Method and system for characterizing and encoding a light detection system

Abstract

Various aspects of the present disclosure include methods for determining a photodetector (e.g., a photodetector in a particle analyzer) parameter. The methods according to certain embodiments include detecting light from a light source with a photodetector for a first predetermined time interval; detecting a step signal with the photodetector, the step signal indicating a change in a parameter of the light source or a parameter of the photodetector; detecting light from the light source for a second predetermined time interval; integrating a data signal over the first predetermined time interval and the second predetermined time interval; and determining one or more parameters of the photodetector based on the integrated data signal. Systems (e.g., particle analyzers) having a light source and a photodetector for practicing the methods of the present subject matter are also described. Non-transitory computer readable storage media having stored thereon instructions for determining a parameter of a photodetector according to the methods of the present subject matter are also provided.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 107,701, filed October 30, 2020, pursuant to 35 U.S. SC §119(e), the disclosure of which is incorporated herein by reference in its entirety.

[0003] introduction

[0004] Characterizing analytes in bodily fluids has become an important part of medical diagnosis and assessment of a patient's overall health. Detecting analytes in bodily fluids (such as human blood or blood derivatives) can provide results that can play a role in determining treatment plans for patients with various diseases.

[0005] Flow cytometry is a technique used to characterize and often sort biological materials, such as cells in blood samples or particles of interest in other types of biological or chemical samples. A flow cytometer typically includes a sample reservoir for receiving liquid samples (e.g., blood samples) and a sheath reservoir containing sheath fluid. The flow cytometer transports particles (including cells) in the liquid sample as a flow stream to the flow unit, while also guiding the sheath fluid into the flow unit. To characterize the components in the flow stream, the flow stream is illuminated at a query point. Changes in the material in the flow stream, such as the presence of morphology or fluorescent labels, can cause changes in the observed light detected by a photodetector, enabling characterization and separation.

[0006] Flow cytometry instruments use various optical detectors, such as photodiodes (PDs), photomultiplier tubes (PMTs), and avalanche photodiodes (APDs), to detect photon emission from the studied samples. The response of these detectors to incident light varies not only depending on the type of photodiode but also across multiple samples using the same type of photodiode. Furthermore, some detectors (such as APDs) exhibit highly nonlinear characteristics.

[0007] Therefore, detector response profiles need to be created for flow cytometers to measure the linearity of each detector's response to light and to calibrate the signal response detected due to detector gain adjustments (e.g., voltage adjustments on photomultiplier tubes or gain adjustments to avalanche photodiodes) in order to optimize system sensitivity for different user panels. Traditionally, this is done by using fluorescent droplets with known scattering distributions (e.g., different sizes) and fluorescence staining intensities and measuring the signal from the detector. The detector gain is also adjusted to explore the gain response of known standard particles.

[0008] Recently, LED technology has begun to be used to generate flashes with known intensity distributions to characterize components of flow cytometry detection systems. These pulses generate detection "events" within the detection system when they exceed its trigger threshold. The height, area, and width of the pulses, as well as the timestamp of the event, can then be measured. In these cases, the LED flash consists of one or more LEDs (possibly emitting with different spectral distributions) that illuminate the optics of the cytometry detection system. These flashes pass through optical fibers, filters, and mirrors and are detected at photodiodes, photomultiplier tubes (PMTs), or avalanche photodiodes (APDs). The intensity of the flashes generated by the LEDs, as well as the frequency of the LED flash, the flash duration, and the total emission spectrum, can be controlled. Summary of the Invention

[0009] Embodiments of the present invention provide a method for increasing reliability and reducing the timing of LED-based protocols by incorporating data stream coding to control the time interval of LED flicker rate. As described in more detail below, the subject matter method according to certain embodiments is capable of determining and calibrating the linearity of the photodetector's response to light illumination and adjusting the detector gain in real time. The methods described herein provide a more accurate and consistent characterization of the photodetector's response and sensitivity. In some embodiments, the parameters of the photodetector can be determined and calibrated without further user intervention. In some embodiments, the parameters of the photodetector can be determined and calibrated in real time without the use of calibration beads or luminescence control particles.

[0010] Various aspects of this disclosure include methods for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). A method according to certain embodiments includes: detecting light from a light source using a photodetector at a first predetermined time interval; detecting a step signal using the photodetector, the step signal indicating a change in parameters of the light source or the photodetector; detecting light from the light source at a second predetermined time interval; integrating a data signal over the first and second predetermined time intervals; and determining one or more parameters of the photodetector based on the integrated data signal. In embodiments, the duration of the first predetermined time interval may be the same as or different from the duration of the second predetermined time interval.

[0011] In some embodiments, the step signal indicates a change in the light intensity of the light source. In some examples, the change in intensity refers to a decrease in the light intensity of the light source. In other examples, the change in intensity refers to an increase in the light intensity of the light source. In other embodiments, the step signal indicates a change in the spectral parameters of the light emitted by the light source. In some examples, the change in spectral parameters refers to a change in the wavelength of the light emitted by the light source. In other examples, the change in spectral parameters refers to an increase in the number of wavelengths of light emitted by the light source. In some examples, an increase in the number of wavelengths of light includes an increase in the spectral width emitted by the light source. In other examples, the change in spectral parameters refers to a decrease in the number of wavelengths of light emitted by the light source. In some examples, a decrease in the number of wavelengths of light includes a decrease in the spectral width emitted by the light source.

[0012] In some embodiments, the light source is a light-emitting diode (LED). In some examples, the LED is a continuous-wave LED. In other examples, the LED is a pulsed LED. In some embodiments, the light source includes one or more monochromatic LEDs. In other embodiments, the light source includes one or more multicolor LEDs. In still other embodiments, the light source includes multiple LEDs.

[0013] In some examples, the step signal includes a change in the frequency of a light pulse from the light source. In some examples, the step signal includes detecting a light pulse from the light source. In other examples, the step signal includes detecting an increase in the frequency of a light pulse compared to a light pulse detected from the light source during a first predetermined time interval. In other examples, the step signal includes detecting a decrease in the frequency of a light pulse compared to a light pulse detected from the light source during the first predetermined time interval. In other examples, the step signal includes detecting an increase in the duration of a light pulse compared to a light pulse detected from the light source during the first predetermined time interval. In other examples, the step signal includes detecting a decrease in the duration of a light pulse compared to a light pulse detected from the light source during the first predetermined time interval. In some examples, the step signal includes detecting an absence of light from the light source.

[0014] In some embodiments, the step signal indicates a change in the detector gain of the photodetector. In some examples, the change in detector gain refers to a decrease in the detector gain of the photodetector. In other examples, the change in detector gain refers to an increase in the detector gain of the photodetector. In some embodiments, the change in detector gain is indicated by a step signal that includes a change in the frequency of light pulses from the light source. In other embodiments, the change in detector gain is indicated by a step signal that includes a absence of light from the light source.

[0015] In some embodiments, a tag signal is inserted into a data signal over one or more predetermined time intervals. In some examples, the tag signal is a frequency-modulated data signal that can be detected and processed during integration of the data signal from the photodetector at each predetermined time interval. In some embodiments, a step signal indicating a change in the parameters of a light source or photodetector is a tag signal inserted into a measurement data signal over one or more predetermined time intervals.

[0016] In some embodiments, the method includes detecting a synchronization signal before detecting light from a light source prior to a first predetermined time interval. In some embodiments, the synchronization signal includes detecting light from the light source having a predetermined pulse frequency. In other embodiments, the synchronization signal includes detecting light from the light source having an intensity exceeding a predetermined intensity threshold. In still other embodiments, the synchronization signal includes detecting light from the light source at its maximum intensity.

[0017] In some embodiments, the method includes detecting light from a light source at multiple predetermined time intervals. In some examples, the intensity of the light source increases after each predetermined time interval (i.e., a second illumination intensity is greater than a first illumination intensity). At each light intensity, the time interval for the photodetector to detect light from the light source can vary. In some examples, the various time intervals are the same. In other examples, the various time intervals are different.

[0018] In one embodiment, the method includes integrating a data signal from a photodetector over a period of time, the period of time including at least the illumination time interval at each different light intensity. In some embodiments, the data signal from the photodetector is integrated over a period of time including a first time interval during which the light source illuminates the photodetector at a first intensity and a second time interval during which the light source illuminates the photodetector at a second intensity. In other embodiments, the data signal from the photodetector is integrated over a period of time including multiple time intervals, wherein the light source illuminates the photodetector at an increasing light intensity during each of the multiple time intervals.

[0019] In some embodiments, integrating the data signal from the photodetector includes calculating the signal amplitude over the time period. In some examples, calculating the signal amplitude includes calculating the median signal amplitude. In some examples, the method further includes comparing the calculated signal amplitude with the light intensity of the light source. Based on one or more calculated signal amplitudes 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 of the photodetector (e.g., detector sensitivity, minimum detection threshold, maximum detection threshold, detector dynamic range, detector signal-to-noise ratio, or number of photoelectrons per unit output). Detector parameters can be determined within the operating voltage range of the photodetector, for example, across the entire operating voltage range of the photodetector. In some embodiments, the photodetector is located within a particle analyzer, for example, where the photodetector is part of the light detection module of the particle analyzer. In some examples, the particle analyzer is incorporated into a flow cytometer, where the photodetector detects the light emitted by particles in the flowing stream.

[0020] Various aspects of this disclosure also include a system having a light source, a photodetector, and a processor, wherein the photodetector is configured to detect light from the light source in a first predetermined time interval, detect a step signal indicating a change in a parameter of the light source or a parameter of the photodetector, and detect light from the light source in a second predetermined time interval. The processor has a memory operatively coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to integrate data signals from the photodetector in the first and second predetermined time intervals, and determine one or more parameters of the photodetector based on the integrated data signals. 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 examples, the particle analyzer is incorporated into a flow cytometer.

[0021] In some embodiments, the light source is a light-emitting diode (LED). In some examples, the LED is a continuous-wave LED. In other examples, the LED is a pulsed LED. In some embodiments, the light source includes one or more monochromatic LEDs. In other embodiments, the light source includes one or more multicolor LEDs. In still other embodiments, the light source includes multiple LEDs.

[0022] In some embodiments, the light detection system includes a photodetector. In some examples, the photodetector is a photomultiplier tube. In other examples, the photodetector is a photodiode. In still other examples, the photodetector is an avalanche photodiode. In some examples, the photodetector includes one or more amplifier components, such as a transimpedance amplifier.

[0023] The system of interest includes a processor having memory operatively coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to detect a step signal indicating a change in parameters of a light source or a photodetector. In some examples, the step signal includes a change in the frequency of a light pulse from the light source. In some examples, the step signal includes detecting a light pulse from the light source. In other examples, the step signal includes detecting an increase in the frequency of a light pulse from the light source compared to a light pulse detected during a first predetermined time interval. In other examples, the step signal includes detecting a decrease in the frequency of a light pulse from the light source compared to a light pulse detected during the first predetermined time interval. In other examples, the step signal includes detecting an increase in the duration of a light pulse from the light source compared to a light pulse detected during the first predetermined time interval. In other examples, the step signal includes detecting a decrease in the duration of a light pulse from the light source compared to a light pulse detected during the first predetermined time interval. In some examples, the step signal includes detecting a lack of light from the light source.

[0024] In some embodiments, the step signal is a change in the detector gain of the photodetector. In some examples, the change in detector gain refers to a decrease in the detector gain of the photodetector. In other examples, the change in detector gain refers to an increase in the detector gain of the photodetector. In some embodiments, the change in detector gain is indicated by a step signal that includes a change in the frequency of light pulses from the light source. In other embodiments, the change in detector gain is indicated by a step signal that includes a lack of light from the light source.

[0025] In some instances, the memory includes instructions for detecting light from a light source at multiple predetermined time intervals. In some examples, the intensity of the light source increases after each predetermined time interval. At each light intensity, the time interval for the photodetector to detect light from the light source can vary. In some examples, the individual time intervals are the same. In other examples, the individual time intervals are different. In these examples, the memory includes instructions for integrating the data signal from the photodetector over a time period including the multiple illumination time intervals.

[0026] In some embodiments, the memory includes instructions for detecting a step signal indicating a change in the light intensity from the light source. In some examples, the change in intensity refers to a decrease in the light intensity of the light source. In other examples, the change in intensity refers to an increase in the light intensity of the light source. In other embodiments, the memory includes instructions for detecting a step signal indicating a change in the spectral parameters of the light from the light source. In some examples, the change in the spectral parameters refers to a change in the wavelength of the light emitted by the light source. In other examples, the change in the spectral parameters refers to an increase in the number of wavelengths of light emitted by the light source. In some examples, an increase in the number of wavelengths of light includes an increase in the spectral width emitted by the light source. In other examples, the change in the spectral parameters refers to a decrease in the number of wavelengths of light emitted by the light source. In some examples, a decrease in the number of wavelengths of light includes a decrease in the spectral width emitted by the light source.

[0027] In some embodiments, the memory includes instructions for detecting a tag signal inserted into a data signal over one or more predetermined time intervals. In some examples, the tag signal is a frequency-modulated data signal that can be detected and processed during the integration of the data signal from the photodetector at each predetermined time interval.

[0028] In some embodiments, the memory includes instructions for detecting a synchronization signal. In some embodiments, the memory includes instructions for detecting a synchronization signal indicating that light from the light source has a predetermined pulse frequency. In other embodiments, the memory includes instructions for detecting a synchronization signal indicating that light from the light source has an intensity exceeding a predetermined intensity threshold. In still other embodiments, the memory includes instructions for detecting a synchronization signal indicating that light from the light source is at its maximum light intensity.

[0029] In some embodiments, the memory includes instructions stored thereon for integrating data signals from the photodetector. In some embodiments, the memory includes instructions for calculating the signal amplitude of the photodetector at 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 the illumination intensity at each predetermined time interval.

[0030] 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 calculated signal amplitudes and comparisons between the calculated signal amplitudes and the light intensity of a light source. For example, the memory may include instructions for determining detector sensitivity, minimum detection threshold, maximum detection threshold, detector dynamic range, detector signal-to-noise ratio, or number of photoelectrons per unit output. The system can be configured to determine detector parameters over the operating voltage range of the photodetector, for example, over the entire operating voltage range of the photodetector.

[0031] Various aspects 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 detecting light from a light source in a particle analyzer using a photodetector during a first predetermined time interval; an algorithm for detecting a step signal indicating a change in a parameter of the light source or a parameter of the photodetector; an algorithm for detecting light from the light source using a photodetector during a second predetermined time interval; an algorithm for integrating a data signal from the photodetector during 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.

[0032] In some examples, the non-transitory computer-readable storage medium includes an algorithm for detecting light from a light source at multiple predetermined time intervals. In some examples, the intensity of the light source increases after each predetermined time interval. At each light intensity, the time interval for the photodetector to detect light from the light source can vary. In some examples, the individual time intervals are the same. In other examples, the individual time intervals are different. In these examples, the non-transitory computer-readable storage medium includes an algorithm for integrating the data signal from the photodetector over a time period comprising multiple illumination time intervals.

[0033] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal indicating a change in light intensity from a light source. In some examples, the change in intensity refers to a decrease in the light intensity of the light source. In other examples, the change in intensity refers to an increase in the light intensity of the light source. In other embodiments, the memory includes instructions for detecting a step signal indicating a change in the spectral parameters of light from the light source. In some examples, the change in spectral parameters refers to a change in the wavelength of light emitted by the light source. In other examples, the change in spectral parameters refers to an increase in the number of wavelengths of light emitted by the light source. In some examples, an increase in the number of wavelengths of light includes an increase in the spectral width emitted by the light source. In other examples, the change in spectral parameters refers to a decrease in the number of wavelengths of light emitted by the light source. In some examples, a decrease in the number of wavelengths of light includes a decrease in the spectral width emitted by the light source.

