensity. In some cases, the method further includes calculating the detector efficiency (Q) of the photodetector for each particle based on the statistical photoelectrons and the determined average fluorescence intensity of the particles. det In some embodiments, the method includes determining the detector efficiency of one or more detector channels of a photodetector, such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 12 or more, such as 16 or more, such as 20 or more, such as 24 or more, such as 36 or more, such as 48 or more, such as 72 or more, and includes instructions for determining the detector efficiency of 96 or more detector channels of the photodetector based on statistical photoelectrons and the determined average fluorescence intensity of the particles. In some cases, the method includes determining the detector efficiency of all detector channels of the photodetector for each particle based on statistical photoelectrons and the determined average fluorescence intensity of each particle. In some embodiments, the detector efficiency for the photodetector is determined according to:

[0075]

[0076] SPE stands for Statistical Photoelectrons, MFI for Average Fluorescence Intensity, and ABD for the specified unit per channel per batch of particles.

[0077] In some embodiments, the method further includes determining a background signal for one or more photodetectors. In some cases, the background signal is determined at one or more illumination intensities, such as 2 or greater, such as 3 or greater, such as 4 or greater, such as 5 or greater, such as 6 or greater, such as 7 or greater, such as 8 or greater, such as 9 or greater, such as 10 or greater, and includes different illumination intensities of 10 or greater. In some cases, the background signal is determined at all applied illumination intensities. Background signals can also be determined in one or more detector channels of a photodetector, such as in two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, sixteen or more, twenty or more, twenty-four or more, thirty-six or more, forty-eight or more, seventy-two or more, and include determining background signals in 96 or more detector channels of a photodetector, wherein in some cases, background signals are determined in all detector channels of the photodetector. In some cases, background signals are determined based on statistical optoelectronics and the detector efficiency of the photodetector. In some cases, the background signal is determined according to the following:

[0078] B SD =B SD,MFI ×Q led

[0079]

[0080] In some embodiments, the method further includes determining the electronic noise of one or more photodetectors. In some cases, the electronic noise of the photodetector is determined at one or more illumination intensities, such as 2 or greater, such as 3 or greater, such as 4 or greater, such as 5 or greater, such as 6 or greater, such as 7 or greater, such as 8 or greater, such as 9 or greater, such as 10 or greater, and includes 10 or more different illumination intensities. In some cases, the electronic noise of the photodetector is determined at all applied illumination intensities. Electronic noise can also be determined in one or more detector channels of a photodetector, such as in two or more, such as in three or more, such as in four or more, such as in five or more, such as in six or more, such as in seven or more, such as in eight or more, such as in nine or more, such as in ten or more, such as in twelve or more, such as in sixteen or more, such as in twenty or more, such as in twenty-four or more, such as in thirty-six or more, such as in forty-eight or more, such as in seventy-two or more, and includes determining electronic noise in ninety-six or more detector channels of a photodetector, wherein in some cases, electronic noise is determined in all detector channels of a photodetector. In some cases, electronic noise is determined based on statistical photoelectrons and the detector efficiency of the photodetector. In some cases, the electronic noise is determined according to the following:

[0081] EN SD =EN SD,MFI ×Q led

[0082]

[0083] In some embodiments, the method further includes determining the detection limit of one or more photodetectors. In some cases, the detection limit of the photodetectors is determined in one or more detector channels of the photodetectors, such as in two or more, such as in three or more, such as in four or more, such as in five or more, such as in six or more, such as in seven or more, such as in eight or more, such as in nine or more, such as in ten or more, such as in twelve or more, such as in sixteen or more, such as in twenty or more, such as in twenty-four or more, such as in thirty-six or more, such as in forty-eight or more, such as in seventy-two or more, and includes determining the detection limit of the photodetectors in 96 or more detector channels of the photodetectors, wherein in some cases, the detection limit is determined in all detector channels of the photodetectors. In some instances, the detection limit of each photodetector is determined according to the following:

[0084] 2+2SD=4(1 SD )

[0085] In some embodiments, the method further includes determining the detector photosensitivity of one or more photodetectors. In some embodiments, determining the detector photosensitivity of the photodetectors includes establishing an initial detector gain for the photodetectors. In some cases, the method includes illuminating the photodetectors with a light source (as described in detail above) at multiple different light intensities, generating data signals from the photodetectors for the multiple light intensities at one or more detector gains of the photodetectors, and determining the minimum light intensity at each detector gain that produces a data signal resolvable from a background data signal. In some cases, the method includes determining the minimum light intensity that generates a data signal that differs from a background data signal by two standard deviations at each detector gain. In some cases, the method includes setting the detector gain to a gain that reaches a plateau when plotted as a function of light intensity, resulting in a data signal resolvable from a background data signal. Figure 3B A graph depicting the initial detector gain for a photodetector according to certain embodiments is shown. For example... Figure 3B As shown, the detector gain of the photodetector is plotted as a function of the illumination intensity of light (e.g., LED) from two different fluorophores (e.g., fluorophores associated with particle stability, as described in more detail below). When setting the initial detector gain for the photodetector, the detector gain is determined where the lowest illumination intensity that produces a data signal resolvable from the background data signal platform is... Figure 3B The voltage is approximately 575 volts.

[0086] System for determining the parameters of a photodetector

[0087] Various embodiments of this disclosure also include systems having a light source and a photodetector configured to detect light from the light source at different illumination intensities over predetermined time intervals. In some embodiments, the light source is a continuous-wave light source. The term "continuous-wave light source" as used herein refers to a light source that provides an uninterrupted luminous flux and maintains illumination of the photodetector with little or no undesirable variation in light intensity. In some embodiments, the continuous light source emits non-pulsed or non-flicker illumination. In some embodiments, the continuous light source provides a substantially constant emitted light intensity. For example, a continuous light source may provide a luminous intensity that varies by 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, such as 0.00001% or less, and includes a luminous intensity variation of 0.000001% or less during the irradiation time interval.

[0088] In some cases, the light source is a pulsed light source. The term "pulsed light source" is used herein in its conventional sense to refer to a light source that provides luminous flux at predetermined time intervals, such as by stroboscopic illumination. The pulse duration can vary depending on the type of light source and can be 0.001 ns or more, such as 0.005 ns or more, such as 0.01 ns or more, such as 0.05 ns or more, such as 0.1 ns or more, such as 0.5 ns or more, such as 1 ns or more, such as 2 ns or more, such as 3 ns or more, such as 5 ns or more, such as 10 ns or more, such as 25 ns or more, such as 50 ns or more, such as 100 ns or more, such as 500 ns or more, such as 1000 ns or more, and includes pulse durations of 5 μs or more. For example, the pulse duration of a pulsed light source can range from 0.00001 μs to 1000 μs, such as from 0.00005 μs to 900 μs, such as from 0.0001 μs to 800 μs, such as from 0.0005 μs to 700 μs, such as from 0.001 μs to 600 μs, such as from 0.005 μs to 500 μs, such as from 0.01 μs to 400 μs, such as from 0.05 μs to 300 μs, such as from 0.1 μs to 200 μs, and includes pulse durations ranging from 1 μs to 100 μs.

[0089] Continuous wave light sources can be any convenient light source and can include both laser and non-laser sources. In some embodiments, the light source is a non-laser source, such as a narrowband source emitting a specific wavelength or a narrow wavelength range. In some cases, narrowband light sources emit light with a narrow wavelength range, such as 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less, and include sources emitting light of a specific wavelength (i.e., monochromatic light). Any convenient narrowband light source protocol can be used, such as narrow-wavelength LEDs.

[0090] In other embodiments, the light source is a broadband light source, such as a broadband light source coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof. In some cases, the broadband light source emits light with a wide range of wavelengths, such as spanning 50 nm or more, such as 100 nm or more, such as 150 nm or more, such as 200 nm or more, such as 250 nm or more, such as 300 nm or more, such as 350 nm or more, such as 400 nm or more, and including spanning 500 nm or more. For example, a suitable broadband light source emits light with wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with wavelengths from 400 nm to 1000 nm. Any convenient broadband light source protocol can be used, such as halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with a continuous spectrum, superluminescent diodes, semiconductor light-emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, and other broadband light sources or any combination thereof. In some embodiments, the light source used to illuminate the flow of particles passing through an opening in the particle sorting module during image capture includes an infrared LED array.

[0091] In some embodiments, the light source is a laser, such as a continuous-wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser may be a helium-neon laser. In some cases, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) stimulated excimer laser, a krypton-fluorine (KrF) stimulated excimer laser, a xenon-chlorine (XeCl) stimulated excimer laser, or a xenon-fluorine (XeF) stimulated excimer laser, or a combination thereof. In other cases, the system includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In other cases, preferred lasers include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof. In other cases, the subject system includes solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers, and combinations thereof.

[0092] In some embodiments, the light source is a narrow-bandwidth light source. In some cases, the light source is a light source that outputs a specific wavelength ranging from 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm, such as from 350 nm to 900 nm, and including 400 nm to 800 nm. In some embodiments, the light source emits light having wavelengths of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm, or 850 nm. In some embodiments, the wavelength of the light output by the light source is matched to optical adjustment components (such as bandpass filters or dichroic mirrors) used when illuminating the photodetector. In some cases, the wavelength of the light output by the light source is matched to the spectral bandwidth of the bandpass filter used for optical communication with the photodetector.

[0093] The light source can be located at any suitable distance from the photodetector, such as a distance of 0.005 mm or longer, such as 0.01 mm or longer, such as 0.05 mm or longer, such as 0.1 mm or longer, such as 0.5 mm or longer, such as 1 mm or longer, such as 5 mm or longer, such as 10 mm or longer, such as 25 mm or longer, and including distances of 100 mm or longer. Additionally, the light source can be positioned at any suitable angle to the photodetector, such as 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including angles of 30° to 60°, for example, 90°.

[0094] According to some embodiments, the light source may also include one or more optical adjustment components. The term "optical adjustment" is used herein in its conventional sense to refer to any means capable of altering the spatial width of illumination from the light source or some other characteristic of the illumination (such as illumination direction, wavelength, beam width, beam intensity, and focus). An optical adjustment protocol can be any convenient means of adjusting one or more characteristics of the light source, including but not limited to lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimation protocols, and combinations thereof. In some embodiments, a preferred system includes one or more focusing lenses. In one example, the focusing lens may be a reducing lens. In another example, the focusing lens is a magnifying lens. In other embodiments, a preferred system includes one or more mirrors. In other embodiments, a preferred system includes optical fibers.

