Apparatus and method for quantitative characterization of photodetectors - Patents.com

Abstract

Aspects of the present disclosure include methods for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). The method, according to certain embodiments, includes illuminating a photodetector positioned in the particle analyzer with a light source (e.g., a continuous wave light source) at a first intensity for a first predetermined time interval, illuminating the photodetector with the light source at a second intensity for a second predetermined time interval, integrating a data signal from the photodetector over a period including 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 performing the subject methods are also described. A non-transitory computer-readable storage medium having stored thereon instructions for determining parameters of a photodetector according to the subject methods is also provided.

Description

[Background technology]

[0001] Characterization of analytes in biological fluids has become an important component of medical diagnosis and evaluation of a patient's overall health and well-being. Detection of analytes in biological fluids (e.g., human blood or blood-derived products) can provide results that may play a role in determining treatment protocols for patients with various disease states.

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

[0003] A flow cytometer transports particles (including cells) in a fluid sample as a cell stream into a flow cell while simultaneously directing a sheath fluid into the flow cell. To characterize the components of the flow stream, the flow stream is illuminated with light. Variations in the materials in the flow stream (e.g., morphology or the presence of fluorescent labels) result in variations in the observed light, and these variations allow for characterization and separation.

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

[0005] Aspects of the present disclosure include a method for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). The method according to certain embodiments includes illuminating the photodetector with a light source at a first intensity for a first predetermined time interval, illuminating the photodetector with a light source at a second intensity for a second predetermined time interval, integrating a data signal from the photodetector over a period including 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. In some examples, the light source is a continuous wave light source. In some examples, the light source is a pulsed light source. In some embodiments, the light source is a light emitting diode. In certain examples, the light source is a narrow band light source, such as a light source that emits light having a wavelength ranging from 20 nm or less.

[0006] In carrying out the subject methods according to certain embodiments, the intensity of the light source increases in intensity after each predetermined time interval (i.e., the second illumination intensity is greater than the first illumination intensity). The time interval for illuminating the light detector with each light intensity can vary. In some examples, each time interval is the same. In other examples, each time interval is different. In certain embodiments, the method includes illuminating the light detector with a continuous wave light source for multiple predetermined time intervals at multiple intensities. In these embodiments, some of the time intervals may be the same and some of the time intervals may be different. In some examples, the method includes increasing the intensity of light from the light source over a period of time that includes at least the first predetermined time interval and the second predetermined time interval. In some examples, the intensity of light from the light source increases linearly over a period of time that includes at least the first predetermined time interval and the second predetermined time interval. In other examples, the intensity of light from the light source increases exponentially over a period of time that includes at least the first predetermined time interval and the second predetermined time interval.

[0007] In embodiments, the method includes integrating a data signal from the photodetector over a period that includes at least a time interval of illumination at each of the different light intensities. In some embodiments, the data signal from the photodetector is integrated over a period that includes 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 that includes multiple time intervals during which the light source illuminates the photodetector with increasing light intensities during each of the multiple time intervals.

[0008] In certain embodiments, integrating the data signal from the photodetector includes calculating a signal amplitude over a period of time. In some examples, calculating the signal amplitude includes calculating one or more of a median signal amplitude, a mean signal amplitude, a standard deviation of the signal amplitude, and a variance and a coefficient of variation of the signal amplitude. In certain examples, the method also includes comparing the calculated signal amplitude to a light intensity of the light source. Based on one or more of the calculated signal amplitude and a comparison of the calculated signal amplitude to the light intensity of the light source, a parameter of the photodetector is calculated. For example, the method may include determining a parameter such as a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, or a number of photoelectrons per unit output for the photodetector. The detector parameter may be determined over a range of operating voltages of the photodetector, such as where the parameter of the photodetector is determined over the entire operating voltage range of the photodetector. In certain embodiments, the photodetector is positioned within a particle analyzer, e.g., the photodetector is part of a light detection module of the particle analyzer. In some examples, the particle analyzer is incorporated into a flow cytometer and the photodetector is positioned to detect light from particles in the flow stream.