[0034] In some examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, the step signal comprising a change in the frequency of a light pulse from a light source. In some examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, the step signal comprising detecting a light pulse from a light source. In other examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, the step signal comprising detecting an increase in the frequency of a light pulse from a light source compared to a light pulse from a light source detected during a first predetermined time interval. In other examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, the step signal comprising detecting a decrease in the frequency of a light pulse from a light source compared to a light pulse from a light source detected during a first predetermined time interval. In other examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, the step signal comprising detecting an increase in the duration of a light pulse from a light source compared to a light pulse from a light source detected during a first predetermined time interval. In other examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, the step signal comprising detecting a decrease in the duration of a light pulse from a light source compared to a light pulse from a light source detected during a first predetermined time interval. In some instances, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, which includes detecting the absence of light from a light source.

[0035] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal indicating a change in the gain of a photodetector. In some examples, the change in detector gain refers to a decrease in the detector gain of the photodetector. In other examples, the change in detector gain refers to an increase in the detector gain of the photodetector. In some embodiments, the change in detector gain is indicated by a step signal that includes a change in the frequency of a light pulse from a light source. In other embodiments, the change in detector gain is indicated by a step signal that includes a loss of light from the light source.

[0036] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting a tag signal inserted into a data signal over one or more predetermined time intervals. In some examples, the tag signal is a frequency-modulated data signal that can be detected and processed during the integration of the data signal from the photodetector at each predetermined time interval.

[0037] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting a synchronization signal prior to detecting light from a light source before a first predetermined time interval. In some embodiments, the synchronization signal includes detecting light from the light source having a predetermined pulse frequency. In other embodiments, the synchronization signal includes detecting light from the light source having an intensity exceeding a predetermined intensity threshold. In still other embodiments, the synchronization signal includes detecting light from the light source at its maximum intensity.

[0038] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating a signal amplitude. In some examples, the non-transitory computer-readable storage medium includes an algorithm for calculating a median signal amplitude. In some examples, 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 examples, the non-transitory computer-readable storage medium includes an algorithm for determining parameters of a photodetector based on one or more calculated signal amplitudes 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 detector sensitivity, minimum detection threshold, maximum detection threshold, detector dynamic range, detector signal-to-noise ratio, or 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, for example, determining the parameters of the photodetector over the entire operating voltage range of the photodetector. Attached Figure Description

[0039] The best understanding of the invention can be obtained from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following images:

[0040] Figure 1A The invention describes the detection of a step signal according to certain embodiments, the step signal comprising a change in pulse frequency that indicates a change in the light intensity of a light source.

[0041] Figure 1B The invention describes the detection of a step signal according to certain embodiments, the step signal comprising a change in pulse duration indicating a change in the light intensity of a light source.

[0042] Figure 1CThe invention describes the detection of a step signal according to certain embodiments, the step signal comprising a change in pulse frequency that indicates a change in the detector gain of a photodetector.

[0043] Figure 1D The invention describes the detection of a step signal according to certain embodiments, the step signal comprising a change in pulse duration that indicates a change in the detector gain of a photodetector.

[0044] Figure 2A and Figure 2B A method is described, according to certain embodiments, for detecting step signals to indicate changes in the parameters of a light source or a photodetector. Figure 2A The detection of a step signal is described, in which a photodetector detects a change in the pulse frequency of light. Figure 2B The step signal in which no light signal was detected was described.

[0045] Figures 3A to 3C A flowchart is depicted to determine one or more parameters of a photodetector according to certain embodiments.

[0046] Figure 4A A functional block diagram of a particle analysis system according to certain embodiments is depicted. Figure 4B A flow cytometer according to certain embodiments is described.

[0047] Figure 5 A functional block diagram of an example particle analyzer control system according to certain embodiments is depicted.

[0048] Figure 6 A block diagram of a computing system according to certain embodiments is depicted. Detailed Implementation

[0049] Various aspects of this disclosure include methods for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). A method according to certain embodiments includes: detecting light from a light source with a photodetector at a first predetermined time interval; detecting a step signal with the photodetector, the step signal indicating a change in parameters of the light source or the photodetector; detecting light from the light source at a second predetermined time interval; integrating a data signal over the first and second predetermined time intervals; and determining one or more parameters of the photodetector based on the integrated data signal. A system (e.g., a particle analyzer) having a light source and a photodetector for practicing the methods of this subject matter is also described herein. A non-transitory computer-readable storage medium having instructions stored thereon for determining parameters of a photodetector according to the methods of this subject matter is also provided herein.

[0050] Before providing a more detailed description of the invention, it should be understood that the invention is not limited to the specific embodiments described, and therefore variations are possible. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0051] When a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range up to one-tenth of the lower limit unit, as well as any other specified value or intermediate value within the specified range, is included in this invention. The upper and lower limits of these smaller ranges may be independently included within that smaller range and also within this invention, but are subject to any explicit exclusions or limitations within the range. If the range includes one or two limits, the range excluding one or both limits is also included in this invention.

[0052] To indicate certain ranges, this article will preface numerical values ​​with the term "approximately". In this article, the term "approximately" is used to provide literal support for the exact number preceding it and for numbers preceding terms of approximation or approximation. In determining whether a number is close to or approximates a particular referenced number, the close to or approximate unreferenced number can be a number that provides a substantially equivalent number to the specific referenced number in the context in which it appears.

[0053] 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 to practice or test the invention, only representative illustrative methods and materials are described here.

[0054] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent were expressly and individually incorporated herein by reference to disclose and describe the methods and / or materials relating to the cited publications. Reference to any publication is made because it was published prior to the filing date and should not be construed as an admission that the invention is not entitled to a prior invention prior to such publication. Furthermore, the publication dates provided may differ from the actual publication dates, which may require separate verification.

[0055] It should be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “the,” and “the” used herein and in the appended claims include plural references. It should also be noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a preliminary basis for the use of exclusive terms such as “only” or “merely” when reciting claim elements or using the “negative” limitation.

[0056] Upon reading this disclosure, those skilled in the art will understand that each individual embodiment described and used herein has discrete components and features that 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 described method can be performed in the order of the described events or any other logically feasible order.

[0057] Although the apparatus and method have been or will be described by functional interpretation for the purpose of grammatical fluency, it should be clearly understood that, unless expressly stated in accordance with 35 U.SC §112, the claims should not be construed as necessarily being limited in any way to “means” or “steps,” but should be given the full meaning and equivalents of the definitions provided in the claims under the principle of equivalents, and, where the claims are expressly stated in accordance with 35 U.SC §112, all their legal equivalents should be given in accordance with 35 U.SC §112.

[0058] As described 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, the method of this subject matter is first described in detail, comprising: detecting light from a light source using a photodetector at a first predetermined time interval; detecting a step signal indicating a change in parameters of the light source or the photodetector; detecting light at a second predetermined time interval; and integrating a data signal from the photodetector over a period of time. Next, a system (e.g., a particle analyzer) having a light source and a photodetector for practicing the method of this subject matter is described. A non-transitory computer-readable storage medium having instructions stored thereon for determining the parameters of the photodetector according to the method of this subject matter is also provided.

[0059] Methods for determining the parameters of a photodetector

[0060] Various aspects of this disclosure include methods for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). In practicing the methods of this subject matter, the photodetector is illuminated with a light source and the light is detected at a first predetermined time interval, a step signal is detected by the photodetector, and the light from the light source is detected at a second predetermined time interval. As described in detail below, the subject matter method according to certain embodiments can provide real-time determination and calibration of the linearity of the photodetector's response to light illumination, as well as adjustment of the detector gain. The methods described herein provide a more accurate and consistent characterization of the photodetector's response and sensitivity. In some embodiments, the parameters of the photodetector can be determined and calibrated without further user intervention. In some embodiments, the parameters of the photodetector can be determined and calibrated in real time without the use of calibration beads or luminescent control particles.

[0061] In some embodiments, the photodetector is illuminated by a pulsed light source for one or more predetermined time intervals. Herein, the term "pulsed light source" refers in the conventional sense to a light source that emits light at predetermined time intervals, each time interval having a predetermined illumination duration (i.e., pulse width). In some embodiments, the pulsed light source is configured to illuminate the photodetector with periodic flashes. For example, the frequency of each light pulse can be 0.0001 kHz or higher, such as 0.0005 kHz or higher, such as 0.001 kHz or higher, such as 0.005 kHz or higher, such as 0.01 kHz or higher, such as 0.05 kHz or higher, such as 0.1 kHz or higher, such as 0.5 kHz or higher, such as 1 kHz or higher, such as 2.5 kHz or higher, such as 5 kHz or higher, such as 10 kHz or higher, such as 25 kHz or higher, such as 50 kHz or higher, and including 100 kHz or higher. In some examples, the pulse irradiation frequency of the light source ranges from 0.00001 kHz to 1000 kHz, for example from 0.00005 kHz to 900 kHz, for example from 0.0001 kHz to 800 kHz, for example from 0.0005 kHz to 700 kHz, for example from 0.001 kHz to 600 kHz, for example from 0.005 kHz to 500 kHz, for example from 0.01 kHz to 400 kHz, for example from 0.05 kHz to 300 kHz, for example from 0.1 kHz to 200 kHz, and includes 1 kHz to 100 kHz. The duration of illumination (i.e., pulse width) of each light pulse can vary and can be 0.000001ms or longer, for example, 0.000005ms or longer, for example, 0.00001ms or longer, for example, 0.00005ms or longer, for example, 0.0001ms or longer, for example, 0.0005ms or longer, for example, 0.001ms or longer, for example, 0.005ms or longer, for example, 0.01ms or longer, for example, 0.05ms or longer, for example, 0.1ms or longer, for example, 0.5ms or longer, for example, 1ms or longer, for example, 2ms or longer, for example, 3ms or longer, for example, 4ms or longer, for example, 5ms or longer, for example, 10ms or longer, for example, 25ms or longer, for example, 50ms or longer, for example, 100ms or longer, and including 500ms or longer.For example, the duration of light irradiation can be from 0.000001ms to 1000ms, for example from 0.000005ms to 950ms, for example from 0.00001ms to 900ms, for example from 0.00005ms to 850ms, for example from 0.0001ms to 800ms, for example from 0.0005ms to 750ms, for example from 0.001ms to 700ms, for example from 0.005ms to 650ms, for example from 0.01ms to 600ms, for example from 0.05ms to 550ms, for example from 0.1ms to 500ms, for example from 0.5ms to 450ms, for example from 1ms to 400ms, for example from 5ms to 350ms, and includes from 10ms to 300ms.

[0062] In some embodiments, the photodetector is illuminated by a continuous-wave light source for one or more predetermined time intervals. Herein, the term "continuous-wave light source" means, in a conventional sense, a light source that provides 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, during the illumination time intervals, the continuous light source can provide emitted light intensity with very small variations, such variations being 10% or less, e.g., 9% or less, e.g., 8% or less, e.g., 7% or less, e.g., 6% or less, e.g., 5% or less, e.g., 4% or less, e.g., 3% or less, e.g., 2% or less, e.g., 1% or less, e.g., 0.5% or less, e.g., 0.1% or less, e.g., 0.01% or less, e.g., 0.001% or less, e.g., 0.0001% or less, e.g., 0.00001% or less, and including 0.000001% or less. It can use any convenient method to measure the intensity of light output, including but not limited to scanning slit profilometers, charge-coupled devices (CCDs) (e.g., 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.

[0063] In embodiments, the light source can be any convenient light source and can include 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 range of wavelengths. In some examples, the narrowband light source emits light with a narrow wavelength range, such as 50 nm or narrower, such as 40 nm or narrower, such as 30 nm or narrower, such as 25 nm or narrower, such as 20 nm or narrower, such as 15 nm or narrower, such as 10 nm or narrower, such as 5 nm or narrower, such as 2 nm or narrower, and includes a light source emitting light of a specific wavelength (i.e., monochromatic light). Any convenient narrowband light source solution can be used, such as a narrow-wavelength LED.

[0064] 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 examples, the broadband light source emits light with a wide wavelength range, for example, 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 is a light source that emits light with wavelengths from 400 nm to 1000 nm. Any convenient broadband light source solution can be used, such as halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with continuous spectrum, superluminescent diodes, semiconductor light-emitting diodes, broad-spectrum 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 includes an LED array. In some examples, the light source includes multiple monochromatic light-emitting diodes, wherein each monochromatic light-emitting diode outputs light with a different wavelength. In some examples, the light source includes multiple multicolor light-emitting diodes (LEDs) that output light with a predetermined spectral width, such as the spectral width of the light jointly output by the multiple multicolor LEDs ranging from 200nm to 1500nm, for example from 225nm to 1475nm, for example from 250nm to 1450nm, for example from 275nm to 1425nm, for example from 300nm to 1400nm, for example from 325nm to 1375nm, for example from 350nm to 1350nm, for example from 375nm to 1325nm, for example from 400nm to 1300nm, for example from 425nm to 1275nm, for example from 450nm to 1250nm, for example from 475nm to 1225nm, and including 500nm to 1200nm.

[0065] In some embodiments, the light source is a laser, such as a pulsed laser or a continuous-wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some examples, 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) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other examples, the subject matter system includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In other examples, lasers of interest 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 examples, the subject matter 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, ytterbium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers, and combinations thereof.

[0066] In some embodiments, the light source is a narrow bandwidth light source. In some examples, the light source is a light source that outputs a specific wavelength, which is from 200nm to 1500nm, for example from 250nm to 1250nm, for example from 300nm to 1000nm, for example from 350nm to 900nm, and includes wavelengths from 400nm to 800nm. In some embodiments, the continuous wave light source emits light with wavelengths of 365nm, 385nm, 405nm, 460nm, 490nm, 525nm, 550nm, 580nm, 635nm, 660nm, 740nm, 770nm, or 850nm.

[0067] In some embodiments, the light source emits light with overlapping wavelengths, for example, the output spectra of one or more components of the light source overlap by 1 nm or more, such as 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, 6 nm or more, 7 nm or more, 8 nm or more, 9 nm or more, 10 nm or more, and including 20 nm or more. In some embodiments, the wavelengths of the light emitted by the light source do not overlap. For example, the output spectra of the light source may be spaced 1 nm or more apart, such as 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, 6 nm or more, 7 nm or more, 8 nm or more, 9 nm or more, 10 nm or more, and including 20 nm or more.

[0068] The light source can illuminate the photodetector at any suitable distance, such as 0.001 mm or greater, for example, 0.005 mm or greater, for example, 0.01 mm or greater, for example, 0.05 mm or greater, for example, 0.1 mm or greater, for example, 0.5 mm or greater, for example, 1 mm or greater, for example, 5 mm or greater, for example, 10 mm or greater, for example, 25 mm or greater, and including at a distance of 100 mm or greater. Furthermore, the photodetector can be illuminated at any suitable angle, for example, the illumination angle ranges from 10° to 90°, for example, from 15° to 85°, for example, from 20° to 80°, for example, from 25° to 75°, and including from 30° to 60°, for example, at 90°.