[0095] When the optical adjustment component is configured to move, it can be configured to move continuously or at discrete intervals. In some embodiments, the movement of the optical adjustment component is continuous. In other embodiments, the optical adjustment component can move at discrete intervals, such as in increments of 0.01 micrometers or greater, such as 0.05 micrometers or greater, such as 0.1 micrometers or greater, such as 0.5 micrometers or greater, such as 1 micrometer or greater, such as 10 micrometers or greater, such as 100 micrometers or greater, such as 500 micrometers or greater, such as 1 mm or greater, such as 5 mm or greater, such as 10 mm or greater, and including increments of 25 mm or greater.

[0096] The optical adjustment component structure can be moved using any displacement protocol, such as coupling to a movable support stage or directly coupling to a motor-actuated translation stage, a lead screw translation assembly, a gear translation device, or using stepper motors, servo motors, brushless motors, brushed DC motors, micro-stepping drive motors, high-resolution stepper motors, and other types of motors.

[0097] In embodiments, a continuous-wave light source is configured to illuminate two or more discrete time intervals, each time interval being at a different illumination intensity. In some embodiments, the continuous-wave light source is configured to illuminate time intervals of 0.1 ms or more at a specific intensity, such as 0.5 ms or more, such as 1.0 ms or more, such as 5 ms or more, such as 10 ms or more, such as 20 ms or more, such as 30 ms or more, such as 40 ms or more, such as 50 ms or more, such as 60 ms or more, such as 70 ms or more, such as 80 ms or more, such as 90 ms or more, and including 100 ms or more. For example, the continuous-wave light source may be configured to illuminate at a specific light intensity for 50 ms.

[0098] In some embodiments, the light source is configured to maintain a substantially constant light intensity over the duration of each predetermined time interval, such as where the intensity of illumination varies by 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, such as 0.00001% or less, and includes where the intensity of illumination by the light source varies by 0.000001% or less over the duration of the predetermined time interval.

[0099] In some cases, the light source is configured to increase its intensity over a time period including at least a first predetermined time interval and a second predetermined time interval. In some cases, the intensity of the light from the light source increases linearly over a time period including at least a first predetermined time interval and a second predetermined time interval. In other cases, the intensity of the light from the light source increases exponentially over a time period including at least a first predetermined time interval and a second predetermined time interval.

[0100] The photodetector of the subject system can be any convenient light detection protocol, including but not limited to light sensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhancement-coupled devices (ICCDs), light-emitting diodes, photon counters, radiation thermometers, thermoelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or avalanche photodiodes, silicon photomultiplier tubes and combinations thereof, and other photodetectors. In some embodiments, the photodetector is a photomultiplier tube, such as one with an active detection surface area of ​​0.01 cm² per region. 2 -10cm 2 Such as 0.05cm 2 -9cm2 Such as 0.1cm 2 -8cm 2 Such as 0.5cm 2 -7cm 2 And including 1cm 2 -5cm 2 The photomultiplier tube. In other embodiments, the photodetector is an avalanche photodiode, such as an avalanche photodiode, with an effective detection surface area of ​​0.01 cm² per region. 2 Up to 10cm 2 Within a range, such as within 0.05cm 2 Up to 9cm 2 Within a range, such as within 0.1cm 2 Up to 8cm 2 Within a range, such as within 0.5cm 2 Up to 7cm 2 Within the range, and including 1cm 2 up to 5cm 2 Within the range. In some cases, light is detected by a photodetector array, such as a photodetector array having 2 or more photodetectors, such as 3 or more photodetectors, such as 5 or more photodetectors, such as 10 or more photodetectors, such as 25 or more photodetectors, such as 50 or more photodetectors, such as 75 or more photodetectors, such as 100 or more photodetectors, such as 500 or more photodetectors, and including a photodetector array having 1000 or more photodetectors. In some cases, light is detected by an array of avalanche photodiodes, such as a photodetector array having two or more avalanche photodiodes, such as three or more avalanche photodiodes, such as five or more avalanche photodiodes, such as ten or more avalanche photodiodes, such as 25 or more avalanche photodiodes, such as 50 or more avalanche photodiodes, such as 75 or more avalanche photodiodes, such as 100 or more avalanche photodiodes, such as 500 or more avalanche photodiodes, and including a photodetector array having 1000 or more avalanche photodiodes.

[0101] In embodiments of this disclosure, the photodetector may be configured to detect light at one or more wavelengths, such as at two or more wavelengths, such as at five or more different wavelengths, such as at ten or more different wavelengths, such as at 25 or more different wavelengths, such as at 50 or more different wavelengths, such as at 100 or more different wavelengths, such as at 200 or more different wavelengths, such as at 300 or more different wavelengths, and includes measuring light from particles in a flowing stream at 400 or more different wavelengths.

[0102] In embodiments, the photodetector can be configured to measure light continuously or at discrete intervals. In some cases, the preferred detector is configured to measure light continuously. In other cases, the preferred detector is configured to measure light at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0103] During each discrete time interval, one or more measurements of light from the light source can be performed, such as two or more, three or more, five or more, and including ten or more. In some embodiments, the light from the light source is measured two or more times by a photodetector, and in some cases the data are averaged.

[0104] In some embodiments, the system further includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to: integrate the data signal from the photodetector over a time period including each of the discrete illumination intervals, and determine one or more parameters of the photodetector based on the integrated data signal. In some embodiments, the memory includes instructions that, when executed by the processor, cause the processor to calculate the median signal amplitude. In other cases, the memory includes instructions that, when executed by the processor, cause the processor to calculate the average signal amplitude. In some cases, the memory includes instructions that, when executed by the processor, cause the processor to also calculate the standard deviation of the signal amplitude. In other cases, the memory includes instructions that, when executed by the processor, cause the processor to also calculate the variance and coefficient of variation (e.g., CV = standard deviation / mean) of the signal amplitude. In some embodiments, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to compare the calculated signal amplitude with the light intensity of the light source.

[0105] In some embodiments, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to compare a calculated signal amplitude with the light intensity of a light source. Based on one or more of the calculated signal amplitude and the comparison between the calculated signal amplitude and the light intensity of the light source, a preferred system includes a memory having instructions for calculating parameters of a photodetector. For example, the memory may include instructions for determining parameters for the photodetector, such as a minimum detection threshold, a maximum detection threshold, a detector sensitivity (i.e., the ratio of detector output to detector input), a detector dynamic range (the range of the detector signal from the minimum to the maximum detection threshold), a detector signal-to-noise ratio, or the number of photoelectrons per unit output. In some embodiments, the memory includes instructions for determining photodetector parameters within a range of the photodetector's operating voltage. In some embodiments, the memory includes instructions for calculating the photodetector's signal amplitude at 10% or more of the photodetector's operating voltage, such as 15% or more, such as 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and determining parameters at 99% or more of the photodetector's operating voltage. In some cases, the memory includes instructions for calculating the signal amplitude of the photodetector over the entire operating voltage range of the photodetector.

[0106] In some embodiments, the system includes a processor having a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following processes: determine one or more parameters of a photodetector, wherein the memory includes instructions to irradiate particles in a flowing stream, wherein the particles (e.g., multispectral beads as described below) comprise one or more fluorophores. In some cases, the memory includes instructions that, when executed by the processor, cause the processor to perform the following processes: determine parameters of the photodetector, including relative fluorescence units (e.g., ABD units) specified for each photodetector, robust coefficient of variation (rCV) of one or more photodetectors, maximum and minimum linearity of each photodetector, relative change of rCV relative to a baseline, relative change of detector gain relative to a baseline, and imaging specifications of the photodetector, such as RF power or axial optical loss.

[0107] In some cases, the memory includes instructions for determining parameters of a photodetector, the instructions including irradiating a flow of particles comprising one or more fluorophores at a first intensity for a first predetermined time interval and irradiating the flow of particles comprising one or more fluorophores at a second intensity for a second predetermined time interval, the photodetector having a light source detecting light from the flow, generating a data signal from the photodetector at the first irradiation intensity, generating a data signal from the photodetector at the second irradiation intensity, and determining one or more parameters of the photodetector based on the data signals generated at the first and second intensities.

[0108] In some embodiments, the memory includes instructions for determining the average fluorescence intensity (M) from the particles under a first irradiation intensity and a second irradiation intensity. In some cases, the memory includes instructions for determining the change in average fluorescence intensity (V(M)) under the first irradiation intensity and the second irradiation intensity. In some cases, the memory includes instructions for determining the %rCV (robust coefficient of variation) of the photodetector. In some embodiments, the memory includes instructions that, when executed by the processor, cause the process to calculate a linear fit to the variance according to:

[0109]

[0110] Q led Given by 1 / c1, and is the statistical photoelectrons per unit average fluorescence intensity (M) (i.e., SPE / MFI). In some embodiments, the memory includes instructions for plotting the variance to determine a linear fit of the variance based on the following:

[0111] y = c1x + c0

[0112] In embodiments, the average fluorescence intensity and variance can be determined for multiple different irradiation intensities, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, and including 15 or more different irradiation intensities.

[0113] In some embodiments, the memory includes instructions for determining statistical photoelectrons (SPEs) at one or more illumination intensities, such as at least a first illumination intensity and a second illumination intensity. In other cases, the memory includes instructions for calculating the detector efficiency (Q) of the photodetector for each particle based on the statistical photoelectrons and the determined average fluorescence intensity of the particle. detIn some embodiments, the memory includes instructions for determining the detector efficiency of one or more detector channels of a photodetector, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, sixteen or more, twenty or more, twenty-four or more, thirty-six or more, forty-eight or more, seventy-two or more, and seventy-two or more, and instructions for determining the detector efficiency of 96 or more detector channels of a photodetector based on statistical photoelectrons and the determined average fluorescence intensity of the particles. In some cases, the memory includes instructions for determining the detector efficiency of all detector channels of a photodetector for each particle based on statistical photoelectrons and the determined average fluorescence intensity of each particle. In some embodiments, the memory includes instructions that, when executed by the processor, cause the processor to perform the following processing: determining the detector efficiency, which is determined according to:

[0114]

[0115] SPE stands for Statistical Photoelectrons, MFI for Average Fluorescence Intensity, and ABD for the specified unit per channel per batch of particles.