[0069] In practicing the methods of this subject, a photodetector is illuminated by a light source for two or more discrete time intervals. Herein, the term "discrete time interval" refers to a period of time following a predetermined duration of illumination of the photodetector by a light source, during which parameters of the light source or detector change, followed by subsequent discrete illumination time intervals. In some embodiments, the method includes illuminating the photodetector at discrete time intervals, which are independently 0.1 ms or longer, such as 0.5 ms or longer, such as 1.0 ms or longer, such as 5 ms or longer, such as 10 ms or longer, such as 20 ms or longer, such as 30 ms or longer, such as 40 ms or longer, such as 50 ms or longer, such as 60 ms or longer, such as 70 ms or longer, such as 80 ms or longer, such as 90 ms or longer, and including 100 ms or longer. In some embodiments, the duration of each predetermined time interval for illuminating the photodetector is the same. For example, each predetermined time interval according to the methods of this subject may be 50 ms. In other embodiments, each predetermined time interval is different. In some embodiments, the method includes illuminating a photodetector with a light source at multiple discrete time intervals, such as three or more discrete time intervals, for example, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, twenty-five or more, fifty or more, seventy-five or more, and including 100 or more discrete time intervals. In some embodiments, the duration of each of the multiple time intervals is the same. In other embodiments, the duration of each of the multiple time intervals is different. In other embodiments, some time intervals have the same duration, while some time intervals have different durations.

[0070] In some embodiments, the illumination intensity of the light source is substantially constant over the duration of each predetermined time interval, for example, wherein the variation in illumination intensity is 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 variation of 0.000001% or less in the illumination intensity of the light source over the duration of each predetermined time interval.

[0071] In practicing the methods of this subject, the parameters of the light source or photodetector may change after each discrete illumination interval. In embodiments, the method includes detecting a step signal with a photodetector, wherein the step signal indicates that the parameters of the light source or the photodetector have changed. In some examples, the step signal includes a change in the frequency of the light irradiated by the light source. In some embodiments, the light source is a continuous-wave light source, and the step signal includes irradiating the light source with one or more light pulses. For example, when the photodetector is irradiated by a continuous light source at predetermined time intervals, the step signal may include irradiating the light source with two or more light pulses (e.g., three or more pulses, four or more pulses, five or more pulses, six or more pulses, seven or more pulses, eight or more pulses, nine or more pulses, and including ten or more pulses) to indicate a change in the parameters of the light source or the photodetector at the next predetermined time interval.

[0072] In other embodiments, the photodetector is illuminated by a pulsed light source at predetermined time intervals, and the step signal includes a change in the frequency of the light pulses from the light source. In some examples, the frequency of the light pulses is increased, for example, by 5% or more, by 10% or more, by 25% or more, by 50% or more, by 75% or more, by 90% or more, by 95% or more, by 1.5 times or more, by 2 times or more, by 3 times or more, by 4 times or more, and includes an increase in the frequency of the light pulses by 5 times or more. For example, the frequency of the light pulse can be increased by 0.0001 kHz or more, for example, by 0.0005 kHz or more, for example, by 0.001 kHz or more, for example, by 0.005 kHz or more, for example, by 0.01 kHz or more, for example, by 0.05 kHz or more, for example, by 0.1 kHz or more, for example, by 0.5 kHz or more, for example, by 1 kHz or more, for example, by 2.5 kHz or more, for example, by 5 kHz or more, for example, by 10 kHz or more, for example, by 25 kHz or more, for example, by 10 kHz or more, for example, by 25 kHz or more, for example, by 50 kHz or more, for example, by 75 kHz or more, for example, by 90 kHz or more, for example, by 95 kHz or more, for example, by 2 / 3 or less, for example, by 1 / 2 or less, for example, by 1 / 3 or less, for example, by 1 / 4 or less, and includes the light pulse frequency to 1 / 5 or less. For example, the frequency of the optical pulse can be reduced by 0.0001 kHz or more, such as 0.0005 kHz or more, such as 0.001 kHz or more, such as 0.005 kHz or more, such as 0.01 kHz or more, such as 0.05 kHz or more, such as 0.1 kHz or more, such as 0.5 kHz or more, such as 1 kHz or more, such as 2.5 kHz or more, such as 5 kHz or more, such as 10 kHz or more, such as 25 kHz or more, such as 50 kHz or more, and including a reduction of 100 kHz or more in the frequency of the optical pulse.

[0073] In some embodiments, the step signal includes a change in the duration of a light pulse from the light source. In some examples, the duration of the light pulse is increased, for example, by 5% or more, by 10% or more, by 25% or more, by 50% or more, by 75% or more, by 90% or more, by 95% or more, by 1.5 times or more, by 2 times or more, by 3 times or more, by 4 times or more, and includes increasing the duration of the light pulse by 5 times or more. For example, the duration of the light pulse can be increased by 0.0001 ms or longer, such as by 0.0005 ms or longer, such as by 0.001 ms or longer, such as by 0.005 ms or longer, such as by 0.01 ms or longer, such as by 0.05 ms or longer, such as by 0.1 ms or longer, such as by 0.5 ms or longer, such as by 1 ms or longer, such as by 2.5 ms or longer, such as by 5 ms or longer, such as by 10 ms or longer, such as by 25 ms or longer, such as by 50 ms or longer, and includes increasing the duration of the light pulse by 100 ms or longer. In other examples, the step signal includes a reduction in the duration of the light pulse from the light source, such as a reduction of 5% or more, such as a reduction of 10% or more, such as a reduction of 25% or more, such as a reduction of 50% or more, such as a reduction of 75% or more, such as a reduction of 90% or more, such as a reduction of 95% or more, such as a reduction to 2 / 3 or less, such as a reduction to 1 / 2 or less, such as a reduction to 1 / 3 or less, such as a reduction to 1 / 4 or less, and includes reducing the duration of the light pulse to 1 / 5 or less. For example, the duration of the light pulse can be reduced by 0.0001 ms or more, such as by 0.0005 ms or more, such as by 0.001 ms or more, such as by 0.005 ms or more, such as by 0.01 ms or more, such as by 0.05 ms or more, such as by 0.1 ms or more, such as by 0.5 ms or more, such as by 1 ms or more, such as by 2.5 ms or more, such as by 5 ms or more, such as by 10 ms or more, such as by 25 ms or more, such as by 50 ms or more, and includes reducing the duration of the light pulse by 100 ms or more.

[0074] Figure 1AThe invention describes the detection of a step signal according to certain embodiments, the step signal comprising a change in pulse frequency indicating a change in the light intensity of a light source. During a first illumination interval, a photodetector is illuminated by light with a pulse frequency of v1 (the time between illumination pulses is t1). A step signal with a different pulse frequency v2 (the time between illumination pulses is t2) is detected. In this embodiment, the step signal indicates an increase in the intensity of the light source, and the photodetector detects light with a pulse frequency v3 (the time between illumination pulses is t3) during a second illumination interval.

[0075] Figure 1B The invention describes the detection of a step signal according to certain embodiments, the step signal comprising a change in pulse duration indicating a change in the light intensity of a light source. During a first illumination interval, a photodetector is illuminated by light with a pulse duration of t1. A step signal with a different pulse duration t2 is detected. In this embodiment, the step signal indicates an increase in the intensity of the light source, and the photodetector detects light with a pulse duration of t3 during a second illumination interval.

[0076] Figure 1C The diagram describes the detection of a step signal, comprising a change in pulse frequency indicating a change in the detector gain of a photodetector, according to certain embodiments. A photodetector positioned at a first detector gain voltage V1 is illuminated with light having a pulse frequency v1 (the time between illumination pulses is t1). A step signal with a different pulse frequency v2 (the time between illumination pulses is t2) is detected, indicating that the photodetector gain has changed to voltage V2. The photodetector positioned at detector gain V2 is then illuminated with a light source having a pulse frequency v3 (the time between illumination pulses is t3).

[0077] Figure 1D The diagram describes the detection of a step signal according to certain embodiments, the step signal comprising a change in pulse duration indicating a change in the detector gain of a photodetector. A photodetector positioned at a first detector gain voltage V1 is illuminated with light having a pulse duration of t1. A step signal with a different pulse duration t2 is detected, indicating that the photodetector gain has changed to voltage V2. The photodetector positioned at detector gain V2 is then illuminated with a light source having a pulse duration of t3.

[0078] In some embodiments, the step signal includes detecting a lack of light from the light source. "A lack of light" means that the amount of light detected by the photodetector is indistinguishable from background noise (e.g., electronic noise from the photodetector or the light source) generated by one or more components of the system (described in detail below). In some examples, the light source does not emit light and the photodetector does not detect any light. In other examples, the amount of light emitted by the light source is below the detection threshold of the photodetector, and the light signal cannot be distinguished from the background noise.

[0079] Figure 2A and Figure 2B A method for detecting step signals to indicate changes in light source parameters or photodetector parameters, according to certain embodiments, is described. Figure 2A A step signal is described, wherein a change in the pulse frequency of light detected by a photodetector indicates a change in light source parameters (e.g., an increase in light intensity) or a change in photodetector parameters (e.g., an increase in detector gain voltage). Figure 2B A step signal is described in which no light signal is detected to indicate changes in the parameters of the light source or the photodetector.

[0080] The duration of the step signal can vary. For example, each step signal in the method of this subject can be 0.000001ms or longer, such as 0.000005ms or longer, such as 0.00001ms or longer, such as 0.00005ms or longer, such as 0.0001ms or longer, such as 0.0005ms or longer, such as 0.001ms or longer, such as more than 0.005ms, such as 0.01ms or longer, such as 0.05ms or longer, such as 0.1ms or longer, such as 0.5ms or longer, such as 1ms or longer, such as 2ms or longer, such as 3ms or longer, such as 4ms or longer, such as 5ms or longer, such as 10ms or longer, such as 25ms or longer, such as 50ms or longer, such as 100ms or longer, and including 500ms or longer. For example, the duration of the step signal can be from 0.000001ms to 1000ms, for example from 0.000005ms to 950ms, for example from 0.00001ms to 900ms, for example from 0.00005ms to 850ms, for example from 0.0001ms to 800ms, for example from 0.0005ms to 750ms, for example from 0.001ms to 700ms, for example from 0.005ms to 650ms, for example from 0.01ms to 600ms, for example from 0.05ms to 550ms, for example from 0.1ms to 500ms, for example from 0.5ms to 450ms, for example from 1ms to 400ms, for example from 5ms to 350ms, and includes 10ms to 300ms.

[0081] In some embodiments, the step signal indicates a change in the light intensity of the light source. In some examples, the change in intensity is a decrease in the light intensity of the light source. For example, the step signal may indicate a decrease in the light intensity emitted by the light source, such as a decrease of 5% or more, such as a decrease of 10% or more, such as a decrease of 25% or more, such as a decrease of 50% or more, such as a decrease of 75% or more, such as a decrease of 90% or more, such as a decrease of 95% or more, such as a decrease to 2 / 3 or less, such as a decrease to 1 / 2 or less, such as a decrease to 1 / 3 or less, such as a decrease to 1 / 4 or less, and including a decrease to 1 / 5 or less. In other examples, the change in intensity is an increase in the light intensity of the light source. For example, a step signal can indicate an increase in the intensity of a light source, such as an increase of 5% or more, such as an increase of 10% or more, such as an increase of 25% or more, such as an increase of 50% or more, such as an increase of 75% or more, such as an increase of 90% or more, such as an increase of 95% or more, such as an increase of 1.5 times or more, such as an increase of 2 times or more, such as an increase of 3 times or more, such as an increase of 4 times or more, and including an increase of 5 times or more.

[0082] In other embodiments, the step signal indicates a change in the spectral parameters of the light from the light source. In some examples, the change in spectral parameters is a change in the wavelength of the light emitted by the light source. In one example, the light source is a monochromatic light source, and the step signal indicates that the light from the light source changes from a first wavelength to a second wavelength. In another example, the light source includes multiple monochromatic light sources, and the step signal indicates a change in multiple wavelengths of light from the multiple monochromatic light sources, for example, a change in wavelength of 5% or more, such as 10% or more, such as 20% or more, such as 30% or more, such as 40% or more, such as 50% or more, such as 60% or more, such as 70% or more, such as 80% or more, such as 90% or more, or even a change in all wavelengths of light from the multiple light sources.

[0083] In other examples, the change in spectral parameters is a change in the number of wavelengths of light emitted by the light source. For example, the change in spectral parameters can include an increase in the number of wavelengths emitted by the light source. In some examples, the number of wavelengths emitted by the light source can increase by 5% or more, for example, by 10% or more, for example, by 20% or more, for example, by 30% or more, for example, by 40% or more, for example, by 50% or more, for example, by 60% or more, for example, by 70% or more, for example, by 80% or more, for example, by 90% or more, for example, by 1.5 times or more, for example, by 2 times or more, for example, by 3 times or more, for example, by 4 times or more, for example, by 5 times or more, and including an increase of 10 times or more. In other embodiments, the change in spectral parameters includes a decrease in the number of wavelengths emitted by the light source. In some instances, the number of wavelengths emitted by the light source may be reduced by 5% or more, for example, by 10% or more, for example, by 20% or more, for example, by 30% or more, for example, by 40% or more, for example, by 50% or more, for example, by 60% or more, for example, by 70% or more, for example, by 80% or more, for example, by 90% or more, for example, by 2 / 3 or less, for example, by 1 / 2 or less, for example, by 1 / 3 or less, for example, by 1 / 4 or less, for example, by 1 / 5 or less, and including by 1 / 10 or less.

[0084] In some embodiments, the variation in the number of wavelengths of light emitted by the light source includes a variation in the spectral width emitted by the light source. In some examples, the spectral width emitted by the light source may increase by 5 nm or more, for example, by 10 nm or more, for example, by 25 nm or more, for example, by 50 nm or more, for example, by 100 nm or more, for example, by 250 nm or more, and includes an increase of 500 nm or more. In other examples, the spectral width emitted by the light source may decrease by 5 nm or more, for example, by 10 nm or more, for example, by 25 nm or more, for example, by 50 nm or more, for example, by 100 nm or more, for example, by 250 nm or more, and includes a decrease of 500 nm or more.

[0085] In these embodiments, the number of wavelengths emitted by the light source (e.g., spectral width) can be varied using any convenient method. In some embodiments, the number of wavelengths emitted by the light source is varied by changing the number of monochromatic light emitters (e.g., single-wavelength LEDs or lasers) in the light source. For example, one or more monochromatic light emitters (e.g., LEDs) of the light source can be turned on or off to change the number of wavelengths of light emitted by the light source. In other embodiments, the number of wavelengths detected by the photodetector can be varied, for example by using a bandpass filter, a cutoff filter, or a dichroic mirror. In other embodiments, a monochromator, a prism, or a diffraction grating can be used to vary the number of wavelengths detected by the photodetector.

[0086] In some embodiments, the step signal indicates a change in a parameter of the photodetector. In some embodiments, this parameter is a change in detector gain. In some examples, the change in detector gain is a decrease in the detector gain of the photodetector. For example, the step signal may indicate a decrease in the detector gain of the photodetector, such as a decrease of 5% or more, such as a decrease of 10% or more, such as a decrease of 25% or more, such as a decrease of 50% or more, such as a decrease of 75% or more, such as a decrease of 90% or more, such as a decrease of 95% or more, such as a decrease to 2 / 3 or less, such as a decrease to 1 / 2 or less, such as a decrease to 1 / 3 or less, such as a decrease to 1 / 4 or less, and including a decrease to 1 / 5 or less. In some instances, the step signal indicates a decrease in detector gain of 0.001 mV or more, for example, a decrease of 0.005 mV or more, for example, a decrease of 0.01 mV or more, for example, a decrease of 0.05 mV or more, for example, a decrease of 0.1 mV or more, for example, a decrease of 0.5 mV or more, for example, a decrease of 1 mV or more, for example, a decrease of 5 mV or more, for example, a decrease of 10 mV or more, for example, a decrease of 25 mV or more, for example, a decrease of 50 mV or more, for example, a decrease of 100 mV or more, for example, a decrease of 250 mV or more, for example, a decrease of 500 mV or more, for example, a decrease of 1000 mV or more, for example, a decrease of 2500 mV or more, and a decrease of 5000 mV or more. In some embodiments, the step signal indicates a reduction of detector gain by 1V or more, such as 2V or more, 5V or more, 10V or more, 25V or more, 50V or more, 100V or more, 200V or more, 300V or more, 400V or more, 500V or more, and including a reduction of 600V or more.

[0087] In other embodiments, the step signal indicates an increase in the detector gain of the photodetector, for example, an increase of 5% or more, for example, an increase of 10% or more, for example, an increase of 25% or more, for example, an increase of 50% or more, for example, an increase of 75% or more, for example, an increase of 90% or more, for example, an increase of 95% or more, for example, an increase of 1.5 times or more, for example, an increase of 2 times or more, for example, an increase of 3 times or more, for example, an increase of 4 times or more, and including an increase of 5 times or more. In some instances, the step signal indicates an increase in detector gain of 0.001 mV or more, for example, an increase of 0.005 mV or more, for example, an increase of 0.01 mV or more, for example, an increase of 0.05 mV or more, for example, an increase of 0.1 mV or more, for example, an increase of 0.5 mV or more, for example, an increase of 1 mV or more, for example, an increase of 5 mV or more, for example, an increase of 10 mV or more, for example, an increase of 25 mV or more, for example, an increase of 50 mV or more, for example, an increase of 100 mV or more, for example, an increase of 250 mV or more, for example, an increase of 500 mV or more, for example, an increase of 1000 mV or more, for example, an increase of 2500 mV or more, and includes an increase of 5000 mV or more. In some embodiments, the step signal represents an increase in detector gain of 1V or more, such as an increase of 2V or more, such as an increase of 5V or more, such as an increase of 10V or more, such as an increase of 25V or more, such as an increase of 50V or more, such as an increase of 100V or more, such as an increase of 200V or more, such as an increase of 300V or more, such as an increase of 400V or more, such as an increase of 500V or more, and including an increase of 600V or more.

[0088] In the subject method according to some embodiments, the intensity of the light source changes after each discrete illumination interval. In some embodiments, the illumination intensity of the light source increases. In other embodiments, the intensity of the light source decreases. At each subsequent time interval, the intensity of the light used to illuminate the photodetector may change by 5% or more, for example, by 10% or more, for example, by 25% or more, for example, by 50% or more, for example, by 75% or more, for example, by 90% or more, and including a change of 100% or more. In some examples, the light intensity change is 1.5 times or more, for example, by 2 times or more, for example, by 3 times or more, for example, by 4 times or more, and including a change of 5 times or more. In some embodiments, the method includes increasing the light intensity in each subsequent time interval, for example, by increasing the light intensity in each subsequent time interval by 5% or more, for example, by 10% or more, for example, by 25% or more, for example, by 50% or more, for example, by 75% or more, for example, by 90% or more, and including an increase of 100% or more. In other embodiments, the method includes increasing the light intensity in each subsequent time interval by 1.5 times or more, for example, by 2 times or more, for example, by 3 times or more, for example, by 4 times or more, and including increasing it by 5 times or more.

[0089] In some embodiments, the method of this subject includes a plurality of discrete illumination intervals, each of which may be separated by the aforementioned step signal. In other words, a step signal is detected before the start of another discrete illumination interval. In some embodiments, the step signal indicates a change in parameters of the light source (e.g., wavelength range or intensity) or parameters of the photodetector (e.g., detector gain) between each discrete illumination interval. In other embodiments, the step signal indicates that the parameters of the light source or the parameters of the photodetector do not change. In some embodiments, the method includes detecting the step signal after every two or more discrete illumination intervals, for example, after every three or more discrete illumination intervals, for example, after every four or more discrete illumination intervals, for example, after every five or more discrete illumination intervals, and after every ten or more discrete illumination intervals. In some embodiments, the plurality of discrete illumination intervals are randomly separated by the step signal (i.e., discrete illumination intervals without a regular pattern between the step signals).

[0090] In some embodiments, a tag signal is inserted into a data signal over one or more predetermined time intervals. In some examples, the tag signal is a frequency-modulated (FM) data signal that can be detected and processed during the integration of the data signal of the photodetector at each predetermined time interval. The tag signal can be inserted at any point in time within one or more predetermined time intervals, for example, at the beginning of data signal acquisition for each predetermined time interval. In other embodiments, the tag signal is inserted at the end of data signal acquisition for each predetermined time interval. In other embodiments, the tag signal is inserted at a point in time that is 10% or more, such as 25% or more, such as 50% or more, and including 75% or more of the predetermined time interval. In embodiments, the tag signal can be a unique identifier of the predetermined time interval. For example, the tag signal can indicate a specific detector gain or light source intensity step used to determine one or more parameters of the photodetector. In some embodiments, the step signal indicating a change in the parameters of the light source or the photodetector (as described above) is precisely the tag signal inserted into the measurement data signal over one or more predetermined time intervals.

[0091] In some embodiments, the method includes detecting a synchronization signal before detecting light from the light source, prior to a first discrete illumination time interval. In some embodiments, the synchronization signal includes detecting light from the light source having a predetermined pulse frequency. In other embodiments, the synchronization signal includes detecting light from the light source having an intensity exceeding a predetermined intensity threshold. In still other embodiments, the synchronization signal includes detecting light from the light source at its maximum light intensity.

[0092] As described above, the method of this disclosure includes detecting light using a photodetector. The photodetector used to practice the methods of this subject matter can be any convenient light detection scheme, including but not limited to photoelectric sensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhancement charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or combinations thereof, and other photodetectors. In some embodiments, the photodetector is a photomultiplier tube, such as a photomultiplier tube having an effective detection surface area region, for example, each region ranging from 0.01 cm. 2 up to 10cm 2 For example, from 0.05cm 2 up to 9cm 2 For example, from 0.1cm 2 up to 8cm 2 For example, from 0.5cm 2 up to 7cm2 And including from 1cm 2 up to 5cm 2 .

[0093] In embodiments of this disclosure, light can be measured using 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 including measuring light from particles in a flowing stream at 400 or more different wavelengths.

[0094] In embodiments, light can be measured continuously or at discrete intervals. In some examples, the detector of interest is configured to measure light continuously. In other examples, the detector of interest 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 other intervals.

[0095] At each discrete time interval, the light from the light source can be measured one or more times, such as two or more times, three or more times, five or more times, and including ten or more times. In some embodiments, the photodetector measures the light from the light source two or more times, and in some examples, the data are averaged.

[0096] Figure 3AA flowchart illustrating the determination of one or more parameters of a photodetector according to certain embodiments is depicted. In step 301, the photodetector detects a synchronization signal. In some embodiments, the synchronization signal detected by the photodetector includes detecting light whose intensity exceeds a predetermined threshold. In some examples, the detected synchronization signal is light from a light source operating at maximum intensity. In step 302, the photodetector is illuminated by a light source of a first intensity for a first predetermined discrete time interval. In step 303, a step signal is detected. In some examples, the step signal includes detecting light having a pulse frequency different from the pulse frequency of the light used to illuminate the photodetector in step 302. For example, the photodetector may be illuminated with a first frequency during the first time interval, while the step signal includes detecting light with a frequency different from the first frequency (e.g., a higher frequency). In other examples, the photodetector may be illuminated with a continuous wave light source during the first time interval, while the step signal includes detecting pulsed light. In step 304, the photodetector is illuminated by a light source of a second intensity for a second predetermined discrete time interval. In step 305, the data signal from the photodetector is integrated over a time period comprising at least a portion of the first and second time intervals. In step 306, the signal amplitude from the photodetector is calculated based on the integrated data signal. In step 307, the calculated signal amplitude is used to determine one or more parameters, such as comparing the calculated signal amplitude with the light intensity during each illumination interval. For example, comparing the calculated signal amplitude with the light intensity during each illumination interval can determine a minimum detection threshold 307a, a maximum detection threshold 307b, a detector sensitivity 307c, a detector dynamic range 307d, a detector signal-to-noise ratio 307e, or the number of photoelectrons per unit output 307f.

[0097] Figure 3B A flowchart illustrating the determination of parameters for a photodetector used in a particle analyzer according to certain embodiments is described. In step 311, the system is configured to instruct a light source to illuminate the photodetector to generate an optical signal. In step 312, the light source illuminates the photodetector at multiple step-incremental light intensities and the voltage gain of the photodetector to determine the parameters of the photodetector. In step 312, each illumination cycle may include a step signal indicating a change in the light source intensity or a change in the detector gain of the photodetector. This step signal may be a change in the light source pulse frequency as described above. The system (described in detail below) includes a processor with a memory having software instructions for analyzing each illumination cycle and the determined parameters of the photodetector (step 313). One or more illumination steps may be repeated or re-acquired to optimize desired parameters of the photodetector (e.g., photodetector sensitivity, detector dynamic range, or detector signal-to-noise ratio).

[0098] Figure 3C A flowchart illustrating the determination of parameters for a photodetector in a particle analyzer according to certain embodiments is provided. The detector gain of the photodetector (e.g., an avalanche photodiode APD) is set to a first voltage level, and the light source (LED) is set to a first frequency synchronized with the first detector gain setting. The photodetector detects the synchronization signal from the light source and initiates data acquisition. The light source is programmed to include changes in intensity (e.g., a gradual increase in intensity). Changes in light intensity and changes in light pulse frequency are encoded as step signals. The light source then illuminates the photodetector for a predetermined duration to detect the programmed step signal. The intensity of the light source is changed, and the photodetector detects the light at the new intensity at predetermined time intervals. These steps can be repeated one or more times at multiple light intensities, such as two or more, three or more, four or more, five or more, ten or more, 25 or more, and including repeating these steps 50 or more times at a desired number of light intensities. Each of the above steps can be repeated at different detector gains. In some embodiments, the steps are repeated over the entire operating voltage range of the photodetector. Figure 3C The steps described herein allow for the determination of photodetector parameters based on data signals acquired at multiple light intensities and measured detector gains.

[0099] In some embodiments, the method includes integrating a data signal from a photodetector. In some embodiments, integrating the data signal from the photodetector includes integrating the data signal for 10% or more of the duration of each discrete illumination interval, such as 15% or more, 20% or more, 25% or more, 50% or more, 75% or more, 90% or more, and more than 99% of the duration of each discrete illumination interval. In some embodiments, the data signal from the photodetector is integrated over the entire duration of each illumination time interval according to the method of this subject matter.

[0100] In some embodiments, the method includes integrating the data signal from the photodetector over a time period comprising at least each discrete time interval of illumination at each different light intensity. For example, when the photodetector is illuminated by a light source for more than 50 or more discrete intervals, the method includes integrating the data signal from the photodetector over a time period comprising at least a portion of the duration of those 50 discrete time intervals. In some embodiments, the method includes integrating the data signal from the photodetector over a time period comprising the duration prior to illuminating the photodetector according to the method of this subject matter, in order to measure the noise component of the photodetector signal. In these embodiments, the method includes integrating the data signal from the photodetector at a time 0.001 ms or earlier before illuminating the photodetector, for example, 0.005 ms or longer, for example, 0.01 ms or longer, for example, 0.05 ms or longer, for example, 0.1 ms or longer, for example, 0.5 ms or longer, for example, 1 ms or longer, for example, 2 ms or longer, for example, 3 ms or longer, for example, 4 ms or longer, for example, 5 ms or longer, for example, 10 ms or longer, for example, 25 ms or longer, for example, 50 ms or longer, for example, 100 ms or longer, and 250 ms or earlier before illuminating the photodetector. In other embodiments, the method includes integrating the data signal from the photodetector after the last discrete illumination time interval, for example, 0.005 ms or longer, for example, 0.01 ms or longer, for example, 0.05 ms or longer, for example, 0.1 ms or longer, for example, 0.5 ms or longer, for example, 1 ms or longer, for example, 2 ms or longer, for example, 3 ms or longer, for example, 4 ms or longer, for example, 5 ms or longer, for example, 10 ms or longer, for example, 25 ms or longer, for example, 50 ms or longer, for example, 100 ms or longer, and including 250 ms or longer.

[0101] In some embodiments, integrating the data signal from the photodetector includes calculating the signal amplitude over a time period. In some examples, calculating the signal amplitude includes calculating the median signal amplitude. In some examples, the method further includes comparing the calculated signal amplitude with the light intensity of the light source. Based on one or more calculated signal amplitudes 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 determining 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 detector signals from the minimum detection threshold to the maximum detection threshold), a detector signal-to-noise ratio, or the number of photoelectrons per unit output.

[0102] Individual detector parameters can be determined within the operating voltage range of the photodetector. In some embodiments, the parameters are determined based on the signal amplitude calculated 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, 90% or more, and 99% or more. In some examples, the individual parameters can be determined across the entire operating voltage range of the photodetector.

[0103] In some examples, the parameters of the photodetector can be adjusted based on one or more calculated signal amplitudes or a comparison between the calculated signal amplitude and the illumination intensity at each discrete time interval. Here, the term "adjustment" refers to changing one or more functional parameters of the photodetector in a conventional sense. For example, adjusting the photodetector may include increasing or decreasing the voltage gain of the photodetector. In some embodiments, adjusting one or more parameters of the photodetector based on calculated signal amplitudes or a comparison between the calculated signal amplitude and the illumination intensity at each discrete time interval of interest can be fully automated, requiring minimal user intervention or manual input.

[0104] System for determining the parameters of a photodetector

[0105] Various aspects of this disclosure also include a system having a light source and a photodetector, wherein the photodetector is configured to detect light from the light source at different illumination intensities at predetermined time intervals. In an embodiment, the system includes a light source, a photodetector, and a processor, wherein the photodetector is configured to detect light from the light source at a first predetermined time interval, detect a step signal indicating a change in parameters of the light source or a change in parameters of the photodetector, and detect light from the light source at a second predetermined time interval. The processor has 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 data signals from the photodetector at the first and second predetermined time intervals, and determine one or more parameters of the photodetector based on the integrated data signals.

[0106] In some embodiments, the system is configured to illuminate a photodetector with a pulsed light source at one or more predetermined time intervals. In embodiments, the light source can be any convenient light source and can include 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 range of wavelengths. In some examples, the narrowband source emits light with a narrow wavelength range, such as 50 nm or narrower, 40 nm or narrower, 30 nm or narrower, 25 nm or narrower, 20 nm or narrower, 15 nm or narrower, 10 nm or narrower, 5 nm or narrower, 2 nm or narrower, and includes a source emitting light of a specific wavelength (i.e., monochromatic light). Any convenient narrowband source solution can be used, such as a narrow-wavelength LED.

[0107] 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 examples, the broadband light source emits light with a wide wavelength range, for example, 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 is a light source that emits light with wavelengths from 400 nm to 1000 nm. Any convenient broadband light source solution can be used, such as halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with continuous spectrum, superluminescent diodes, semiconductor light-emitting diodes, broad-spectrum 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 includes an LED array. In some examples, the light source includes multiple monochromatic light-emitting diodes, wherein each monochromatic light-emitting diode outputs light with a different wavelength. In some examples, the light source includes multiple multicolor light-emitting diodes (LEDs) that output light with a predetermined spectral width, such as the spectral width of the light jointly output by the multiple multicolor LEDs ranging from 200nm to 1500nm, for example from 225nm to 1475nm, for example from 250nm to 1450nm, for example from 275nm to 1425nm, for example from 300nm to 1400nm, for example from 325nm to 1375nm, for example from 350nm to 1350nm, for example from 375nm to 1325nm, for example from 400nm to 1300nm, for example from 425nm to 1275nm, for example from 450nm to 1250nm, for example from 475nm to 1225nm, and including 500nm to 1200nm.