[0116] In some embodiments, the memory includes instructions for determining a background signal for one or more photodetectors. In some cases, the background signal is determined at one or more illumination intensities, such as 2 or greater, such as 3 or greater, such as 4 or greater, such as 5 or greater, such as 6 or greater, such as 7 or greater, such as 8 or greater, such as 9 or greater, such as 10 or greater, and includes different illumination intensities of 10 or greater. In some cases, the memory includes instructions for determining the background signal at all applied illumination intensities. In some embodiments, the memory includes instructions for determining background signals in one or more photodetectors, such as in two or more, such as in three or more, such as in four or more, such as in five or more, such as in six or more, such as in seven or more, such as in eight or more, such as in nine or more, such as in ten or more, such as in twelve or more, such as in sixteen or more, such as in twenty or more, such as in twenty-four or more, such as in thirty-six or more, such as in forty-eight or more, such as in seventy-two detector channels, and includes instructions for determining background signals in ninety-six or more detector channels of a photodetector. In some cases, the memory includes instructions for determining background signals in all detector channels of a photodetector. In some cases, the memory includes instructions that, when executed by a processor, cause the processor to perform the following processing: determining the background signal based on statistical photoelectrons and the detector efficiency of the photodetector. In some cases, the memory contains instructions for determining the background signal according to:

[0117] B SD =B SD,MFI ×Q led

[0118]

[0119] In some embodiments, the memory includes instructions for determining the electronic noise of one or more photodetectors. In some cases, the electronic noise of the photodetector is determined at one or more illumination intensities, such as 2 or greater, such as 3 or greater, such as 4 or greater, such as 5 or greater, such as 6 or greater, such as 7 or greater, such as 8 or greater, such as 9 or greater, such as 10 or greater, and includes 10 or more different illumination intensities. In some cases, the electronic noise of the photodetector is determined at all applied illumination intensities. Electronic noise can also be determined in one or more detector channels of a photodetector, such as in two or more, such as in three or more, such as in four or more, such as in five or more, such as in six or more, such as in seven or more, such as in eight or more, such as in nine or more, such as in ten or more, such as in twelve or more, such as in sixteen or more, such as in twenty or more, such as in twenty-four or more, such as in thirty-six or more, such as in forty-eight or more, such as in seventy-two or more, and includes determining electronic noise in ninety-six or more detector channels. In some cases, the memory includes instructions for determining electronic noise in all detector channels of the photodetector. In some cases, the memory includes instructions for determining electronic noise based on statistical photoelectrons and the detector efficiency of the photodetector. In some cases, the memory includes instructions for determining electronic noise according to the following:

[0120] EN SD =EN SD,MFI ×Q led

[0121]

[0122] In some embodiments, the memory includes instructions for determining the detection limits of one or more photodetectors. In some cases, the memory includes instructions for determining the detection limits of the photodetectors in one or more detector channels of the photodetector, such as in two or more, such as in three or more, such as in four or more, such as in five or more, such as in six or more, such as in seven or more, such as in eight or more, such as in nine or more, such as in ten or more, such as in twelve or more, such as in sixteen or more, such as in twenty or more, such as in twenty-four or more, such as in thirty-six or more, such as in forty-eight or more, such as in seventy-two or more, and includes instructions for determining the detection limits of the photodetectors in 96 or more detector channels of the photodetector. In some cases, the memory includes instructions for determining the detection limits in all detector channels of the photodetector. In some examples, the memory includes instructions for determining the detection limit of each photodetector according to the following:

[0123] 2 + 2SD = 4(1 + B) SD )

[0124] In some embodiments, the memory includes instructions for determining the detector photosensitivity of one or more photodetectors. In some embodiments, the memory includes instructions for setting an initial detector gain for the photodetectors. In some cases, the memory includes instructions for illuminating the photodetectors with a light source (as described in detail above) at multiple different light intensities, instructions for generating data signals from the photodetectors for multiple light intensities at one or more detector gains, and instructions for determining the minimum light intensity required to generate a data signal resolvable from a background data signal at each detector gain. In some cases, the memory includes instructions for determining the minimum light intensity required to generate a data signal that differs from the background data signal by two standard deviations at each detector gain. In some cases, the memory includes instructions for setting the detector gain to a gain that reaches a plateau when plotted as a function of light intensity, resulting in a data signal resolvable from a background data signal.

[0125] In some embodiments, the photodetector is a photodetector located in a particle analyzer, such as a particle sorter. In some embodiments, the subject system is a flow cytometry system that includes a photodetector as part of a light detection system for detecting light emitted by a sample in a flowing stream. Suitable flow cytometry systems may include, but are not limited to, those described in Ormerod (ed.): Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt 1): 17-28; Linden, et al., Semin 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 CarrierSyst. 24(3):203-255; its publication is incorporated herein by reference. In some cases, preferred flow cytometry systems include BD Biosciences FACSCanto. TM II flow cytometer, BD Accuri TM Flow cytometer, BDBiosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortess TM X-20 flow cytometer and BD Biosciences FACSCalibur TMCell sorter, aBD Biosciences FACSCount TM Cell sorting instrument, BD Biosciences FACSLyric TM Cell sorter and BD Biosciences Via TM Cell sorting instruments include BD Biosciences Influx™ Cell Sorter, BD Biosciences Jazz™ Cell Sorter, BD Biosciences Aria™ Cell Sorter, and BD Biosciences FACSMelody™ Cell Sorter.

[0126] In some embodiments, the subject particle sorting system is a flow cytometry system, such as those described in U.S. Patent Nos. 10,006,852; 9,952,076; 9,933,341; 9,784,661; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,23 Those described in 3,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; the disclosures thereof are incorporated herein by reference in their entirety.

[0127] In some embodiments, the subject system is a flow cytometry system with an excitation module that uses radio frequency multiplex excitation to generate multiple frequency-shifted beams. In other cases, the subject system is a flow cytometry system as described in U.S. Patent Nos. 9,423,353 and 9,784,661 and U.S. Patent Publications Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0128] In some embodiments, the subject system is a particle sorting system configured to sort particles using enclosed particle sorting modules, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, a sorting decision unit having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as those described in U.S. Provisional Patent Application No. 62 / 803,264, filed February 8, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a method for sorting components of a sample includes sorting particles (e.g., cells in a biological sample) using a particle sorting module having deflection plates, such as those described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0129] In some embodiments, a preferred system includes a particle analysis system that can be used to analyze and characterize particles, wherein particles may or may not be physically classified into a collection container. Figure 4A A functional block diagram of an example particle analysis system is shown. In some embodiments, particle analysis system 401 is a flow system. Figure 4A The particle analysis system 401 shown is configured to perform the methods described herein, either wholly or partially. The particle analysis system 401 includes a fluid dynamics system 402. The fluid dynamics system 402 may include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, wherein particles 403 (e.g., cells) of the sample move along a common sampling path 409.

[0130] The particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as it passes through one or more detection stations along a common sampling path. Detection station 408 typically refers to a monitoring area 407 along the common sampling path. In some implementations, detection may include detecting the light or one or more other characteristics of particles 403 as they cross the monitoring area 407. Figure 4A The image shows a detection station 408 with a monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Furthermore, some detection stations may monitor more than one area.

[0131] Each signal is assigned a signal value to form a data point for each particle. As mentioned above, this data can be referred to as event data. The data point can be a multidimensional data point that includes values ​​of the corresponding properties measured for the particle. The detection system 404 is configured to collect a series of such data points in a first time interval.

[0132] The particle analysis system 401 may also include a control system 306. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system is operatively associated with the fluid dynamics system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on a Poisson distribution and the number of data points collected by the detection system 404 during the first time interval. The control system 406 may also be configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. The control system 406 may also compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0133] Figure 4B A system 400 for flow cytometry according to an illustrative embodiment of the present invention is shown. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scattering detector 430, a side scattering detector 435, a fluorescence collecting lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long-pass (“LP”) filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0134] The 115a-c laser is excited to emit light in the form of a laser beam. Figure 4B In the exemplary system, the laser beams emitted from excitation lasers 415a-415c have wavelengths of 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first guided through one or more beam splitters 445a and 445b. Beam splitter 445a transmits 488 nm light and reflects 633 nm light. Beam splitter 445b transmits UV light (light with wavelengths in the range of 10 to 400 nm) and reflects both 488 nm and 633 nm light.

[0135] The laser beam is then directed to a focusing lens 420, which focuses the beam onto the portion of the fluid flow within a flow chamber 425 where the sample particles are located. The flow chamber is part of a fluid system that typically directs particles in the flow one at a time to the focused laser beam for probing. The flow chamber may include a flow cell in a benchtop cytometer or a nozzle head in a gas flow cytometer.

[0136] Light from the laser beam interacts with particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various wavelengths depending on the characteristics of the particles, such as their size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present in the particles. The fluorescence emission, along with the diffracted, refracted, reflected, and scattered light, can be routed through one or more of beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collecting lens 440 to one or more of forward scattering detector 430, side scattering detector 435, and one or more fluorescence detectors 460a-460f.

[0137] A fluorescence collecting lens 440 collects light emitted from the particle-laser beam interaction and directs the light to one or more beam splitters and filters. Bandpass filters (such as bandpass filters 450a-450e) allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number indicates the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short-pass filters transmit light wavelengths equal to or shorter than a specified wavelength. Long-pass filters, such as long-pass filters 455a-455b, transmit light wavelengths equal to or longer than a specified wavelength. For example, long-pass filter 455a, as a 670 nm long-pass filter, transmits light equal to or longer than 670 nm. Filters are typically selected to optimize the detector's specificity for a particular fluorescent dye. Filters can be configured such that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0138] A beam splitter directs light of different wavelengths in different directions. A beam splitter can be characterized by filter characteristics such as short-pass and long-pass. For example, beam splitter 445g is a 620Sp beam splitter, meaning that beam splitter 445g transmits light wavelengths of 620 nm or less and reflects light wavelengths longer than 620 nm in different directions. In one embodiment, beam splitters 445a-445g may include optical mirrors, such as dichroic mirrors.

[0139] A forward scattering detector 430 is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light, i.e., excitation light that travels primarily in the forward direction through or around the particle. The intensity of the light detected by the forward scattering detector depends on the overall size of the particle. The forward scattering detector may include a photodiode. A side scattering detector 435 is configured to detect refracted and reflected light from the surface and internal structures of the particle, and tends to increase with increasing particle structural complexity. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 460a-460f. The side scattering detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected at the forward scattering detector 430, the side scattering detector 435, and the fluorescence detector can be converted into electrical signals (voltages) by the detectors. This data provides information about the sample.

[0140] Those skilled in the art will recognize that the flow cytometer according to embodiments of the present invention is not limited to Figure 4B The flow cytometer described herein may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors with various wavelengths and various different configurations.