[0108] In some embodiments, the light source is a laser, such as a pulsed laser or a continuous-wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some examples, 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) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other examples, the system includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In other examples, lasers of interest 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 examples, the subject matter 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, ytterbium YAG lasers, ytterbium₂O₃ lasers, or cerium-doped lasers, and combinations thereof.

[0109] In some embodiments, the light source is a pulsed light source. As described above, the term "pulsed light source" herein refers in the conventional sense to a light source that emits light at predetermined time intervals, each time interval having a predetermined illumination duration (i.e., pulse width). In some embodiments, the pulsed light source is configured to illuminate a photodetector with periodic flashes. For example, the frequency of each light pulse can be 0.0001 kHz or higher, such as 0.0005 kHz or higher, such as 0.001 kHz or higher, such as 0.005 kHz or higher, such as 0.01 kHz or higher, such as 0.05 kHz or higher, such as 0.1 kHz or higher, such as 0.5 kHz or higher, such as 1 kHz or higher, such as 2.5 kHz or higher, such as 5 kHz or higher, such as 10 kHz or higher, such as 25 kHz or higher, such as 50 kHz or higher, and including 100 kHz or higher. In some examples, the pulse irradiation frequency of the light source ranges from 0.00001 kHz to 1000 kHz, for example from 0.00005 kHz to 900 kHz, for example from 0.0001 kHz to 800 kHz, for example from 0.0005 kHz to 700 kHz, for example from 0.001 kHz to 600 kHz, for example from 0.005 kHz to 500 kHz, for example from 0.01 kHz to 400 kHz, for example from 0.05 kHz to 300 kHz, for example from 0.1 kHz to 200 kHz, and includes 1 kHz to 100 kHz. The duration of illumination (i.e., pulse width) of each light pulse can vary and can be 0.000001ms or longer, for example, 0.000005ms or longer, for example, 0.00001ms or longer, for example, 0.00005ms or longer, for example, 0.0001ms or longer, for example, 0.0005ms or longer, for example, 0.001ms or longer, for example, 0.005ms or longer, for example, 0.01ms or longer, for example, 0.05ms or longer, for example, 0.1ms or longer, for example, 0.5ms or longer, for example, 1ms or longer, for example, 2ms or longer, for example, 3ms or longer, for example, 4ms or longer, for example, 5ms or longer, for example, 10ms or longer, for example, 25ms or longer, for example, 50ms or longer, for example, 100ms or longer, and including 500ms or longer.For example, the duration of light irradiation can be from 0.000001ms to 1000ms, for example from 0.000005ms to 950ms, for example from 0.00001ms to 900ms, for example from 0.00005ms to 850ms, for example from 0.0001ms to 800ms, for example from 0.0005ms to 750ms, for example from 0.001ms to 700ms, for example from 0.005ms to 650ms, for example from 0.01ms to 600ms, for example from 0.05ms to 550ms, for example from 0.1ms to 500ms, for example from 0.5ms to 450ms, for example from 1ms to 400ms, for example from 5ms to 350ms, and includes from 10ms to 300ms.

[0110] In some embodiments, the light source is a continuous-wave light source. The term "continuous-wave light source" refers to a light source that provides 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 substantially constant emitted light intensity. For example, during the interval of illumination, the continuous light source can provide emitted light intensity with very small variations, such variations being 10% or less, for example 9% or less, for example 8% or less, for example 7% or less, for example 6% or less, for example 5% or less, for example 4% or less, for example 3% or less, for example 2% or less, for example 1% or less, for example 0.5% or less, for example 0.1% or less, for example 0.01% or less, for example 0.001% or less, for example 0.0001% or less, for example 0.00001% or less, and including 0.000001% or less.

[0111] In some embodiments, the light source is a narrow bandwidth light source. In some examples, the light source is a light source that outputs a specific wavelength, which is from 200nm to 1500nm, for example from 250nm to 1250nm, for example from 300nm to 1000nm, for example from 350nm to 900nm, and includes wavelengths from 400nm to 800nm. In some embodiments, the continuous wave light source emits light with wavelengths of 365nm, 385nm, 405nm, 460nm, 490nm, 525nm, 550nm, 580nm, 635nm, 660nm, 740nm, 770nm, or 850nm.

[0112] In some embodiments, the light emitted by the light source has overlapping wavelengths, for example, the output spectra of one or more components of the light source overlap by 1 nm or more, for example, overlap by 2 nm or more, for example, overlap by 3 nm or more, for example, overlap by 4 nm or more, for example, overlap by 5 nm or more, for example, overlap by 6 nm or more, for example, overlap by 7 nm or more, for example, overlap by 8 nm or more, for example, overlap by 9 nm or more, for example, overlap by 10 nm or more, and including overlap by 20 nm or more. In some embodiments, the wavelengths of the light emitted by the light source do not overlap. For example, the output spectra of the light source may be spaced by 1 nm or more, for example, spaced by 2 nm or more, for example, spaced by 3 nm or more, for example, spaced by 4 nm or more, for example, spaced by 5 nm or more, for example, spaced by 6 nm or more, for example, spaced by 7 nm or more, for example, spaced by 8 nm or more, for example, spaced by 9 nm or more, for example, spaced by 10 nm or more, and including overlap by 20 nm or more.

[0113] In the system of interest, the light source can be located at any suitable distance from the photodetector, such as at a distance of 0.001 mm or greater, for example, 0.005 mm or greater, for example, 0.01 mm or greater, for example, 0.05 mm or greater, for example, 0.1 mm or greater, for example, 0.5 mm or greater, for example, 1 mm or greater, for example, 5 mm or greater, for example, 10 mm or greater, for example, 25 mm or greater, and including distances of 100 mm or greater. Furthermore, the photodetector can be positioned at any suitable angle to the light source, for example, angles in the range of 10° to 90°, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and including 30° to 60°, for example, at 90°.

[0114] According to some embodiments, the light source may also include one or more optical adjustment components. Hereinafter, the term "optical adjustment" refers in the conventional sense to any device capable of altering the spatial width of illumination or other characteristics of the light source's illumination (e.g., illumination direction, wavelength, beam width, beam intensity, and focal spot). Optical adjustment schemes can be any convenient device used to adjust 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, the system of interest includes one or more focusing lenses. In one example, the focusing lens may be a non-magnifying lens. In another example, the focusing lens is a magnifying lens. In other embodiments, the system of interest includes one or more mirrors. In other embodiments, the system of interest includes optical fibers.

[0115] When the optical adjustment component is configured to move, it can be configured to move continuously or at discrete intervals. In some embodiments, the optical adjustment component moves continuously. In other embodiments, the optical adjustment component moves at discrete intervals, such as in increments of 0.01 μm or greater, such as 0.05 μm or greater, such as 0.1 μm or greater, such as 0.5 μm or greater, such as 1 μm or greater, such as 10 μm or greater, such as 100 μm or greater, such as 500 μm 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.

[0116] Any displacement scheme can be used to move the optical adjustment component structure, such as coupling to a movable support platform or directly coupling to a motor-driven translation platform, lead screw translation component, gear transmission translation device, etc. These motors are, for example, stepper motors, servo motors, brushless motors, brushed DC motors, micro-stepper drive motors, high-resolution stepper motors, etc.

[0117] The photodetector of this system can be any convenient light detection scheme, including but not limited to photoelectric sensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), enhancement charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or combinations thereof, and other photodetectors. In some embodiments, the photodetector is a photomultiplier tube, which is a photomultiplier tube with an effective detection surface area region, for example, each region ranging from 0.01 cm. 2 up to 10cm 2 For example, from 0.05cm 2 up to 9cm 2 For example, from 0.1cm 2 up to 8cm 2 For example, from 0.5cm 2 up to 7cm 2 And including from 1cm 2 up to 5cm 2 .

[0118] In embodiments of this disclosure, the photodetector can be configured to detect light of one or more wavelengths, such as two or more wavelengths, such as five or more different wavelengths, such as ten or more different wavelengths, such as 25 or more different wavelengths, such as 50 or more different wavelengths, such as 100 or more different wavelengths, such as 200 or more different wavelengths, such as 300 or more different wavelengths, and includes measuring light of 400 or more different wavelengths.

[0119] In embodiments, the photodetector can be configured to measure light continuously or at discrete intervals. In some examples, the detector of interest is configured to measure light continuously. In other examples, the detector of interest 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 other intervals.

[0120] A photodetector can be configured to measure light from a light source one or more times at each discrete time interval, such as two or more times, three or more times, five or more times, and including ten or more times. In some embodiments, the photodetector measures light from the light source two or more times, and in some examples, averages the data.

[0121] In some embodiments, the light source is configured to illuminate two or more discrete time intervals, each time interval having a different illumination intensity. In some embodiments, the light source is configured to illuminate a certain time interval at a specific intensity, the time interval being 0.1 ms or longer, for example 0.5 ms or longer, for example 1.0 ms or longer, for example 5 ms or longer, for example 10 ms or longer, for example 20 ms or longer, for example 30 ms or longer, for example 40 ms or longer, for example 50 ms or longer, for example 60 ms or longer, for example 70 ms or longer, for example 80 ms or longer, for example 90 ms or longer, and including 100 ms or longer. For example, the light source may be configured to illuminate for 50 ms at a specific light intensity.

[0122] In some embodiments, the duration of each predetermined time interval used to illuminate the photodetector is the same. In other embodiments, each predetermined time interval is different. In some embodiments, the system is configured to illuminate the photodetector with a light source at multiple discrete time intervals, such as three or more discrete time intervals, for example, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, twenty-five or more, fifty or more, seventy-five or more, and including 100 or more discrete time intervals. In some embodiments, the duration of each of the multiple time intervals is the same. In other embodiments, the duration of each of the multiple time intervals is different. In other embodiments, some time intervals have the same duration, while some time intervals have different durations.

[0123] In some embodiments, the illumination intensity of the light source is substantially constant over the duration of each predetermined time interval, for example, wherein the variation in illumination intensity is 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 0.000001% or less.

[0124] The system of interest includes a processor having memory operatively coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to detect a step signal indicating a change in parameters of a light source or a photodetector. In some examples, the step signal comprises a change in the frequency of light pulses from the light source. In some embodiments, the light source is a continuous wave source, and the step signal comprises one or more light pulses.

[0125] In some embodiments, the step signal comprises a change in the frequency of a light pulse from a light source. In some examples, the frequency of the light pulse increases, for example, by 5% or more, by 10% or more, by 25% or more, by 50% or more, by 75% or more, by 90% or more, by 95% or more, by 1.5 times or more, by 2 times or more, by 3 times or more, by 4 times or more, and includes an increase of 5 times or more. For example, the frequency of the light pulse may increase by 0.0001 kHz or more, for example, by 0.0005 kHz or more, by 0.001 kHz or more, by 0.005 kHz or more, by 0.01 kHz or more, by 0.05 kHz or more, by 0.1 kHz or more, by 0.5 kHz or more, by 1 kHz or more, by 2.5 kHz or more, by 5 kHz or more, by 10 kHz or more, by 25 kHz or more, by 50 kHz or more, and includes an increase of 100 kHz or more. In other examples, the step signal includes a reduction in the frequency of the light pulse from the light source, such as a reduction of 5% or more, such as a reduction of 10% or more, such as a reduction of 25% or more, such as a reduction of 50% or more, such as a reduction of 75% or more, such as a reduction of 90% or more, such as a reduction of 95% or more, such as a reduction to 2 / 3 or less, such as a reduction to 1 / 2 or less, such as a reduction to 1 / 3 or less, such as a reduction to 1 / 4 or less, and includes a reduction in the light pulse frequency to 1 / 5 or less. For example, the frequency of the optical pulse can be reduced by 0.0001 kHz or more, such as 0.0005 kHz or more, such as 0.001 kHz or more, such as 0.005 kHz or more, such as 0.01 kHz or more, such as 0.05 kHz or more, such as 0.1 kHz or more, such as 0.5 kHz or more, such as 1 kHz or more, such as 2.5 kHz or more, such as 5 kHz or more, such as 10 kHz or more, such as 25 kHz or more, such as 50 kHz or more, and including a reduction of 100 kHz or more in the frequency of the optical pulse.

[0126] In some embodiments, the step signal includes a change in the duration of a light pulse from the light source. In some examples, the duration of the light pulse is increased, for example, by 5% or more, by 10% or more, by 25% or more, by 50% or more, by 75% or more, by 90% or more, by 95% or more, by 1.5 times or more, by 2 times or more, by 3 times or more, by 4 times or more, and includes increasing the duration of the light pulse by 5 times or more. For example, the duration of the light pulse can be increased by 0.0001 ms or longer, for example, by 0.0005 ms or longer, for example, by 0.001 ms or longer, for example, by 0.005 ms or longer, for example, by 0.01 ms or longer, for example, by 0.05 ms or longer, for example, by 0.1 ms or longer, for example, by 0.5 ms or longer, for example, by 1 ms or longer, for example, by 2.5 ms or longer, for example, by 5 ms or longer, for example, by 10 ms or longer, for example, by 25 ms or longer, for example, by 50 ms or longer, and the duration of the light pulse can be increased by 100 ms or longer. In other examples, the step signal includes a reduction in the duration of the light pulse from the light source, such as a reduction of 5% or more, such as a reduction of 10% or more, such as a reduction of 25% or more, such as a reduction of 50% or more, such as a reduction of 75% or more, such as a reduction of 90% or more, such as a reduction of 95% or more, such as a reduction to 2 / 3 or less, such as a reduction to 1 / 2 or less, such as a reduction to 1 / 3 or less, such as a reduction to 1 / 4 or less, and includes reducing the duration of the light pulse to 1 / 5 or less. For example, the duration of the light pulse can be reduced by 0.0001 ms or more, such as by 0.0005 ms or more, such as by 0.001 ms or longer, such as by 0.005 ms or longer, such as by 0.01 ms or longer, such as by 0.05 ms or longer, such as by 0.1 ms or longer, such as by 0.5 ms or longer, such as by 1 ms or longer, such as by 2.5 ms or longer, such as by 5 ms or longer, such as by 10 ms or longer, such as by 25 ms or longer, such as by 50 ms or longer, and includes a reduction of 100 ms or longer in the duration of the light pulse. In some embodiments, the step signal represents the absence of light from the light source.

[0127] In some embodiments, the step signal indicates a change in the light intensity of the light source. In some examples, the change in intensity is a decrease in the light intensity of the light source. For example, the step signal may indicate a decrease in the light intensity emitted by the light source, such as a decrease of 5% or more, such as a decrease of 10% or more, such as a decrease of 25% or more, such as a decrease of 50% or more, such as a decrease of 75% or more, such as a decrease of 90% or more, such as a decrease of 95% or more, such as a decrease to 2 / 3 or less, such as a decrease to 1 / 2 or less, such as a decrease to 1 / 3 or less, such as a decrease to 1 / 4 or less, and including a decrease to 1 / 5 or less. In other examples, the change in intensity is an increase in the light intensity of the light source. For example, a step signal can indicate an increase in the intensity of a light source, such as an increase of 5% or more, such as an increase of 10% or more, such as an increase of 25% or more, such as an increase of 50% or more, such as an increase of 75% or more, such as an increase of 90% or more, such as an increase of 95% or more, such as an increase of 1.5 times or more, such as an increase of 2 times or more, such as an increase of 3 times or more, such as an increase of 4 times or more, and including an increase of 5 times or more.

[0128] In other embodiments, the step signal indicates a change in the spectral parameters of the light emitted by the light source. In some examples, the change in spectral parameters is a change in the wavelength of the light emitted by the light source. In one example, the light source is a monochromatic light source, and the step signal indicates that the light from the light source changes from a first wavelength to a second wavelength. In another example, the light source includes multiple monochromatic light sources, and the step signal indicates a change in multiple wavelengths of light from the multiple monochromatic light sources, for example, the wavelength change is 5% or more, such as 10% or more, such as 20% or more, such as 30% or more, such as 40% or more, such as 50% or more, such as 60% or more, such as 70% or more, such as 80% or more, such as 90% or more, or even includes a change in all wavelengths of light from the multiple light sources.