[0141] In operation, the blood cell counter is controlled by a controller / processor 490, and measurement data from the detector can be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive output signals from it, and may also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control the laser, fluid flow parameters, etc. Input / output (I / O) capabilities 497 may also be provided in the system. Memory 495, controller / processor 490, and I / O 497 may be provided entirely as part of the flow cytometer 410. In such embodiments, a display may also form part of the I / O capability 497 for presenting experimental data to a user of the blood cell counter 400. Alternatively, some or all of the memory 495, controller / processor 490, and I / O capabilities may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 495 and controller / processor 490 may communicate wirelessly or wired with the blood cell counter 410. The controller / processor 490, together with the memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0142] Figure 4BThe system shown comprises six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as “filter windows” for a given detector), as defined by the configuration of filters and / or splitters in the beam path from flow cell 425 to each detector. Different fluorescent molecules used in flow cytometry experiments will emit light in their own characteristic wavelength bands. Specific fluorescent labels used in the experiment and their associated fluorescence emission bands can be selected to generally coincide with the filter windows of the detectors. However, due to the availability of more detectors and the use of more labels, a perfect correspondence between filter windows and fluorescence emission spectra is not possible. Typically, although the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of a particular detector, some emission spectra of that label will also overlap with the filter windows of one or more other detectors. This can be referred to as overflow. I / O 497 can be configured to receive data on a flow cytometry experiment having a fluorescent label panel and multiple cell populations with multiple labels, each cell population having a subset of multiple labels. I / O 497 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data assigning markers to one or more markers, and cytometer configuration data. Flow cytometry experimental data, such as marker spectral characteristics and flow cytometry configuration data, can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more label-to-marker assignments.

[0143] Figure 5 A functional block diagram of an example particle analyzer control system, such as an analysis controller 500, is shown for analyzing and displaying biological events. The analysis controller 500 can be configured to implement various processes for controlling graphical displays of biological events.

[0144] The particle analyzer or classification system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or classification system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0145] Analysis controller 500 may be configured to receive biological event data from particle analyzer or classification system 502. The biological event data received from particle analyzer or classification system 502 may include flow cytometry event data. Analysis controller 500 may be configured to provide a graphical display of a first graph including the biological event data to display device 506. Analysis controller 500 may also be configured to present preferred regions as, for example, gates overlaid on the first graph around populations of biological event data displayed by display device 506. In some embodiments, the gates may be a logical combination of one or more preferred graphical regions plotted on a single-parameter histogram or bivariate graph. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0146] The analysis controller 500 can also be configured to display bio-event data that differs from other events in the bio-event data outside the gating on the display device 506 within the gating. For example, the analysis controller 500 can be configured to make the color of the bio-event data included within the gating different from the color of the bio-event data outside the gating. The display device 506 can be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0147] The analysis controller 500 can be configured to receive a gating selection signal identifying a gating from a first input device. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a gating selection signal to the analysis controller 500 that will be displayed on or manipulated via a display device 506 (e.g., by clicking the desired gating when the cursor is positioned on it or by clicking within the desired gating). In some embodiments, the first device can be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, stylus, optical detector, or voice recognition system. Some input devices may include multiple input functions. In such implementations, each input function can be considered an input device. For example, such as... Figure 5 As shown, mouse 510 may include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0148] Triggering events can cause the analysis controller 500 to change how the data is displayed, which parts of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selecting preferred groups for particle sorting.

[0149] In some embodiments, the analysis controller 500 can be configured to detect when a gating selection is initiated by the mouse 510. The analysis controller 500 can also be configured to automatically modify the plotting visualization to facilitate the gating process. This modification can be based on a specific distribution of the biological event data received by the analysis controller 500.

[0150] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can also be configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0151] Display device 506 can be configured to receive display data from analysis controller 500. The display data may include gating of portions of graphs and delineations of biological event data. Display device 506 can also be configured to change the presented information based on input received from analysis controller 500 and input from particle analyzer 502, storage device 504, keyboard 508 and / or mouse 510.

[0152] In some implementations, the analysis controller 500 may generate a user interface to receive exemplary events for classification. For example, the user interface may include a controller for receiving exemplary events or exemplary images. Exemplary events or images, or exemplary strobes, may be provided before collecting event data for a sample, or based on an initial set of events for a portion of the sample.

[0153] In some embodiments, a preferred system includes a particle sorting system. Figure 6A This is a schematic diagram of a particle classifier system 600 (e.g., a particle analyzer or classification system 502) according to one embodiment presented herein. In some embodiments, the particle classifier system 600 is a cell classifier system. Figure 6A As shown, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, include, or may be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically concentrates a sample fluid 606, including particles 609, into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, particles 609 (e.g., cells) align in a single file to pass through a monitoring area 611 (e.g., where the laser streams intersect) irradiated by an irradiation source 612 (e.g., a laser). Vibration of the droplet-forming transducer 602 causes the moving fluid column 608 to break into more droplets 610, some of which contain particles 609.

[0154] In operation, a detection station 614 (e.g., an event detector) identifies when a preferred particle (or preferred cell) crosses a monitoring area 611. The detection station 614 is fed to a timing circuit 628, which in turn feeds to a flash charging circuit 630. At a droplet interruption point notified by a timing droplet delay (Δt), a flash charge can be applied to a moving fluid column 608, causing the preferred droplet to carry a charge. The preferred droplet may comprise one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect the droplet into a container such as a collection tube or a porous or microporous sample plate, where a pore or micropore may be associated with a particularly preferred droplet. Figure 6A As shown, the droplets can be collected in the discharge container 638.

[0155] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a preferred particle passes through the monitor 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 allows the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may be fed an amplitude signal 620 and / or a phase signal 618, which in turn (via amplifier 622) feeds into an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624, in turn, control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included in a control system.

[0156] In some implementations, the classification electronics (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory configured to store detected events and classification decisions based thereon. The sorting decisions may be included in the event data of the particles. In some embodiments, detection system 616 and detection station 614 may be implemented as a single detection unit or communicatively coupled, such that event measurements can be collected by one of detection system 616 or detection station 614 and provided to a non-collecting element.

[0157] Figure 6B This is a schematic diagram of a particle sorter system based on an embodiment presented herein. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge can be applied via a current-charged wire in the barbs. This generates a stream of microdroplets 610 containing particles 610 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to produce light scattering and fluorescence information. This can be achieved through sorting electronics or other detection systems (such as sorting electronics or other detection systems). Figure 6B(Not shown in the image) to analyze particle information. Deflection plates 652 and 654 can be independently controlled to attract or repel charged droplets, thereby guiding the droplets toward a destination collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflector plates 652 and 654 can be controlled to guide particles along a first path 662 to container 674 or along a second path 668 to container 678. If the particles are not preferred (e.g., do not present scattering or illumination information within a specified classification range), the deflector plates can allow the particles to continue along flow path 664. Such uncharged droplets can enter the waste container, for example, via aspirator 670.

[0158] It may include sorting electronics to begin collecting measurement results, receive the fluorescence signal of the particles, and determine how to adjust the deflection plate to induce particle sorting. Figure 6B An exemplary implementation of the illustrated embodiment includes the BDFACSAria™ linear flow cytometer, commercially available from Becton, Dickinson, and Franklin Lakes (NJ).

[0159] Computer-controlled systems

[0160] Various embodiments of this disclosure also include computer-controlled systems, wherein the systems further include one or more computers for full or partial automation. In some embodiments, the system includes a computer having a computer-readable storage medium on which a computer program is stored, wherein when the computer program is loaded onto the computer, the computer program includes instructions for: illuminating a photodetector with a first intensity at a first predetermined time interval using a light source; illuminating the photodetector with a second intensity at a second predetermined time interval using the light source; integrating a data signal from the photodetector over a time period including the first and second predetermined time intervals; and determining one or more parameters of the photodetector based on the integrated data signal. In some embodiments, the system includes a computer having a computer-readable storage medium having a computer program stored thereon, wherein the computer program, when loaded onto the computer, includes instructions for increasing the intensity of light from the light source over a time period including at least the first and second predetermined time intervals. In some cases, the computer program includes instructions for linearly increasing the intensity of light from the light source. In some cases, the computer program includes instructions for exponentially increasing the intensity of light from the light source.

[0161] In some embodiments, the computer program includes instructions to calculate a signal amplitude from an integrated data signal and compare the calculated signal amplitude with the illumination intensity of a light source. In some embodiments, the computer program includes instructions for determining one or more of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and the number of photoelectrons per unit output of the illuminated photodetector. In some cases, the computer program includes instructions for determining parameters of the photodetector within the operating voltage range of the photodetector, such as across the entire operating voltage range of the photodetector.

[0162] In an embodiment, the system includes an input module, a processing module, and an output module. The subject system may include hardware and software components, wherein the hardware components may take the form of one or more platforms, for example, as a server, such that functional elements, i.e., those elements in the system that perform specific tasks of the system (such as managing the input and output of information, processing information, etc.), can be executed by running software applications on one or more computer platforms representing the system and across those one or more computer platforms.

[0163] The system may include a display and operator input devices. Operator input devices may be, for example, a keyboard, mouse, etc. The processing module includes a processor that can access memory containing instructions stored thereon for executing steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor, or it may be one of other processors that are currently available or will become available. The processor executes the operating system in a known manner, and the operating system interfaces with firmware and hardware, facilitating the processor to coordinate and execute 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, all according to known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow a user to manually align the light source with the flow 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.

[0164] System memory can be any of a variety of known or future memory storage devices. Examples include any generally available random access memory (RAM), magnetic media such as resident hard disks or magnetic tapes, optical media such as optical discs for reading and writing, flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, removable hard disk drives, or floppy disk drives. Such types of memory storage devices typically read from and / or write to program storage media (not shown) such as optical discs, magnetic tapes, removable hard disks, or floppy disks. Any of these program storage media, or other media currently in use or that may be developed in the future, can be considered a computer program product. It is understood that these program storage media typically store computer software programs and / or data. Computer software programs (also known as computer control logic) are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0165] In some embodiments, a computer program product is described, comprising a computer-usable medium storing control logic (computer software program, including program code). When executed by a computer's processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, using, for example, a hardware state machine. Implementing a hardware state machine to perform the functions described herein will be apparent to those skilled in the art.

[0166] The memory can be any suitable device in which a processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including disks or optical discs or magnetic tapes or RAM, or any other suitable fixed or portable device). The processor can include a general-purpose digital microprocessor suitable for programming from a computer-readable medium carrying the necessary program code. Programming can be provided to the processor remotely via a communication channel, or it can be pre-stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium, using any of those memory-associated devices. For example, a disk or optical disc can carry programs and can 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 implementing the methods described above. Programming according to the invention can be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; portable flash drives; and mixtures of these kinds, such as magnetic / optical storage media.

[0167] The processor can also access communication channels to communicate with users at remote locations. Remote locations mean that users do not directly contact the system and relay input information from external devices to the input manager. External devices include computers connected to wide area networks (“WANs”), telephone networks, satellite networks, or any other suitable communication channels, including mobile phones (i.e., smartphones).

[0168] In some embodiments, the system according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., RFID, Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth communication protocol and cellular communication, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0169] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces, such as USB ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the subject system and other external devices, such as computer terminals configured for similar supplementary data communication (e.g., in a physician's office or in a hospital environment).

[0170] In one embodiment, the communication interface is configured for infrared communication, Bluetooth communication, or any other suitable wireless communication protocol to enable the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user can use in conjunction with them.

[0171] In one embodiment, the communication interface is configured to provide connectivity for data transmission via Internet Protocol (IP) over a cellular telephone network, Short Message Service (SMS), wireless connectivity to a personal computer (PC) on a local area network (LAN) connected to the Internet, or WiFi connectivity to the Internet at a WiFi hotspot.

[0172] In one embodiment, the topic system is configured, for example, to use 802.11 or The common standards of RF protocol or IrDA infrared protocol wirelessly communicate with the server device via a communication interface. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop computer; or a larger device such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, keyboard, mouse, or touch screen.

[0173] In some embodiments, the communication interface is configured to use one or more of the above-described communication protocols and / or mechanisms to automatically or semi-automatically transmit data stored in the subject system to a network or server device, for example, in an optional data storage unit.

[0174] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, that information may typically be logically and / or physically organized as an array of image elements. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. The functional elements of the computer may communicate with each other via a system bus. In alternative embodiments, some of these communications may be implemented using a network or other types of remote communication. According to known technologies, the output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network. The presentation of data by the output manager may be implemented according to a variety of known technologies. As some examples, the data may include SQL, HTML, or XML documents, emails, or other files, or other forms of data. The data may include an Internet URL address that allows the user to retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the subject system may be any type of known computer platform or a type to be developed in the future, although they are typically a class of computers commonly referred to as servers. However, they can also be host computers, workstations, or other computer types. They can be connected via any known or future type of cable or other communication system, including wireless systems (networking or otherwise). They can be located in the same place or they can be physically separated. Depending on the type and / or architecture of the computer platform chosen, a variety of operating systems can be used on any computer platform. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, etc.

[0175] Figure 7 A general architecture of an example computing device 700 according to certain embodiments is depicted. Figure 7 The general architecture of the computing device 700 depicted includes the arrangement of computer hardware and software components. The computing device 700 may include... Figure 7More (or fewer) components are shown. However, it is not necessary to show all of these generally conventional components in order to provide a disclosure of what can be achieved. As shown, computing device 700 includes processing unit 710, network interface 720, computer-readable media drive 730, input / output device interface 740, display 750, and input device 760, all of which can communicate with each other via a communication bus. Network interface 720 can provide connectivity to one or more networks or computing systems. Processing unit 710 can therefore receive information and instructions from other computing systems or services via the network. Processing unit 710 can also communicate with memory 770 and also provide output information for optional display 750 via input / output device interface 740. Input / output device interface 740 can also accept input from optional input device 760 such as keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input devices.

[0176] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments) that the processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other permanent, auxiliary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of the computing device 700. Memory 770 may also include computer program instructions and other information for implementing the embodiments of this disclosure.

[0177] Computer-readable storage media

[0178] The scheme of this disclosure also includes a non-transitory computer-readable storage medium having instructions for practicing the subject methods. The computer-readable storage medium can be used on one or more computers to achieve full or partial automation of the system used for practicing the methods described herein. In some embodiments, instructions according to the methods described herein can be encoded onto a computer-readable medium in a “programmed” form, wherein the term “computer-readable medium” as used herein refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network attached storage (NAS), whether such devices are internal or external to a computer. Files containing information can be “stored” on a computer-readable medium, where “stored” means recording the information so that it can be accessed and retrieved by a computer at a later date. The computer-implemented methods described herein can be executed using a program that can be written in one or more of any number of computer programming languages. Such languages ​​include, for example, Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as any other language.

[0179] In some embodiments, a preferred computer-readable storage medium includes a computer program stored thereon, wherein when the computer program is loaded onto a computer, the computer program includes instructions having: an algorithm for illuminating a photodetector with a continuous wave light source of a first intensity for a first predetermined time interval; and an algorithm for illuminating the photodetector with a light source of a second intensity for a second predetermined time interval.

[0180] An algorithm for integrating data signals from a photodetector over a time period including a first predetermined time interval and a second predetermined time interval; and an algorithm for determining one or more parameters of the photodetector based on the integrated data signals.

[0181] In some cases, non-transitory computer-readable storage media include algorithms for illuminating a photodetector with multiple light intensities over multiple time intervals. In these cases, the non-transitory computer-readable storage medium includes algorithms for integrating data signals from the photodetector over a time period including multiple illumination time intervals.

[0182] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the signal amplitude. In some examples, the non-transitory computer-readable storage medium includes an algorithm for calculating the median signal amplitude. In some cases, the non-transitory computer-readable storage medium includes an algorithm for comparing the calculated signal amplitude with the light intensity of a light source. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining parameters of a photodetector based on one or more of the calculated signal amplitude and a comparison between the calculated signal amplitude and the light intensity of the light source. For example, the non-transitory computer-readable storage medium may include algorithms for determining a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, or the number of photoelectrons per unit output. The non-transitory computer-readable storage medium may include algorithms for determining detector parameters over the operating voltage range of the photodetector, such as wherein the parameters of the photodetector are determined over the entire operating voltage range of the photodetector.

[0183] Computer-readable storage media can be used on one or more computer systems having a display and operator input devices. Operator input devices may, for example, be a keyboard, mouse, etc. The processing module includes a processor that can access memory having instructions stored thereon for executing steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor, or it may be one of other processors that are currently available or will become available. The processor executes the operating system in a known manner, and the operating system interfaces with firmware and hardware, facilitating the processor to coordinate and execute 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 known in the art. The operating system typically cooperates 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, all according to known techniques.

[0184] Multispectral fluorescent particles

[0185] As outlined above, the schemes of this disclosure also include particles (e.g., beads) having one or more fluorophores for implementing certain methods described herein. Preferred particles according to certain embodiments may include single-peak multifluorophore beads that provide a bright photodetector signal at all light source wavelengths (e.g., at all LEDs or lasers in the system) and at the detection wavelength of the photodetector.

[0186] In an embodiment, subject particles (e.g., in a fluid composition) are formulated to flow in a flow stream irradiated by a light source as described above. Each particle may have one or more different types of fluorophores, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more, or 25 or more, or 30 or more, or 35 or more, or 40 or more, or 45 or more, or 50 or more different types of fluorophores. For example, each particle may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different types of fluorophores.

[0187] In some embodiments, each fluorophore is stably associated with a particle. Stable association means that the fluorophore does not readily dissociate from the particle to come into contact with a liquid medium, such as an aqueous medium. In some embodiments, one or more fluorophores are covalently attached to the particle. In other embodiments, one or more fluorophores are physically associated with the particle (i.e., non-covalently coupled). In other embodiments, one or more fluorophores are covalently attached to the particle, and one or more fluorophores are physically associated with the particle.

[0188] In some embodiments, each particle contains two or more different types of fluorophores. Any two fluorophores are considered different if they differ from each other in one or more aspects of molecular formula, excitation maximum, and emission maximum. Therefore, different or distinct fluorophores may differ from each other in terms of chemical composition or in one or more properties of the fluorophores. For example, different fluorophores may differ from each other in at least one aspect of excitation maximum and emission maximum. In some cases, different fluorophores differ from each other due to their excitation maximum. In some cases, different fluorophores have different emission maximums. In some cases, different fluorophores have different excitation maximums and emission maximums. Therefore, in embodiments including a first fluorophore and a second fluorophore, the first fluorophore and the second fluorophore may differ from each other in at least one of excitation maximum and emission maximum. For example, the first fluorophore and the second fluorophore may differ from each other by excitation maximum, by emission maximum, or by both excitation and emission maximum. If a given set of fluorophores differs from each other in terms of excitation or emission maxima, they can be considered to be different, where in some cases the size of such difference is 5 nm or more, such as 10 nm or more, including 15 nm or more, where in some cases the size of the difference ranges from 5 to 400 nm, such as 10 to 200 nm, including 15 to 100 nm, such as 25 to 50 nm.

[0189] Preferred fluorophores according to certain embodiments have an excitation maximum ranging from 100 nm to 800 nm, such as from 150 nm to 750 nm, such as from 200 nm to 700 nm, such as from 250 nm to 650 nm, such as from 300 nm to 600 nm, and including an excitation maximum ranging from 400 nm to 500 nm. Preferred fluorophores according to certain embodiments have an excitation maximum ranging from 400 nm to 1000 nm, such as from 450 nm to 950 nm, such as from 500 nm to 900 nm, such as from 550 nm to 850 nm, and including a maximum emission ranging from 600 nm to 800 nm. In some cases, the fluorophore is a luminescent dye, such as a fluorescent dye having a peak emission wavelength of 200 nm or more, such as 250 nm or more, such as 300 nm or more, such as 350 nm or more, such as 400 nm or more, such as 450 nm or more, such as 500 nm or more, such as 550 nm or more, such as 600 nm or more, such as 650 nm or more, such as 700 nm or more, such as 750 nm or more, such as 800 nm or more, such as 850 nm or more, such as 900 nm or more, such as 950 nm or more, such as 1000 nm or more, and including fluorescent dyes with a peak emission wavelength of 1050 nm or more. For example, the fluorophore can be a fluorescent dye having a peak emission wavelength in the range of 200 nm to 1200 nm, such as 300 nm to 1100 nm, such as 400 nm to 1000 nm, such as 500 nm to 900 nm, and including fluorescent dyes with a peak emission wavelength of 600 nm to 800 nm. In some embodiments, the subject multispectral particle provides stable excitation of a laser at or near wavelengths of 349 nm (UV laser), 488 nm (blue laser), 532 nm (Nd:YAG solid-state laser), 640 nm (red laser), and 405 nm (violet laser). In other cases, the subject multispectral particle provides stable excitation by a light source across the entire spectral detection band, such as from 350 nm to 850 nm.