[0129] In other examples, the change in spectral parameters is a change in the number of wavelengths of light emitted by the light source. For example, the change in spectral parameters can include an increase in the number of wavelengths emitted by the light source. In some examples, the number of wavelengths emitted by the light source can increase by 5% or more, for example, by 10% or more, for example, by 20% or more, for example, by 30% or more, for example, by 40% or more, for example, by 50% or more, for example, by 60% or more, for example, by 70% or more, for example, by 80% or more, for example, by 90% or more, for example, by 1.5 times or more, for example, by 2 times or more, for example, by 3 times or more, for example, by 4 times or more, for example, by 5 times or more, and including an increase of 10 times or more. In other embodiments, the change in spectral parameters includes a decrease in the number of wavelengths emitted by the light source. In some instances, the number of wavelengths emitted by the light source may be reduced by 5% or more, for example, by 10% or more, for example, by 20% or more, for example, by 30% or more, for example, by 40% or more, for example, by 50% or more, for example, by 60% or more, for example, by 70% or more, for example, by 80% or more, for example, by 90% or more, for example, by 2 / 3 or less, for example, by 1 / 2 or less, for example, by 1 / 3 or less, for example, by 1 / 4 or less, for example, by 1 / 5 or less, and including by 1 / 10 or less.

[0130] In some embodiments, the variation in the number of wavelengths of light emitted by the light source includes a variation in the spectral width emitted by the light source. In some examples, the spectral width emitted by the light source may increase by 5 nm or more, for example, by 10 nm or more, for example, by 25 nm or more, for example, by 50 nm or more, for example, by 100 nm or more, for example, by 250 nm or more, and includes an increase of 500 nm or more. In other examples, the spectral width emitted by the light source may decrease by 5 nm or more, for example, by 10 nm or more, for example, by 25 nm or more, for example, by 50 nm or more, for example, by 100 nm or more, for example, by 250 nm or more, and includes a decrease of 500 nm or more.

[0131] In some embodiments, the step signal indicates a change in a parameter of the photodetector. In some embodiments, this parameter is a change in the detector gain of the photodetector. In some examples, the change in detector gain is a decrease in the detector gain of the photodetector. For example, the step signal may indicate a decrease in the detector gain of the photodetector, such as a decrease of 5% or more, such as a decrease of 10% or more, such as a decrease of 25% or more, such as a decrease of 50% or more, such as a decrease of 75% or more, such as a decrease of 90% or more, such as a decrease of 95% or more, such as a decrease to 2 / 3 or less, such as a decrease to 1 / 2 or less, such as a decrease to 1 / 3 or less, such as a decrease to 1 / 4 or less, and including a decrease to 1 / 5 or less. In some instances, the step signal indicates a decrease in detector gain of 0.001 mV or more, for example, a decrease of 0.005 mV or more, for example, a decrease of 0.01 mV or more, for example, a decrease of 0.05 mV or more, for example, a decrease of 0.1 mV or more, for example, a decrease of 0.5 mV or more, for example, a decrease of 1 mV or more, for example, a decrease of 5 mV or more, for example, a decrease of 10 mV or more, for example, a decrease of 25 mV or more, for example, a decrease of 50 mV or more, for example, a decrease of 100 mV or more, for example, a decrease of 250 mV or more, for example, a decrease of 500 mV or more, for example, a decrease of 1000 mV or more, for example, a decrease of 2500 mV or more, and a decrease of 5000 mV or more. In some embodiments, the step signal indicates a reduction of detector gain by 1V or more, such as 2V or more, 5V or more, 10V or more, 25V or more, 50V or more, 100V or more, 200V or more, 300V or more, 400V or more, 500V or more, and including a reduction of 600V or more.

[0132] In other embodiments, the step signal indicates an increase in the detector gain of the photodetector, for example, an increase of 5% or more, for example, an increase of 10% or more, for example, an increase of 25% or more, for example, an increase of 50% or more, for example, an increase of 75% or more, for example, an increase of 90% or more, for example, an increase of 95% or more, for example, an increase of 1.5 times or more, for example, an increase of 2 times or more, for example, an increase of 3 times or more, for example, an increase of 4 times or more, and including an increase of 5 times or more. In some instances, the step signal indicates an increase in detector gain of 0.001 mV or more, for example, an increase of 0.005 mV or more, for example, an increase of 0.01 mV or more, for example, an increase of 0.05 mV or more, for example, an increase of 0.1 mV or more, for example, an increase of 0.5 mV or more, for example, an increase of 1 mV or more, for example, an increase of 5 mV or more, for example, an increase of 10 mV or more, for example, an increase of 25 mV or more, for example, an increase of 50 mV or more, for example, an increase of 100 mV or more, for example, an increase of 250 mV or more, for example, an increase of 500 mV or more, for example, an increase of 1000 mV or more, for example, an increase of 2500 mV or more, and includes an increase of 5000 mV or more. In some embodiments, the step signal represents an increase in detector gain of 1V or more, such as an increase of 2V or more, such as an increase of 5V or more, such as an increase of 10V or more, such as an increase of 25V or more, such as an increase of 50V or more, such as an increase of 100V or more, such as an increase of 200V or more, such as an increase of 300V or more, such as an increase of 400V or more, such as an increase of 500V or more, and including an increase of 600V or more.

[0133] In some embodiments, the memory includes instructions for detecting a synchronization signal. In some embodiments, the memory includes instructions for detecting a synchronization signal indicating that light from the light source has a predetermined pulse frequency. In other embodiments, the memory includes instructions for detecting a synchronization signal indicating that light from the light source has an intensity exceeding a predetermined intensity threshold. In still other embodiments, the memory includes instructions for detecting a synchronization signal indicating that light from the light source is at its maximum intensity.

[0134] In some embodiments, the system further includes a processor having a memory operatively coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to: integrate the data signal from the photodetector over a time period comprising at least a portion of 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 a median 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.

[0135] 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 calculated signal amplitudes and the comparison between the calculated signal amplitudes and the light intensity of the light source, systems of interest include a memory having instructions for calculating parameters of a photodetector. For example, the memory may include instructions for determining parameters of 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 detector signals 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 parameters of the photodetector within the operating voltage range of the photodetector. In some embodiments, the memory includes instructions for calculating parameters of the photodetector at an operating voltage that is 10% or more, for example 15% or more, for example 20% or more, for example 25% or more, for example 50% or more, for example 75% or more, for example 90% or more, and 99% or more. In some examples, the memory includes instructions for calculating the signal amplitude of the photodetector over the entire operating voltage range of the photodetector.

[0136] In some embodiments, the photodetector is a photodetector located in a particle analyzer (e.g., 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 from a sample in a flowing stream. Suitable flow cytometry systems may include, but are not limited to, Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Hmmmmana Press 10 (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt 1): 17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30(5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222(4): 335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier The flow cytometry systems described in Syst. 24(3):203-255, the contents of which are incorporated herein by reference. In some instances, the flow cytometry systems of interest include BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortess TM X-20 flow cytometer and BD Biosciences 20FACSCalibur TMCell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorting instrument, BDBiosciences Aria TM Cell sorter, BD Biosciences FACSMelody TM Cell sorting instruments, etc.

[0137] 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, 8,753,57, 8,233,146, and N. The flow cytometry systems described in No. 8,140,300, No. 7,544,326, No. 7,201,875, No. 7,129,505, No. 6,821,740, No. 6,813,017, No. 6,809,804, No. 6,372,506, No. 5,700,692, No. 5,643,796, No. 5,627,040, No. 5,620,842, and No. 5,602,039, the disclosures of which are incorporated herein by reference in their entirety.

[0138] In some embodiments, the subject matter system is a flow cytometry system with an excitation module that uses radio frequency multiplexed excitation to generate multiple frequency-shifted beams. In some examples, the subject matter system is the flow cytometry system described in U.S. Patent Nos. 9,423,353 and 9,784,661 and U.S. Patent Publications Nos. 2017 / 0133857 and 2017 / 0350803, which are incorporated herein by reference.

[0139] In some embodiments, the subject matter system is a particle sorting system configured to sort particles using a closed particle sorting module, such as the particle sorting system described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, which is incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as the sorting decision unit described in U.S. Provisional Patent Application No. 62 / 803,264, filed February 8, 2019, which is incorporated herein by reference. In some embodiments, a method for sorting sample components includes sorting particles (e.g., cells in a biological sample) using a particle sorting module with deflection plates, as described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, which is incorporated herein by reference.

[0140] In some embodiments, the system is a particle analyzer, wherein the particle analysis system 401 ( Figure 4A It can be used to analyze and characterize particles, whether or not the particles are physically sorted into collection containers. Figure 4A A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization is shown. In some embodiments, the particle analysis system 401 is a flow system. Figure 4A The particle analysis system 401 shown can be configured to perform all or part of the methods described herein. The particle analysis system 401 includes a fluid system 402. The fluid system 402 can include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, through which particles 403 (e.g., cells) of the sample move along a common sample path 409 within the sample tube 405.

[0141] The particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as each particle passes through one or more detection stations along a common sample path. Detection station 408 typically refers to a monitoring area 407 along the common sample path. In some implementations, detection can include detecting light or detecting one or more other characteristics of the particle as particle 403 passes through monitoring area 407. Figure 4A The diagram shows a detection station 408 with a monitoring area 407. A partial implementation of the particle analysis system 401 can include multiple detection stations. Furthermore, some detection stations can monitor multiple areas.

[0142] 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, which includes values ​​of various characteristics of the measured particle. The detection system 404 is configured to continuously collect a series of such data points in a first time interval.

[0143] The particle analysis system 401 may also include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The control system shown may be operationally associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency based on the number of data points collected by the detection system 404 within a first time interval, for at least a portion of the first time interval. The control system 406 may also be configured to generate an experimental signal frequency based on the number of data points in that portion of the first time interval. The control system 406 may also compare the experimental signal frequency with the calculated signal frequency or a predetermined signal frequency.

[0144] 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 scatter detector 430, a side scatter detector 435, a fluorescence collecting lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long-pass (“LP”) filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0145] The laser 115a-c is excited to emit light in the form of a laser beam. Figure 4B In the example system, the laser beams emitted by excitation lasers 415a-415c have wavelengths of 488 nm, 633 nm, and 325 nm, respectively. First, the laser beams are guided through one or more beams splitters 445a and 445b. Beam splitter 445a transmits 488 nm light and reflects 633 nm light. Beam splitter 445b transmits ultraviolet light (wavelength range of 10 to 400 nm) and reflects both 488 nm and 633 nm light.

[0146] The laser beam is then guided to a focusing lens 420, which focuses the beam onto the fluid flow portion of the flow chamber 425 containing the sample particles. The flow chamber is part of a fluid system that guides particles (typically one at a time) in the flow to the focused laser beam for querying. The flow chamber can include a flow cell in a benchtop cytometer or a nozzle tip in an airflow cytometer.

[0147] Light from the laser beam interacts with particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at different wavelengths depending on the characteristics of the particles (e.g., their size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles). The fluorescence emission, along with the diffracted, refracted, reflected, and scattered light, can be guided by one or more of the beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collecting lens 440 to one or more of the forward scattering detector 430, the side scattering detector 435, and one or more fluorescence detectors 460a-460f.

[0148] A fluorescence collecting lens 440 collects light emitted from the particle-laser beam interaction and directs this light to one or more beam splitters and filters (bandpass filters, such as bandpass filters 450a-450e), allowing a narrow range of wavelengths to pass through the filters. For example, bandpass filter 450a is a 510 / 20 filter. The first number indicates the center of the spectral band, and the second number provides the range of the spectral band. Thus, the 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short-pass filters transmit light with wavelengths equal to or less than a specified wavelength. Long-pass filters, such as long-pass filters 455a-455b, transmit light with wavelengths equal to or greater than a specified wavelength. For example, long-pass filter 455a, as a 670 nm long-pass filter, transmits light with wavelengths equal to or greater than 670 nm. Filters are typically selected to optimize the detector's specificity for a particular fluorescent dye. The filter 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.

[0149] Beam splitters direct light of different wavelengths in different directions. Beam splitters can be characterized by filter properties such as short-pass and long-pass filters. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 455g transmits light with wavelengths of 620 nm or shorter and directs light with wavelengths greater than 620 nm in different directions. In one embodiment, beam splitters 445a-445g can include optical mirrors, such as dichroic mirrors.

[0150] The forward scattering detector 430 is positioned slightly off-center from the axis of the direct beam passing through the flow cell and is configured to detect diffracted light, i.e., excitation light that propagates 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 can include a photodiode. The side scattering detector 435 is configured to detect refracted and reflected light from the particle surface and internal structure, and tends to increase with increasing particle structural complexity. Fluorescence emission of particle-associated fluorescent molecules can be detected by one or more fluorescence detectors 460a-460f. The side scattering detector 435 and the fluorescence detector can include photomultiplier tubes. The signals detected at the forward scattering detector 430, the side scattering detector 435, and the fluorescence detector can be converted into electronic signals (voltages) by the detectors. This data can provide information about the sample.

[0151] Those skilled in the art will recognize that the flow cytometer according to embodiments of the present invention is not limited to Figure 4B The flow cytometer shown is not limited to any flow cytometer known in the art. For example, a flow cytometer can have any number of lasers, beam splitters, filters, and detectors with different wavelengths and various different configurations.

[0152] During operation, the cytometer is controlled by a controller / processor 490, and measurement data from the detector can be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive output signals from the detector and can also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control the laser, fluid flow parameters, etc. Input / output (I / O) functionality 497 can also be provided in the system. Memory 495, controller / processor 490, and I / O 497 can be provided as a whole as part of the flow cytometer 410. In such embodiments, a display can also be part of the I / O functionality 497 for presenting experimental data to a user of the cytometer 400. Alternatively, some or all of the memory 495, controller / processor 490, and I / O functionality can be part of one or more external devices (e.g., a general-purpose computer). In some embodiments, some or all of the memory 495 and controller / processor 490 can communicate wirelessly or wiredly with the cytometer 410. The controller / processor 490, together with the memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0153] The system shown in Figure 4 includes six different detectors that detect fluorescence in six different wavelength bands (here referred to as the “filter window” of a given detector) defined by the configuration of filters and / or beam splitters in the beam path from flow cell 425 to each detector. Different fluorescent molecules used in flow cytometry experiments emit light in their own characteristic wavelength bands. Specific fluorescent labels used in experiments and their associated fluorescence emission bands can be selected to generally coincide with the filter window of the detector. However, as more detectors are provided and more labels are used, a perfect correspondence between filter windows and fluorescence emission spectra becomes impossible. Generally, although the emission spectral peak of a particular fluorescent molecule may lie within the filter window of a particular detector, some emission spectra of that label may also overlap with the filter windows of one or more other detectors. This can be referred to as spillover. I / O 497 can be configured to receive data on flow cytometry experiments having a set of fluorescent labels and multiple cell populations with multiple labels, each cell population having a subset of multiple labels. I / O 497 can also be configured to receive biological data for assigning one or more markers to one or more cell populations, marker density data, emission spectral data, data for assigning tags to one or more markers, and cytometer configuration data. Flow cytometry experimental data, such as tag spectral characteristics and flow cytometry configuration data, can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more tag-to-marker assignments.

[0154] Figure 5 A functional block diagram of an example particle analyzer control system (e.g., analyzer controller 500) for analyzing and displaying biological events is shown. Analyzer controller 500 can be configured to implement various processes for controlling graphical displays of biological events.

[0155] The particle analyzer 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 502 and the analysis controller 500. Biological event data can be provided to the analysis controller 500 through the data communication channel.

[0156] Analysis controller 500 can be configured to receive biological event data from particle analyzer 502. The biological event data received from particle analyzer 502 can include flow cytometry event data. Analysis controller 500 can be configured to provide a graphical display of a first plot including the biological event data to display device 506. Analysis controller 500 can also be configured to present regions of interest as gates surrounding the population of biological event data displayed by display device 506, for example, overlaid on the first plot. In some embodiments, the gate can be a logical combination of one or more regions of interest plotted on a single-parameter histogram or bivariate plot. In some embodiments, the display can be used to display particle parameters or saturation detector data.

[0157] The analysis controller 500 can also be configured to display biological event data that differs from other events outside the door on the display device 506 inside the door. For example, the analysis controller 500 can be configured to render the colors of the biological event data inside the door to distinguish them from the colors of the biological event data outside the door. The display device 1006 can be a monitor, tablet computer, smartphone, or other electronic device configured to display a graphical interface.

[0158] The analysis controller 500 can be configured to receive a door selection signal from a first input device to identify a door. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a door selection signal to the analysis controller 500 to identify a door to be displayed on or manipulated via the display device 506 (e.g., by clicking on or inside the desired door when the cursor is positioned there). In some implementations, the first input device can be implemented as a keyboard 508 or other device, such as a touchscreen, stylus, optical detector, or voice recognition system, for providing input signals to the analysis controller 500. Some input devices can include multiple input functions. In such implementations, each input function is considered an input device. For example, as... Figure 5 As shown, mouse 510 can include a right mouse button and a left mouse button, each of which can generate trigger events.

[0159] 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 subsequent processing, such as selecting groups of interest for particle classification.

[0160] In some embodiments, the analysis controller 500 can be configured to detect when the mouse 510 initiates gate selection. The analysis controller 500 can also be configured to automatically modify the visualization to facilitate the gate selection process. This modification can be based on a specific distribution of the biological event data received by the analysis controller 500.

[0161] The analysis controller 500 can be connected to the 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 data, such as flow cytometry event data.

[0162] Display device 506 can be configured to receive display data from analysis controller 500. The display data can include gates for plotting and summarizing biological event data. Display device 506 can also be configured to change the presented information based on input received from analysis controller 500 and in combination with input from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0163] In some implementations, the analysis controller 500 can generate a user interface to receive example events for classification. For example, the user interface can include controls for receiving example events or example images. Example events, example images, or example gates can be provided before collecting event data for the samples, or based on an initial set of events from a sample portion.

[0164] Computer control system

[0165] Various aspects of this disclosure also include a computer control system, wherein the system further includes one or more computers for full or partial automation. In some embodiments, the system includes a computer having a computer-readable storage medium on which a computer program is stored, wherein the computer program, when loaded onto the computer, includes instructions for: detecting light from a light source using a photodetector in a particle analyzer at a first predetermined time interval; detecting a step signal indicating a change in a parameter of the light source or a parameter of the photodetector; detecting light from the light source using a photodetector at a second predetermined time interval; integrating a data signal from the photodetector at the first and second predetermined time intervals; and determining one or more parameters of the photodetector based on the integrated data signal.

[0166] In this embodiment, the system includes an input module, a processing module, and an output module. The system may include hardware components and software components, wherein the hardware components may take the form of one or more platforms, for example, as servers, so that functional elements performing specific tasks (e.g., managing the input and output of information, processing information, etc.) can be implemented by executing software applications on one or more computer platforms representing the system or across platforms.

[0167] 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 capable of accessing memory storing instructions for performing the steps of the methods of this subject. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, caches, data backup units, and many other devices. The processor may be a commercially available processor or one of other processors that are already available or will become available in the future. The processor executes the operating system, which interacts with firmware and hardware in well-known ways and helps the processor coordinate and execute the functions of various computer programs, which may be written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is well known in the art. The operating system typically works with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all conforming 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 a light source with a flow based on a first optical signal and a second optical signal. In some embodiments, the processor includes analog electronics that provide feedback control (e.g., negative feedback control).

[0168] System memory can be any of a variety of known or future memory storage devices. Examples include any commonly used random access memory (RAM), magnetic media (such as resident hard disks or magnetic tapes), optical media (such as optical discs), flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, removable hard disk drives, or floppy disk drives. This type of memory storage device typically reads and / or writes 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 to be developed in the future, can be considered a computer program product. As will be understood, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or in program storage devices used with memory storage devices.

[0169] In some embodiments, the described computer program product includes a computer-usable medium having control logic (computer software program, including program code) stored therein. When the control logic is executed by a computer's processor, it causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, such as a hardware state machine. The implementation of a hardware state machine for performing the functions described herein will be readily apparent to those skilled in the art.

[0170] The memory can be any suitable device in which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including disks, optical discs, magnetic tapes, RAM, or any other suitable device, fixed or portable). The processor can include a general-purpose digital microprocessor that is appropriately programmed according to a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel, or the programming, pre-stored in a computer program product (e.g., memory or a portion of other portable or fixed computer-readable storage media), can be provided to the processor using any device connected to the memory. For example, a disk or optical disc can carry the programming and can be read by a disk writer / reader. The system of the present invention also includes programming (e.g., in the form of a computer program product) and algorithms for practicing the methods described above. The programming according to the present invention can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; portable flash drives; and mixtures of these categories, such as magnetic / optical storage media.

[0171] The processor can also access communication channels to communicate with users in remote locations. A remote location refers to a user who does not have direct contact with the system and relays input information from an external device to the input manager. This external device is, for example, a computer connected to a wide area network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including mobile phones (i.e., smartphones).

[0172] 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 can be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., RFID), Zigbee communication protocol, WiFi, infrared, USB, and UWB. Communication protocols and cellular communications (e.g., Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM)).

[0173] 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 system and other external devices, which are computer terminals configured for similar complementary data communication (e.g., in a doctor's office or hospital environment).

[0174] In one embodiment, the communication interface is configured for infrared communication. The system may use a communication or any other suitable wireless communication protocol to enable it to communicate with other devices, including computer terminals and / or networks, mobile phones that support communication, personal digital assistants, or any other communication devices that the user may use in combination.

[0175] In one embodiment, the communication interface is configured to provide data transmission connectivity via a mobile network, short message service (SMS), a wireless connection to a personal computer (PC) connected to a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot, using the Internet Protocol (IP).

[0176] In one embodiment, the system is configured to communicate wirelessly with a server device via a communication interface, for example, using 802.11 or... The RF protocol is a common standard, or IrDA infrared protocol. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop; 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, a keyboard, a mouse, or a touchscreen.

[0177] In one embodiment, the system is configured to communicate wirelessly with a server device via a communication interface, for example, using 802.11 or... Common standards such as RF protocols or IrDA infrared protocols. The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop; or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display (e.g., a liquid crystal display, LCD) and input devices (e.g., buttons, keyboard, mouse, or touchscreen).

[0178] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate with network or server devices data stored in the subject system (e.g., optional data storage unit) using one or more of the above-described communication protocols and / or mechanisms.

[0179] The output controller may include any of a variety of known display devices for presenting information to a user, whether the user is a person or a machine, local or remote. If one of the display devices provides visual information, it can 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. Functional elements of the computer may communicate with each other via a system bus. In alternative embodiments, some of these communications may be accomplished using a network or other types of remote communication. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite networks, according to known technologies. The data representation of the output manager may be implemented according to a variety of known technologies. As a partial example, the data may include SQL, HTML, or XML documents, emails, or other files, or other forms of data. The data may include an Internet URL address so that the user can retrieve other SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present in the system of this subject may be any type of known computer platform or type to be developed in the future. Although they typically belong to a class of computers known as servers, they can also be mainframes, workstations, or other computer types. They can be connected via any known or future type of cable or other communication system (including wireless systems), whether networked or otherwise. They can be located in the same location or physically separated. A variety of operating systems can be used on any computer platform, depending on the type and / or architecture of the chosen platform. Suitable operating systems include Windows 10, Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, etc.

[0180] Figure 6 A general architecture of an example computing device 600 according to certain embodiments is described. Figure 6 The general architecture of the described computing device 600 includes the arrangement of computer hardware and software components. The computing device 600 may include, but is not limited to, the following: Figure 6The diagram shows more (or fewer) of those components. However, it is not necessary to show all of these generally conventional elements to provide an enabling disclosure. As shown, computing device 600 includes processing unit 610, network interface 620, computer-readable media drive 630, input / output device interface 640, display 650, and input device 660, all of which can communicate with each other via a communication bus. Network interface 620 can provide connectivity to one or more networks or computing systems. Processing unit 610 can therefore receive information and instructions from other computing systems or services via the network. Processing unit 610 can also communicate with memory 670 and further provide output information to optional display 650 via input / output device interface 640. Input / output device interface 640 can also receive input from optional input device 660 (e.g., keyboard, mouse, digital pen, microphone, touchscreen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input device).

[0181] Memory 670 may contain computer program instructions (grouped into modules or components in some embodiments) that the processing unit 610 executes to implement one or more embodiments. Memory 670 typically includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 670 may store an operating system 672 that provides computer program instructions for use by the processing unit 610 in the general management and operation of the computing device 600. Memory 670 may also include computer program instructions and other information for implementing various aspects of this disclosure.

[0182] Computer-readable storage media

[0183] Various aspects of this disclosure also include non-transitory computer-readable storage media having instructions for practicing the methods of this subject matter. Computer-readable storage media can be used on one or more computers to achieve full or partial automation of the system for practicing the methods described herein. In some embodiments, instructions according to the methods described herein can be encoded in a “programmed” form onto a computer-readable medium, wherein the term “computer-readable medium” as used herein refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network attached storage (NAS), whether these devices are internal or external to a computer. Files containing information can be “stored” on a computer-readable medium, where “stored” means recording the information so that it can be accessed and retrieved by a computer later. The computer implementation methods described herein can be implemented using programming, which can be written using one or more of any number of computer programming languages. For example, these languages ​​include Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as many other languages.

[0184] In some embodiments, the computer-readable storage medium of interest includes a computer program stored thereon, wherein the computer program, when loaded onto a computer, includes instructions having the following algorithms: an algorithm for detecting light from a light source using a photodetector in a particle analyzer at a first predetermined time interval; an algorithm for detecting a step signal indicating a change in a parameter of the light source or a parameter of the photodetector; an algorithm for detecting light from the light source using a photodetector at a second predetermined time interval; an algorithm for integrating a data signal from the photodetector at 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.

[0185] In some instances, the non-transitory computer-readable storage medium includes algorithms for detecting light from a light source at multiple predetermined time intervals. In some examples, the intensity of the light source increases after each predetermined time interval. At each light intensity, the time interval for the photodetector to detect light from the light source can vary. In some examples, each time interval is the same. In other examples, each time interval is different. In these examples, the non-transitory computer-readable storage medium includes algorithms for integrating the data signal from the photodetector over a time period comprising multiple illumination time intervals.

[0186] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal indicating a change in the intensity of light from a light source. In some examples, the change in intensity is a decrease in the light intensity of the light source. In other examples, the change in intensity is an increase in the light intensity of the light source. In other embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal indicating a change in the spectral parameters of light from a light source. In some examples, the change in spectral parameters is a change in the wavelength of light emitted by the light source. In other examples, the change in spectral parameters is an increase in the number of wavelengths of light emitted by the light source. In some examples, the increase in the number of wavelengths of light includes an increase in the spectral width emitted by the light source. In other examples, the change in spectral parameters is a decrease in the number of wavelengths of light emitted by the light source. In some examples, the decrease in the number of wavelengths of light includes a decrease in the spectral width emitted by the light source.

[0187] In some examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, which includes a change in the frequency of light pulses from a light source. In some examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, which includes detecting light pulses from a light source. In other examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, which includes detecting an increase in the frequency of light pulses compared to light pulses from a light source detected during a first predetermined time interval. In other examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, which includes detecting a decrease in the frequency of light pulses compared to light pulses from a light source detected during a first predetermined time interval. In some examples, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal, which includes detecting a lack of light from a light source.

[0188] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting a step signal indicating a change in the gain of a photodetector. In some examples, the change in detector gain refers to a decrease in the detector gain of the photodetector. In other examples, the change in detector gain refers to an increase in the detector gain of the photodetector. In some embodiments, the change in detector gain is indicated by a step signal comprising a change in the frequency of a light pulse from a light source. In other embodiments, the change in detector gain is indicated by a step signal comprising a loss of light from the light source.

[0189] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting a synchronization signal before detecting light from a light source prior to a first predetermined time interval. In some embodiments, the synchronization signal includes detecting light from the light source having a predetermined pulse frequency. In other embodiments, the synchronization signal includes detecting light from the light source having an intensity exceeding a predetermined intensity threshold. In still other embodiments, the synchronization signal includes detecting light from the light source at its maximum light intensity.

[0190] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating a signal amplitude. In some examples, the non-transitory computer-readable storage medium includes an algorithm for calculating a median signal amplitude. In some examples, 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 examples, the non-transitory computer-readable storage medium includes an algorithm for determining parameters of a photodetector based on one or more calculated signal amplitudes 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 detector sensitivity, a minimum detection threshold, a maximum detection threshold, detector dynamic range, 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, for example, wherein the parameters of the photodetector are determined over the entire operating voltage range of the photodetector.

[0191] Non-transitory computer-readable storage media can be used in one or more computer systems having a display and operator input devices. Operator input devices may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access memory on which instructions for performing the steps of this method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or one of other processors available now or in the future. The processor executes the operating system, which works in a well-known manner with firmware and hardware and helps the processor coordinate and execute the functions of various computer programs written in various programming languages, such as Java, Perl, C++, other high-level languages ​​or low-level languages, and combinations thereof. 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 conforming to known techniques.

[0192] kit

[0193] Kits comprising one or more components of the system of this subject matter are also provided. Kits according to certain embodiments include one or more light sources, such as narrowband light-emitting diodes and photodetectors (e.g., photomultiplier tubes), wherein analysis of one or more parameters of the photodetectors is required. Kits may also include optical adjustment components, such as lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimation protocols, and combinations thereof.

[0194] In addition to the components described above, (in some embodiments) this subject matter kit may also include instructions for practicing the methods of this subject matter. These instructions may exist in a variety of forms within the subject matter kit, one or more of which may be present in the kit. One form of these instructions may be printing information on a suitable medium or substrate, such as one or more sheets of paper on which information is printed, kit packaging, packaging inserts, etc. Another form of these instructions may be a computer-readable medium containing information, such as a floppy disk, optical disc (CD), portable flash drive, etc. Yet another possible form of these instructions may be a website address that can be used to access information on a deleted site via the Internet.

[0195] Practical Applications

[0196] 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 can also be used with photodetectors for the analysis and sorting of particulate components in samples in fluid media, such as biological samples. This disclosure can be used in flow cytometry to provide a flow cytometer during cell sorting with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced particle deflection capability. In embodiments, this disclosure reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In some embodiments, the methods and systems of this subject matter provide a fully automated scheme, so that adjustments to the flow cytometer during use require almost no manual input.

[0197] Notwithstanding the appended claims, this disclosure may also be defined by the following terms:

[0198] 1. A method comprising:

[0199] Light from the light source is detected by a photodetector in a particle analyzer at a first predetermined time interval;

[0200] The photodetector is used to detect a step signal, wherein the step signal indicates a change in the parameters of the light source or the parameters of the photodetector.

[0201] The photodetector detects light from the light source at a second predetermined time interval.

[0202] The data signal from the photodetector is integrated during the first predetermined time interval and the second predetermined time interval; and

[0203] One or more parameters of the photodetector are determined based on the integrated data signal.