[0190] Preferred fluorophores may include, but are not limited to, two or more dyes such as fluoroboron pyrrole dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazo dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, anthocyanine dyes, asymmetric anthocyanine dyes, quinone-imine dyes, azazine dyes, eurhodin dyes, saffron dyes, indoleamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazinone dyes, thiazine dyes, thiazolium dyes, xanthonium dyes, fluorene dyes, pyronin dyes, fluorine dyes, phenanthridine dyes, squarine cyanine dyes, fluoroboron complexes, squarine roxitanes, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethines, or tetrapyrroles and combinations thereof. In some embodiments, the conjugate may include two or more dyes, such as those selected from fluoroboron pyrrole dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazo dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azazine dyes, eurhodin dyes, saffron dyes, indoleamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazinone dyes, thiazine dyes, thiazolium dyes, xanthonium dyes, fluorene dyes, pyronin dyes, fluorine dyes, phenanthridine dyes, squarine cyanine dyes, fluoroboron complexes, squarine roxitanes, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethines, or tetrapyrroles and combinations thereof.

[0191] In some embodiments, preferred fluorophores may include, but are not limited to, fluorescein isothiocyanate (FITC), phycoerythrin (PE) dye, polydinophyte chlorophyll-cyanin dye (e.g., PerCP-Cy5.5), phycoerythrin-cyanin (PE-Cy) dye (PE-Cy7), allophycocyanin (APC) dye (e.g., APC-R700), allophycocyanin-cyanin dye (e.g., APC-Cy7), and coumarin dye (e.g., V450 or V500). In some cases, the fluorophore may include one or more of the following: 1,4-bis-(o-methylstyryl)-benzene (bis-MSB 1,4-bis[2-(2-methylphenyl)vinyl]-benzene), C510 dye, C6 dye, Nile Red dye, T614 dye (e.g., N-[7-(methanesulfonamide)-4-oxo-6-phenoxychromene-3-yl]formamide), LDS 821 dye ((2-(6-(p-dimethylaminophenyl)-2,4-neopentene-1,3,5-hextrienyl)-3-ethylbenzothiazole perchloric acid), and mFluor dye (e.g., mFluor Red dye, such as mFluor 780NS).

[0192] The particles can be any convenient shape for illumination by the light source as described above. In some cases, the particles are solid supports, and their shape or configuration is disc, sphere, oval, cube, block, cone, etc., as well as irregular shapes. The mass of the particles can vary in some cases from 0.01 mg to 20 mg, such as 0.05 mg to 19.5 mg, such as 0.1 mg to 19 mg, such as 0.5 mg to 18.5 mg, such as 1 mg to 18 mg, such as 1.5 mg to 17.5 mg, such as 2 mg to 15 mg, and including 3 mg to 10 mg. The particles may have, for example, a surface area of ​​0.01 mm² or greater, such as 0.05 mm² or greater, such as 0.1 mm² or greater, such as 0.5 mm² or greater, such as 1 mm² or greater, such as 1.5 mm² or greater, such as 2 mm² or greater, such as 2.5 mm² or greater, such as 3 mm² or greater, such as 3.5 mm² or greater, such as 4 mm² or greater, such as 4.5 mm² or greater, and including 5 mm² or greater.

[0193] The particle size can vary as needed, and in some cases, the longest particle size is 0.01 mm to 10 mm, such as 0.05 mm to 9.5 mm, such as 0.1 mm to 9 mm, such as 0.5 mm to 8.5 mm, such as 1 mm to 8 mm, such as 1.5 mm to 7.5 mm, such as 2 mm to 7 mm, such as 2.5 mm to 6.5 mm, and including 3 mm to 6 mm. In some cases, the particle size is in the range of 0.01 mm to 5 mm, such as from 0.05 mm to 4.5 mm, such as from 0.1 mm to 4 mm, such as from 0.5 mm to 3.5 mm, and including 1 mm to 3 mm.

[0194] In some cases, the preferred particles are porous, such as those having a porosity in the range of 5 μ to 100 μ, such as from 10 μ to 90 μ, such as from 15 μ to 85 μ, such as from 20 μ to 80 μ, such as from 25 μ to 75 μ, and including from 30 μ to 70 μ, such as 50 μ, as determined by a capillary flow porosometer or equivalent.

[0195] The particles can be formed from any readily available material. In some embodiments, particles with low or no autofluorescence, such as beads, are preferred. Suitable materials include, but are not limited to, glass materials (e.g., silicates), ceramic materials (e.g., calcium phosphate), metallic materials, and polymeric materials, such as polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, etc. In some cases, the particles are formed from a solid carrier, such as a porous matrix, as described in U.S. Publication No. 9,797,899, the disclosure of which is incorporated herein by reference. Thus, the surface region of the particles can be any suitable macroporous or microporous substrate, wherein suitable macroporous and microporous substrates include, but are not limited to, ceramic matrices, glass frits such as sintered glass, polymeric matrices, and metal-organic polymeric matrices. In some embodiments, the porous matrix is ​​a frit. The term "frit" is used herein in its conventional sense to refer to a porous composition formed from a sintered particulate solid such as glass. The frit may have a chemical composition that varies depending on the type of sintered particles used to prepare the frit, wherein the frits that can be used include, but are not limited to, frits composed of aluminosilicates, boron trioxide, borophosphosilicate glass, borosilicate glass, ceramic glaze, cobalt glass, blueberry glass, fluorophosphate glass, fluorosilicate glass, woolly quartz, germanium dioxide, metal and sulfide-embedded borosilicates, lead-containing glass, phosphate glass, phosphorus pentoxide glass, phosphosilicate glass, potassium silicate, soda-lime glass, sodium hexametaphosphate glass, sodium silicate, tellurite glass, uranium glass, glass stone, and combinations thereof. In some embodiments, the porous matrix is ​​the frit, such as borosilicate, aluminosilicate, fluorosilicate, potassium silicate, or borophosphosilicate frit.

[0196] In some embodiments, the particles are formed from a porous organic polymer. Preferred porous organic polymers vary depending on the sample volume, the components in the sample, and the analytical reagents present, and may include, but are not limited to, porous polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), ethyl vinyl acetate (EVA), polycarbonate alloys, polyurethane, polyethersulfone, copolymers thereof, and combinations thereof. For example, preferred porous polymers include homopolymers, heteropolymers, and copolymers composed of monomer units, such as styrene, monoalkyleneallyl monomers such as ethylstyrene, α-methylstyrene, vinyltoluene, and vinylethylbenzene; (meth)acrylates, such as methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, isodecyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, cyclohexyl (meth)acrylate, and benzyl (meth)acrylate; chlorinated monomers, such as vinyl chloride, vinylidene chloride, and chloromethylstyrene; acrylonitrile compounds, such as acrylonitrile and methacrylonitrile; and vinyl acetate, vinyl propionate, n-octadecylacrylamide, ethylene, propylene, and butane, and combinations thereof.

[0197] In some embodiments, the particles are formed from a metal-organic polymer matrix, for example, an organic polymer matrix having a main chain structure comprising a metal such as aluminum, barium, antimony, calcium, chromium, copper, erbium, germanium, iron, lead, lithium, phosphorus, potassium, silicon, tantalum, tin, titanium, vanadium, zinc, or zirconium. In some embodiments, the porous metal-organic matrix is ​​an organosiloxane polymer, including but not limited to polymers of methyltrimethoxysilane, dimethyldimethoxysilane, tetraethoxysilane, methacryloxypropyltrimethoxysilane, bis(triethoxysilyl)ethane, bis(triethoxysilyl)butane, bis(triethoxysilyl)pentane, bis(triethoxysilyl)hexane, bis(triethoxysilyl)heptane, bis(triethoxysilyl)octane, and combinations thereof.

[0198] Reagent test kit

[0199] Kits are also provided that include one or more components of a subject system. Kits according to certain embodiments include one or more continuous-wave light sources, such as narrowband light-emitting diodes and photodetectors (e.g., photomultiplier tubes), wherein one or more parameters of the photodetector need to be analyzed. Kits may also include optical adjustment components, such as lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimation protocols, and combinations thereof.

[0200] In addition to the components described above, the kit of the present invention may also include (in some embodiments) instructions for practicing the methods of the subject matter. These instructions may exist in a variety of forms in the kit of the present invention, one or more of which may be present in the subject kit. One form of these instructions may be as printed information on a suitable medium or substrate, such as a sheet or multiple sheets of paper containing printed information, in the packaging of the kit, in a packaging insert, etc. Another form of these instructions is as a computer-readable medium on which information is recorded, such as a disk, optical disc (CD), portable flash drive, etc. Yet another form of these instructions may be as a website address accessible via the Internet to access information on removed sites.

[0201] practicality

[0202] The methods, systems, and computer systems of this subject matter can be used in a variety of applications requiring calibration or optimization of photodetectors, such as in particle analyzers. The methods and systems of this subject matter are also used with photodetectors for analyzing and sorting particulate components in samples in fluid media, such as biological samples. This disclosure also reveals applications in flow cytometry, wherein it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection during cell sorting. In embodiments, this disclosure reduces the need for user input or manual adjustments during sample analysis in a flow cytometer. In some embodiments, the methods and systems of this subject matter provide a fully automated scheme, such that adjustments to the flow cytometer during use require minimal human input, if any.

[0203] The solutions (including embodiments) of the subject matter described herein can be used alone or in combination with one or more other solutions or embodiments, which is advantageous. Without limiting this specification, certain non-limiting solutions of this disclosure numbered 1-115 are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered solutions can be used or combined with any of the previously or subsequently individually numbered solutions. This is intended to support combinations of all such solutions, but not limited to combinations of the solutions explicitly provided below:

[0204] 1. A method for determining parameters of a photodetector in a particle analyzer, the method comprising:

[0205] The light source illuminates the photodetector located in the particle analyzer at a first intensity for a first predetermined time interval.

[0206] The photodetector is illuminated with the light source at a second intensity for a second predetermined time interval;

[0207] Integrate the data signal from the photodetector within a time period including the first predetermined time interval and the second predetermined time interval; and

[0208] One or more parameters of the photodetector are determined based on the integrated data signal.

[0209] 2. The method according to 1, wherein the particle analyzer is integrated into a flow cytometer.

[0210] 3. The method according to any one of 1-2, wherein the photodetector is positioned in the particle analyzer to detect light from particles in the flowing stream.

[0211] 4. The method according to any one of 1-3, wherein the light source is a continuous wave light source.

[0212] 5. The method according to any one of 1-3, wherein the light source is a pulsed light source.