[0204] 2. The method according to Clause 1, wherein the parameter of the light source is the light intensity from the light source.

[0205] 3. The method according to Clause 1, wherein the parameters of the light source are the spectrum from the light source.

[0206] 4. The method according to any one of clauses 1-3, wherein the light source comprises one or more light-emitting diodes.

[0207] 5. The method according to Clause 4, wherein the light source comprises a plurality of monochromatic light-emitting diodes.

[0208] 6. The method according to Clause 4, wherein the light source comprises a plurality of multicolor light-emitting diodes.

[0209] 7. The method according to any one of clauses 1-6, wherein the detection of a step signal indicates a change in the light intensity of the light source.

[0210] 8. The method according to Clause 7, wherein the detection of a step signal indicates that the light intensity of the light source increases from a first intensity to a second intensity.

[0211] 9. The method according to any one of Clauses 7-8, wherein the step signal comprises a change in the frequency of a light pulse from the light source.

[0212] 10. The method according to any one of Clauses 7-8, wherein the step signal comprises a variation in the duration of a light pulse from the light source.

[0213] 11. The method according to any one of Clauses 7-8, wherein the step signal includes the absence of light from the light source.

[0214] 12. The method according to any one of clauses 1-6, wherein the detection of a step signal indicates a change in the detector gain of the photodetector from a first detector gain to a second detector gain.

[0215] 13. The method according to Clause 12, wherein the step signal comprises a change in the frequency of a light pulse from the light source.

[0216] 14. The method according to Clause 13, wherein the step signal comprises a change in the duration of a light pulse from the light source.

[0217] 15. The method according to Clause 12, wherein the step signal includes the absence of light from the light source.

[0218] 16. The method according to any one of Clauses 1-15, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0219] 17. The method according to any one of clauses 1-16, wherein the method further comprises detecting a synchronization signal before the first predetermined time interval.

[0220] 18. The method according to Clause 17, wherein the synchronization signal includes detecting light from the light source at its maximum intensity.

[0221] 19. The method according to any one of clauses 1-18, wherein the method includes detecting a tag signal inserted into one or more data signals of the first predetermined time interval and the second predetermined time interval.

[0222] 20. The method according to Clause 19, wherein the tag signal includes an FM data signal.

[0223] 21. The method according to any one of clauses 1-20, wherein the method further comprises setting the photodetector to a predetermined detector gain before the first predetermined time interval.

[0224] 22. The method according to any one of clauses 1-21, wherein the method includes determining one or more parameters of the photodetector, said one or more parameters being selected from: detector sensitivity, minimum detection threshold, maximum detection threshold, detector sensitivity, detector dynamic range, detector signal-to-noise ratio, and number of photoelectrons per unit output.

[0225] 23. The method according to any one of clauses 1-22, wherein the parameters of the photodetector are determined within the operating voltage range of the photodetector.

[0226] 24. The method according to Clause 23, wherein the parameters of the photodetector are determined over the entire operating voltage range of the photodetector.

[0227] 25. The method according to any one of clauses 1-24 further includes calculating the optimal detector gain of the photodetector based on the determined parameters.

[0228] 26. The method according to any one of clauses 1-25, wherein the photodetector is located in a flow cytometer.

[0229] 27. The method according to Clause 26, wherein the flow cytometer includes a flow cell for propagating particles in a flow stream.

[0230] 28. A particle analyzer, comprising:

[0231] light source;

[0232] The optical detection system located within the housing of the particle analyzer includes a photodetector configured as follows:

[0233] Detect light from the light source at a first predetermined time interval;

[0234] Detect a step signal, wherein the step signal indicates a change in the parameters of the light source or the parameters of the photodetector;

[0235] Detect light from the light source at a second predetermined time interval; and

[0236] 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:

[0237] The data signal from the photodetector is integrated during 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] 29. The particle analyzer as described in Clause 28, wherein the particle analyzer is incorporated into a flow cytometer.

[0240] 30. The particle analyzer according to any one of clauses 28-29, wherein the parameter of the light source is the light intensity of the light source.

[0241] 31. The particle analyzer according to any one of Clauses 28-29, wherein the parameter of the light source is the spectrum of the light source.

[0242] 32. The particle analyzer according to any one of clauses 28-31, wherein the light source comprises one or more light-emitting diodes.

[0243] 33. The particle analyzer according to Clause 32, wherein the light source comprises a plurality of monochromatic light-emitting diodes.

[0244] 34. The particle analyzer according to Clause 33, wherein the light source comprises a plurality of multicolor light-emitting diodes.

[0245] 35. A particle analyzer according to any one of clauses 28-34, wherein the step signal indicates a change in the light intensity of the light source.

[0246] 36. The particle analyzer according to Clause 35, wherein the step signal indicates that the light intensity of the light source increases from a first intensity to a second intensity.

[0247] 37. The particle analyzer according to any one of clauses 35-36, wherein the step signal comprises a change in the frequency of a light pulse from the light source.

[0248] 38. The particle analyzer according to any one of clauses 35-36, wherein the step signal comprises a change in the duration of a light pulse from the light source.

[0249] 39. The particle analyzer according to any one of clauses 35-36, wherein the step signal includes the absence of light from the light source.

[0250] 40. A particle analyzer according to any one of clauses 28-34, wherein the step signal indicates a change in the detector gain of the photodetector from a first detector gain to a second detector gain.

[0251] 41. The particle analyzer according to Clause 40, wherein the step signal comprises a change in the frequency of a light pulse from the light source.

[0252] 42. The particle analyzer according to Clause 41, wherein the step signal comprises a change in the duration of a light pulse from the light source.

[0253] 43. The particle analyzer according to Clause 42, wherein the step signal includes the absence of light from the light source.

[0254] 44. The particle analyzer according to any one of clauses 28-43, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0255] 45. The particle analyzer according to any one of clauses 28-44, wherein the photodetector is further configured to detect a synchronization signal before the first predetermined time interval.

[0256] 46. ​​The particle analyzer according to Clause 45, wherein the synchronization signal includes detecting light from the light source at its maximum intensity.

[0257] 47. The particle analyzer according to any one of clauses 28-46, wherein the memory includes instructions stored thereon that, when the processor executes the instructions, cause the processor to detect a tag signal inserted into one or more data signals of the first predetermined time interval and the second predetermined time interval.

[0258] 48. The particle analyzer as described in Clause 47, wherein the tag signal includes a frequency-modulated data signal.

[0259] 49. A particle analyzer according to any one of clauses 28-48, wherein the memory includes instructions stored thereon that, when the processor executes the instructions, cause the processor to set the photodetector to a predetermined detector gain before the first predetermined time interval.

[0260] 50. A particle analyzer according to any one of clauses 28-49, wherein the memory includes instructions stored thereon that, when the processor executes the instructions, cause the processor to determine one or more parameters of the photodetector, said one or more parameters being selected from detector sensitivity, minimum detection threshold, maximum detection threshold, detector sensitivity, detector dynamic range, detector signal-to-noise ratio, and number of photoelectrons per unit output.

[0261] 51. A particle analyzer according to any one of clauses 28-50, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine parameters of the photodetector within the operating voltage range of the photodetector.

[0262] 52. The particle analyzer according to Clause 51, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine parameters of the photodetector across the entire operating voltage range of the photodetector.

[0263] 53. The particle analyzer according to any one of clauses 28-52, wherein the memory includes instructions stored thereon that, when the processor executes the instructions, cause the processor to calculate the optimal detector gain of the photodetector based on determined parameters.

[0264] 54. The particle analyzer according to any one of clauses 28-53, wherein the photodetector comprises a photomultiplier tube.

[0265] 55. The particle analyzer according to any one of clauses 28-53, wherein the photodetector comprises an avalanche photodiode.

[0266] 56. A non-transitory computer-readable storage medium comprising instructions stored thereon, the instructions comprising:

[0267] An algorithm for detecting light from a light source using a photodetector in a particle analyzer at a first predetermined time interval;

[0268] An algorithm for detecting a step signal using the photodetector, wherein the step signal indicates a change in the parameters of the light source or the parameters of the photodetector;

[0269] An algorithm for detecting light from a light source using the photodetector at a second predetermined time interval;

[0270] An algorithm for integrating the data signal from the photodetector during the first predetermined time interval and the second predetermined time interval; and

[0271] An algorithm for determining one or more parameters of the photodetector based on integrated data signals.

[0272] 57. The non-transitory computer-readable storage medium as described in Clause 56, wherein the light source parameter is the light intensity of the light source.

[0273] 58. The non-transitory computer-readable storage medium as described in Clause 56, wherein the light source parameters are the spectrum of the light source.

[0274] 59. The non-transitory computer-readable storage medium according to any one of clauses 56-58, wherein the light source comprises one or more light-emitting diodes.

[0275] 60. The non-transitory computer-readable storage medium as described in Clause 59, wherein the light source comprises a plurality of monochromatic light-emitting diodes.

[0276] 61. The non-transitory computer-readable storage medium as described in Clause 59, wherein the light source comprises a plurality of multicolor light-emitting diodes.

[0277] 62. The non-transitory computer-readable storage medium according to any one of clauses 56-61, wherein the step signal indicates a change in the light intensity of the light source.

[0278] 63. The non-transitory computer-readable storage medium according to Clause 62, wherein the step signal indicates that the light intensity of the light source increases from a first intensity to a second intensity.

[0279] 64. The non-transitory computer-readable storage medium according to any one of clauses 62-63, wherein the step signal comprises a change in the frequency of a light pulse from the light source.

[0280] 65. The non-transitory computer-readable storage medium according to any one of clauses 62-63, wherein the step signal comprises a variation in the duration of a light pulse from the light source.

[0281] 66. A non-transitory computer-readable storage medium claimed under any of clauses 62-63, wherein the step signal includes the absence of light from the light source.

[0282] 67. A non-transitory computer-readable storage medium according to any one of clauses 56-61, wherein detecting the step signal indicates a change in the detector gain of the photodetector from a first detector gain to a second detector gain.

[0283] 68. The non-transitory computer-readable storage medium as described in Clause 67, wherein the step signal comprises a change in the frequency of a light pulse from the light source.

[0284] 69. The non-transitory computer-readable storage medium as described in Clause 67, wherein the step signal comprises a variation in the duration of a light pulse from the light source.

[0285] 70. The non-transitory computer-readable storage medium as described in Clause 67, wherein the step signal includes the absence of light from the light source.

[0286] 71. The non-transitory computer-readable storage medium according to any one of clauses 56-70, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0287] 72. The non-transitory computer-readable storage medium according to any one of clauses 56-71, wherein the non-transitory computer-readable storage medium further includes an algorithm for detecting a synchronization signal before the first predetermined time interval.

[0288] 73. The non-transitory computer-readable storage medium as described in Clause 62, wherein the synchronization signal includes detecting light from a light source at its maximum intensity.

[0289] 74. The non-transitory computer-readable storage medium according to any one of clauses 56-73, wherein the non-transitory computer-readable storage medium further includes an algorithm for detecting a tag signal inserted into one or more data signals of the first predetermined time interval and the second predetermined time interval.

[0290] 75. The non-transitory computer-readable storage medium as described in Clause 74, wherein the tag signal includes an FM data signal.

[0291] 76. The non-transitory computer-readable storage medium according to any one of clauses 56-73, wherein the non-transitory computer-readable storage medium further includes an algorithm for setting the photodetector to a predetermined detector gain before the first predetermined time interval.

[0292] 77. A non-transitory computer-readable storage medium according to any one of clauses 56-76, wherein the non-transitory computer-readable storage medium includes an algorithm for determining one or more parameters of the photodetector, said one or more parameters being selected from detector sensitivity, minimum detection threshold, maximum detection threshold, detector dynamic range, detector signal-to-noise ratio, and number of photoelectrons per unit output.

[0293] 78. The non-transitory computer-readable storage medium according to any one of clauses 56-77, 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.

[0294] 79. The non-transitory computer-readable storage medium according to Clause 78, wherein the non-transitory computer-readable storage medium includes an algorithm for determining parameters of the photodetector over its entire operating voltage range.

[0295] 80. The non-transitory computer-readable storage medium according to any one of clauses 56-79, wherein the non-transitory computer-readable storage medium further comprises an algorithm for calculating the optimal detector gain of the photodetector based on the determined parameters.

[0296] Although the invention has been described in detail by way of illustration and example for ease of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made to it without departing from the spirit or scope of the appended claims.

[0297] Therefore, the foregoing is merely illustrative of 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 cited herein are primarily intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to promote technological development; these principles and concepts are not limited to the specifically cited examples and situations. Moreover, all statements and specific examples of the principles, aspects, and embodiments of the invention enumerated herein are intended to include their structural and functional equivalents. Furthermore, these equivalents include both currently known equivalents and those developed in the future, i.e., any element developed that performs the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended to be exclusively disclosed to the public, whether or not such disclosure is expressly referenced in the claims.

[0298] 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 referring to the limitation in the claims only if the exact phrase “means for…” or “step for…” is used at the beginning of such limitation in the claims; if the exact phrase is not used in the limitation in the claims, then 35U.SC §112(f) or 35U.SC §112(6) is not referred to.

Claims

1. A method comprising: The photodetector in the particle analyzer is illuminated with light pulses at a first predetermined time interval using a light source; The photodetector is illuminated with a light pulse at a second predetermined time interval; A step signal is detected based on the change in the frequency of the light pulse measured by the photodetector between the first predetermined time interval and the second predetermined time interval; The photodetector is illuminated with a light pulse at a different intensity than that during the first predetermined time interval; and The response linearity of the photodetector is calibrated based on the detected step signal and the light measured during a third predetermined time interval.

2. The method of claim 1, wherein the step signal comprises a change in light intensity from the light source.

3. The method of claim 1, wherein the step signal comprises a change in the duration of a light pulse from the light source.

4. The method according to any one of claims 1-3, wherein the light source comprises one or more light-emitting diodes.

5. The method according to claim 4, wherein the light source comprises a plurality of monochromatic light-emitting diodes or a plurality of multicolor light-emitting diodes.

6. The method according to any one of claims 1-3, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

7. The method according to any one of claims 1-3, wherein the method further comprises detecting a synchronization signal before the first predetermined time interval.

8. The method according to any one of claims 1-3, wherein the method includes detecting a tag signal inserted into one or more data signals of the first predetermined time interval and the second predetermined time interval.

9. The method of claim 8, wherein the tag signal comprises an FM data signal.

10. The method according to any one of claims 1-3, wherein the method further comprises setting the photodetector to a predetermined detector gain before the first predetermined time interval.

11. The method according to any one of claims 1-3, wherein the method comprises determining one or more parameters of the photodetector, said one or more parameters being selected from: detector sensitivity, minimum detection threshold, maximum detection threshold, detector sensitivity, detector dynamic range, detector signal-to-noise ratio, and number of photoelectrons per unit output.

12. The method according to any one of claims 1-3, further comprising calculating the optimal detector gain of the photodetector based on the determined parameters.

13. The method according to any one of claims 1-3, wherein the photodetector is located in a flow cytometer.

14. A particle analyzer, comprising: A light source is configured to be irradiated with light pulses at a first predetermined time interval, irradiated with light pulses at a second predetermined time interval, and irradiated with light pulses at an intensity different from that during the first predetermined time interval at a third predetermined time interval; The optical detection system, located within the housing of the particle analyzer, includes a photodetector configured as follows: A step signal is detected based on the change in the frequency of the light pulse measured by the photodetector between the first predetermined time interval and the second predetermined time interval; as well as A processor, comprising memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to: The response linearity of the photodetector is calibrated based on the detected step signal and the light measured during a third predetermined time interval.

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