[0213] 6. The method according to any one of 1-5, wherein the light source is a light-emitting diode.

[0214] 7. The method according to any one of 1-6, wherein the light source is a narrow bandwidth light source.

[0215] 8. The method according to 7, wherein the light source emits light comprising wavelengths spanning 20 nm or less.

[0216] 9. The method according to any one of 1-8, wherein the method includes irradiating the photodetector with a second intensity greater than the first intensity.

[0217] 10. The method according to any one of 1-9, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0218] 11. The method according to any one of 1-10, wherein the method includes continuously irradiating the photodetector during a time period including the first predetermined time interval and the second predetermined time interval.

[0219] 12. The method according to any one of 1-11, wherein the method includes increasing the light intensity from the light source from the first intensity to the second intensity within a third predetermined time interval.

[0220] 13. The method according to any one of 1-11, wherein the method includes increasing the light intensity from the light source during a time period including the first predetermined time interval and the second predetermined time interval.

[0221] 14. According to the method of 13, the light intensity from the light source increases linearly over a time period including the first predetermined time interval and the second predetermined time interval.

[0222] 15. According to the method of 13, wherein the light intensity from the light source increases exponentially over a time period including the first predetermined time interval and the second predetermined time interval.

[0223] 16. The method according to any one of 1-15, wherein integrating the data signal from the photodetector includes calculating the signal amplitude over the time period.

[0224] 17. The method according to 16, wherein the method includes calculating the median signal amplitude over the time period.

[0225] 18. The method of any one of 16-17, wherein the method includes comparing the calculated signal amplitude with the light intensity of the light source.

[0226] 19. The method of any one of 1-18, wherein the method includes determining one or more parameters of the photodetector, the one or more parameters being selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and the number of photoelectrons per unit output.

[0227] 20. The method according to any one of 1-19, wherein the method includes:

[0228] The photodetector is illuminated by the light source at multiple intensities over multiple predetermined time intervals;

[0229] Integrate the data signal from the photodetector over a time period including the multiple predetermined time intervals; and

[0230] One or more parameters of the photodetector are determined based on the integrated data signal.

[0231] 21. The method according to any one of 1-20, wherein the parameters of the photodetector are determined within the operating voltage range of the photodetector.

[0232] 22. According to the method of 21, wherein the parameters of the photodetector are determined over the entire operating voltage range of the photodetector.

[0233] 23. The method according to any one of 1-22 further includes calculating the optimal detector gain of the photodetector based on the determined parameters.

[0234] 24. A method comprising:

[0235] The photodetector is illuminated with a light source at a first intensity for a first predetermined time interval;

[0236] The photodetector is illuminated with the light source at a second intensity for a second predetermined time interval;

[0237] Integrate the data signal from the photodetector within a time period including the first predetermined time interval and the second predetermined time interval; and

[0238] One or more parameters of the photodetector are determined based on the integrated data signal.

[0239] 25. According to the method in 24, the light source is a continuous wave light source.

[0240] 26. According to the method in 24, wherein the light source is a pulsed light source.

[0241] 27. The method according to any one of 24-26, wherein the light source is a light-emitting diode.

[0242] 28. The method according to any one of 24-27, wherein the light source is a narrow bandwidth light source.

[0243] 29. The method according to 28, wherein the light source emits light comprising wavelengths spanning 20 nm or less.

[0244] 30. The method of any one of 24-29, wherein the method includes irradiating the photodetector with a second intensity greater than the first intensity.

[0245] 31. The method according to any one of 24-30, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0246] 32. The method according to any one of 24-31, wherein the method includes continuously irradiating the photodetector during a time period including the first predetermined time interval and the second predetermined time interval.

[0247] 33. The method according to any one of 24-32, wherein the method includes increasing the light intensity from the light source from the first intensity to the second intensity within a third predetermined time interval.

[0248] 34. The method according to any one of 24-32, wherein the method includes increasing the light intensity from the light source during a time period including the first predetermined time interval and the second predetermined time interval.

[0249] 35. According to the method of 34, the light intensity from the light source increases linearly over a time period including the first predetermined time interval and the second predetermined time interval.

[0250] 36. According to the method of 34, the light intensity from the light source increases exponentially over a time period including the first predetermined time interval and the second predetermined time interval.

[0251] 37. The method according to any one of 24-36, wherein integrating the data signal from the photodetector includes calculating the signal amplitude over the time period.

[0252] 38. The method according to 37, wherein the method includes calculating the median signal amplitude over the time period.

[0253] 39. The method according to any one of 37-38, wherein the method includes comparing the calculated signal amplitude with the light intensity of the light source.

[0254] 40. The method of any one of 24-39, wherein the method includes determining one or more parameters of the photodetector, the one or more parameters being selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and the number of photoelectrons per unit output.

[0255] 41. The method according to any one of 24-40, wherein the method comprises:

[0256] The photodetector is illuminated by the light source at multiple intensities over multiple predetermined time intervals;

[0257] Integrate the data signal from the photodetector over a time period including the multiple predetermined time intervals; and

[0258] One or more parameters of the photodetector are determined based on the integrated data signal.

[0259] 42. The method of any one of 24-41, wherein the parameters of the photodetector are determined within the operating voltage range of the photodetector.

[0260] 43. According to the method of 42, wherein the parameters of the photodetector are determined over the entire operating voltage range of the photodetector.

[0261] 44. The method according to any one of 24-43 further includes calculating the optimal detector gain of the photodetector based on the determined parameters.

[0262] 45. The method according to any one of 24-44, wherein the photodetector is located in a flow cytometer.

[0263] 46. ​​The method of 45, wherein the flow cytometer includes a flow cell for propagating particles in a flow stream.

[0264] 47. A particle analyzer, comprising:

[0265] light source;

[0266] A light detection system located within the housing of the particle analyzer, the light detection system comprising a photodetector configured to:

[0267] Detecting light from the light source at a first intensity for a first predetermined time interval; and

[0268] The light from the light source is detected at a second intensity for a second predetermined time interval; and

[0269] A processor includes memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following processes:

[0270] Integrating the data signal from the photodetector within a time period including the first predetermined time interval and the second time interval; and

[0271] One or more parameters of the photodetector are determined based on the integrated data signal.

[0272] 48. The particle analyzer according to 47, wherein the particle analyzer is incorporated into a flow cytometer.

[0273] 49. A particle analyzer according to any one of 47-48, wherein the light source is a continuous wave source.

[0274] 50. A particle analyzer according to any one of 47-48, wherein the light source is a pulsed light source.

[0275] 51. A particle analyzer according to any one of 47-50, wherein the light source is a light-emitting diode.

[0276] 52. A particle analyzer according to any one of 47-51, wherein the light source is a narrow bandwidth light source.

[0277] 53. The particle analyzer according to 52, wherein the light source emits light comprising wavelengths spanning 20 nm or less.

[0278] 54. A particle analyzer according to any one of 47-53, wherein the second intensity is greater than the first intensity.

[0279] 55. A particle analyzer according to any one of 47-54, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0280] 56. A particle analyzer according to any one of 47-55, wherein the light source is configured to continuously illuminate the photodetector during a time period including the first predetermined time interval and the second predetermined time interval.

[0281] 57. The particle analyzer of 56, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following process: increasing the light intensity from the light source from the first intensity to the second intensity within a third predetermined time interval.

[0282] 58. A particle analyzer according to any one of 47-56, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to increase the light intensity from the light source during a time period including the first predetermined time interval and the second predetermined time interval.

[0283] 59. The particle analyzer according to 58, wherein the light intensity from the light source increases linearly over a time period including the first predetermined time interval and the second predetermined time interval.

[0284] 60. According to the particle analyzer of 58, the light intensity from the light source increases exponentially over a time period including the first predetermined time interval and the second predetermined time interval.

[0285] 61. A particle analyzer according to any one of 47-60, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the signal amplitude during the time period.

[0286] 62. The particle analyzer of 61, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following process: calculate the median signal amplitude over the time period.

[0287] 63. A particle analyzer according to any one of 61-62, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to compare the calculated signal amplitude with the light intensity of the light source.

[0288] 64. A particle analyzer according to any one of 47-63, wherein one or more parameters of the photodetector are selected from the group consisting of minimum detection threshold, maximum detection threshold, detector sensitivity, detector dynamic range, detector signal-to-noise ratio, and number of photoelectrons per unit output.

[0289] 65. A particle analyzer according to any one of 47-64, wherein the light source is configured to illuminate the photodetector at multiple intensities over multiple predetermined time intervals.

[0290] 66. The particle analyzer of 65, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following processes:

[0291] Integrate the data signal from the photodetector over a time period including the multiple predetermined time intervals; and

[0292] One or more parameters of the photodetector are determined based on the integrated data signal.

[0293] 67. A particle analyzer according to any one of 47-66, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following process: determine parameters of the photodetector within the operating voltage range of the photodetector.

[0294] 68. The particle analyzer of 67, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following process: determine the parameters of the photodetector over the entire operating voltage range of the photodetector.

[0295] 69. A particle analyzer according to any one of 47-68, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the optimal detector gain of the photodetector based on the determined parameters.

[0296] 70. A system comprising:

[0297] light source;

[0298] A photodetector system including a photodetector, the photodetector being configured as follows:

[0299] Detecting light from the light source at a first intensity for a first predetermined time interval; and

[0300] The light from the light source is detected at a second intensity for a second predetermined time interval; and

[0301] A processor includes memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following processes:

[0302] Integrating the data signal from the photodetector within a time period including the first predetermined time interval and the second time interval; and

[0303] One or more parameters of the photodetector are determined based on the integrated data signal.

[0304] 71. According to the system of 70, the light source is a continuous wave light source.

[0305] 72. According to the system of 70, the light source is a pulsed light source.

[0306] 73. A system according to any one of 70-72, wherein the continuous wave light source is a light-emitting diode.

[0307] 74. A system according to any one of 70-73, wherein the light source is a narrow bandwidth light source.

[0308] 75. The system according to 70, wherein the light source emits light comprising wavelengths spanning 20 nm or less.

[0309] 76. A system according to any one of 70-75, wherein the second strength is greater than the first strength.

[0310] 77. The system according to any one of 70-76, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0311] 78. The system according to any one of 70-77, wherein the light source is configured to continuously illuminate the photodetector during a time period including the first predetermined time interval and the second predetermined time interval.

[0312] 79. The system according to 78, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following process: increasing the light intensity from the light source from the first intensity to the second intensity within a third predetermined time interval.

[0313] 80. A system according to any one of 70-78, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to increase the light intensity from the light source during a time period including the first predetermined time interval and the second predetermined time interval.

[0314] 81. The system according to 80, wherein the light intensity from the light source increases linearly over a time period including the first predetermined time interval and the second predetermined time interval.

[0315] 82. The system according to 80, wherein the light intensity from the light source increases exponentially over a time period including the first predetermined time interval and the second predetermined time interval.

[0316] 83. The system according to any one of 70-82, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the signal amplitude during the time period.

[0317] 84. The system according to 83, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following process: calculate the median signal amplitude over the time period.

[0318] 85. The system according to any one of 83-84, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to compare the calculated signal amplitude with the light intensity of the light source.

[0319] 86. A system according to any one of 83-85, wherein one or more parameters of the photodetector are selected from the group consisting of minimum detection threshold, maximum detection threshold, detector sensitivity, detector dynamic range, detector signal-to-noise ratio, and number of photoelectrons per unit output.

[0320] 87. The system according to any one of 85-86, wherein the light source is configured to illuminate the photodetector at multiple intensities over multiple predetermined time intervals.

[0321] 88. The system of claim 87, wherein the processor includes memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following processes:

[0322] Integrate the data signal from the photodetector over a time period including the multiple predetermined time intervals; and

[0323] One or more parameters of the photodetector are determined based on the integrated data signal.

[0324] 89. A system according to any one of 70-88, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following process: determining parameters of the photodetector within the operating voltage range of the photodetector.

[0325] 90. The system according to 89, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following process: determining parameters of the photodetector over the entire operating voltage range of the photodetector.

[0326] 91. A system according to any one of 70-90, wherein the processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate an optimal detector gain for the photodetector based on the determined parameters.

[0327] 92. A system according to any one of 70-91, wherein the system is a flow cytometer.

[0328] 93. The system according to 92, wherein the flow cytometer includes a flow cell for propagating particles in a flow stream.

[0329] 94. The system according to 93, wherein the photodetector is positioned to detect light from particles in the flow.

[0330] 95. A non-transitory computer-readable storage medium comprising instructions stored thereon, the instructions comprising:

[0331] An algorithm that uses a light source to illuminate a photodetector with a first intensity for a first predetermined time interval;

[0332] An algorithm for illuminating a photodetector with a second intensity for a second predetermined time interval using the light source;

[0333] An algorithm for integrating data signals from the photodetector over a time period including the first predetermined time interval and the second predetermined time interval; and

[0334] An algorithm for determining one or more parameters of the photodetector based on the integrated data signal.

[0335] 96. A non-transitory computer-readable storage medium according to 95, wherein the light source is a continuous wave light source.

[0336] 97. According to 95, a non-transitory computer-readable storage medium wherein the light source is a pulsed light source.

[0337] 98. A non-transitory computer-readable storage medium according to any one of 95-97, wherein the continuous wave light source is a light-emitting diode.

[0338] 99. A non-transitory computer-readable storage medium according to any one of 95-98, wherein the light source is a narrow bandwidth light source.

[0339] 100. A non-transitory computer-readable storage medium according to 99, wherein the light source emits light comprising wavelengths spanning 20 nm or less.

[0340] 101. A non-transitory computer-readable storage medium according to any one of claims 95-100, wherein the non-transitory computer-readable storage medium includes an algorithm for irradiating the photodetector with a second intensity greater than the first intensity.

[0341] 102. A non-transitory computer-readable storage medium according to any one of 95-101, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0342] 103. A non-transitory computer-readable storage medium according to any one of claims 95-102, wherein the non-transitory computer-readable storage medium includes an algorithm for continuously illuminating the photodetector during a time period including the first predetermined time interval and the second predetermined time interval.

[0343] 104. The non-transitory computer-readable storage medium according to 103, wherein the non-transitory computer-readable storage medium includes an algorithm for increasing the light intensity from the light source from the first intensity to the second intensity within a third predetermined time interval.

[0344] 105. The non-transitory computer-readable storage medium according to 103, wherein the non-transitory computer-readable storage medium includes an algorithm for increasing the light intensity from the light source during a time period including the first predetermined time interval and the second predetermined time interval.

[0345] 106. The non-transitory computer-readable storage medium according to 105, wherein the light intensity from the light source increases linearly over a time period including the first predetermined time interval and the second predetermined time interval.

[0346] 107. The non-transitory computer-readable storage medium according to 105, wherein the light intensity from the light source increases exponentially over a time period including the first predetermined time interval and the second predetermined time interval.

[0347] 108. A non-transitory computer-readable storage medium according to any one of claims 95-107, wherein the non-transitory computer-readable storage medium includes an algorithm for calculating the signal amplitude during the time period.

[0348] 109. The non-transitory computer-readable storage medium of 108, wherein the non-transitory computer-readable storage medium includes an algorithm for calculating the median signal amplitude over the time period.

[0349] 110. A non-transitory computer-readable storage medium according to any one of 108-109, wherein the non-transitory computer-readable storage medium includes an algorithm for comparing the calculated signal amplitude with the light intensity of the light source.

[0350] 111. A non-transitory computer-readable storage medium according to any one of 95-110, wherein the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of the photodetector, the one or more parameters being selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and the number of photoelectrons per unit output.

[0351] 112. The non-transitory computer-readable storage medium according to any one of claims 95-111, wherein the non-transitory computer-readable storage medium comprises:

[0352] An algorithm for illuminating the photodetector with the light source at multiple intensities over multiple predetermined time intervals;

[0353] An algorithm for integrating data signals from a photodetector over a time period comprising multiple predetermined time intervals; and

[0354] An algorithm for determining one or more parameters of the photodetector based on the integrated data signal.

[0355] 113. A non-transitory computer-readable storage medium according to any one of 95-112, wherein the non-transitory computer-readable storage medium includes an algorithm for determining parameters of the photodetector within the operating voltage range of the photodetector.

[0356] 114. The non-transitory computer-readable storage medium of claim 113, wherein the non-transitory computer-readable storage medium includes an algorithm for determining parameters of the photodetector over its entire operating voltage range.

[0357] 115. A non-transitory computer-readable storage medium according to any one of 95-114, the non-transitory computer-readable storage medium including an algorithm for calculating the optimal detector gain of the photodetector based on the determined parameters.

[0358] Although the foregoing invention has been described in considerable detail by way of illustration and examples for purposes of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims.

[0359] Therefore, the foregoing illustrations merely illustrate the principles of the invention. It should be understood that those skilled in the art will be able to devise various arrangements, although not explicitly described or shown herein, that embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language listed herein are primarily intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to the field, and should be interpreted as not being limited to such specifically listed examples and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to cover both their structural and functional equivalents. Additionally, these equivalents are intended to include both currently known equivalents and those developed in the future, i.e., any element developed that performs the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether such disclosure is expressly stated in the claims.

[0360] Therefore, the scope of the invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35U.SC§112(f) or 35U.SC§112(6) is explicitly defined as being invoked only for limitation in the claims if the exact phrase “means for…” or the exact phrase “step for…” is stated at the beginning of such limitation in the claims; if such exact phrase is not used to limit the claims, then 35U.SC§112(f) or 35U.SC§112(6) is not invoked.

Claims

1. A method for determining parameters of a photodetector in a particle analyzer, the method comprising: The light source illuminates the photodetector located in the particle analyzer at a first intensity for a first predetermined time interval. The photodetector is illuminated by the light source at a second intensity different from the first intensity for a second predetermined time interval; The data signals generated by the photodetector during the first predetermined time interval and the second predetermined time interval are integrated together; as well as One or more parameters of the photodetector are determined based on the integrated data signal.

2. The method according to claim 1, wherein The light source is a continuous wave light source.

3. The method according to claim 1 or 2, wherein The light source is a light-emitting diode (LED).

4. The method according to claim 1, wherein The method includes irradiating the photodetector with a second intensity greater than the first intensity.

5. The method according to claim 1, wherein The method includes increasing the light intensity of the light source from the first intensity to the second intensity within a third predetermined time interval.

6. The method according to claim 1, wherein The method includes increasing the light intensity of the light source within a time period including the first predetermined time interval and the second predetermined time interval.

7. The method according to claim 1, wherein Integrating the data signals generated by the photodetector together includes: The signal amplitude within a time period is calculated, and the calculated signal amplitude is compared with the light intensity of the light source.

8. The method according to claim 1, wherein The method includes determining one or more parameters of the photodetector, the one or more parameters being selected from a group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and the number of photoelectrons per unit output.

9. The method according to claim 1, wherein The method includes: The parameters of the photodetector are determined within the operating voltage range of the photodetector; as well as The optimal detector gain of the photodetector is calculated based on the determined parameters.

10. A particle analyzer, comprising: light source; A light detection system, located within the housing of the particle analyzer, includes a photodetector configured to: detect light from the light source at a first intensity for a first predetermined time interval; and detect light from the light source at a second intensity different from the first intensity for a second predetermined time interval. as well as A processor, including memory operatively coupled to the processor, wherein the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to perform the following processes: Integrate the data signals generated by the photodetector during the first predetermined time interval and the second predetermined time interval; and One or more parameters of the photodetector are determined based on the integrated data signal.

11. The particle analyzer according to claim 10, wherein The light source is a continuous wave light source.

12. The particle analyzer according to claim 10 or 11, wherein The processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to increase the light intensity of the light source from the first intensity to the second intensity within a third predetermined time interval.

13. The particle analyzer according to claim 10, wherein The processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to increase the light intensity of the light source during a time period including the first predetermined time interval and the second predetermined time interval.

14. The particle analyzer according to claim 10, wherein The processor includes a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following processes: a) Calculate the signal amplitude within the time period; and Determine one or more parameters of the photodetector, wherein the one or more parameters are selected from a group consisting of minimum detection threshold, maximum detection threshold, detector sensitivity, detector dynamic range, detector signal-to-noise ratio, and number of photoelectrons per unit output; or b) Calculate the signal amplitude during the time period; The parameters of the photodetector are determined within the operating voltage range of the photodetector; and The optimal detector gain of the photodetector is calculated based on the determined parameters.

15. A non-transitory computer-readable storage medium, comprising instructions stored thereon, The instructions include: An algorithm that uses a light source to illuminate a photodetector with a first intensity for a first predetermined time interval; An algorithm for illuminating the photodetector with the light source at a second intensity different from the first intensity for a second predetermined time interval; An algorithm for integrating the data signals generated by the photodetector during the first predetermined time interval and the second predetermined time interval; as well as An algorithm for determining one or more parameters of the photodetector based on the integrated data signal.

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