[0009] In some embodiments, the method includes determining one or more parameters of a photodetector (e.g., a photodetector in a particle analyzer) by illuminating particles in a flow stream, the particles comprising one or more fluorophores. In some examples, the particles are beads (e.g., polystyrene beads). In some examples, a method for determining parameters of a photodetector includes illuminating a flow stream having particles comprising one or more fluorophores at a first intensity for a first predetermined time interval and at a second intensity for a second predetermined time interval, detecting light from the flow stream with a photodetector having a light source, generating a data signal from the photodetector at the first illumination intensity and generating a data signal from the photodetector at the second illumination intensity, and determining one or more parameters of the photodetector based on the data signals generated at the first intensity and the second intensity. In some examples, the method includes determining an average fluorescence intensity from the particles at the first illumination intensity and the second illumination intensity. In some examples, the method includes determining a variance of the average fluorescence intensity at the first illumination intensity and the second illumination intensity. In some examples, the method includes determining a statistical photoelectron (SPE) at the first illumination intensity and the second illumination intensity. In particular examples, the method determines a detector efficiency (Q) of the photodetector for each fluorophore on the particle based on the statistical photoelectron and the determined average fluorescence intensity of the fluorophore. det In certain embodiments, the method further comprises determining a detector efficiency for each detector channel of the photodetector. In some embodiments, the method further comprises determining a background signal for each photodetector. In some embodiments, the method further comprises determining electronic noise from each photodetector. In certain embodiments, the method further comprises determining a detection limit of the photodetector.

[0010] Aspects of the present disclosure also include a system having a light source and a photodetector configured to detect light from the light source at a first intensity over a first predetermined time interval and detect light from the light source at a second intensity over a second predetermined time interval, and a processor having a memory operatively coupled to the processor such that the memory stores instructions that, when executed by the processor, cause the processor to integrate a data signal from the photodetector over a period including the first predetermined time interval and the second predetermined time interval and determine one or more parameters of the photodetector based on the integrated data signal. In some embodiments, the system is a particle analyzer. In some examples, the photodetector is part of a photodetection module of the particle analyzer. In certain examples, the particle analyzer is incorporated into a flow cytometer.

[0011] The light source of the subject system is, in some embodiments, a continuous wave light source. In other embodiments, the light source is a pulsed light source. In certain embodiments, the light source is a light emitting diode. In some examples, the light source is a narrow bandwidth light source, such as a light emitting diode that emits light having a wavelength that spans 20 nm or less. The light source is configured to illuminate the photodetector at two or more different intensities over a predetermined time interval. In some embodiments, the light source is configured to illuminate the photodetector at a first intensity over a first predetermined time interval and at a second intensity over a second predetermined time interval. In other embodiments, the light source is configured to illuminate the photodetector for a plurality of predetermined time intervals at a plurality of intensities. In embodiments, each time interval can be of the same duration or of different duration. In some embodiments, the light source is configured to increase the intensity of illumination after each predetermined time interval (i.e., the intensity of the light increases for each successive interval of illumination). In some embodiments, the light source is configured to increase the intensity over a period that includes at least the first predetermined time interval and the second predetermined time interval. In some examples, the intensity of the light source is configured to increase linearly. In another example, the intensity of the light source is configured to increase exponentially.

[0012] The subject systems include 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 a data signal from a photodetector. In some embodiments, the memory includes instructions for calculating a signal amplitude from the photodetector over each time interval. In other embodiments, the memory includes instructions for calculating a median signal amplitude. In certain embodiments, the memory includes instructions for comparing the calculated signal amplitude to an intensity of illumination during each predetermined time interval.

[0013] In an embodiment, the system includes a memory operably coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to determine a parameter of the photodetector based on one or more of the calculated signal amplitude and a comparison between the calculated signal amplitude and the light intensity of the light source. For example, the memory may include instructions for determining a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, or a number of photoelectrons per unit output. The subject systems may be configured to determine the detector parameters over a range of operating voltages of the photodetector, such as over the entire operating voltage range of the photodetector.

[0014] Aspects of the present disclosure also include a non-transitory computer readable storage medium for determining one or more parameters of a photodetector. In an embodiment, the non-transitory computer readable storage medium includes an algorithm for illuminating the photodetector with a light source at a first intensity for a first predetermined time interval, an algorithm for illuminating the photodetector with a light source at a second intensity for a second predetermined time interval, an algorithm for integrating a data signal from the photodetector over a period that includes the first predetermined time interval and the second predetermined time interval, and an algorithm for determining one or more parameters of the photodetector based on the integrated data signal. In certain examples, the non-transitory computer readable storage medium includes an algorithm for illuminating the photodetector with multiple light intensities over multiple time intervals. In these examples, the non-transitory computer readable storage medium includes an algorithm for integrating a data signal from the photodetector over a period of time that includes multiple illumination time intervals.

[0015] In some embodiments, the non-transitory computer readable storage medium includes an algorithm for calculating the signal amplitude. In some examples, the non-transitory computer readable storage medium includes an algorithm for calculating one or more of the median signal amplitude, the mean signal amplitude, the standard deviation of the signal amplitude, and the variance and the coefficient of variation of the signal amplitude. In certain examples, the non-transitory computer readable storage medium includes an algorithm for comparing the calculated signal amplitude to the light intensity of the light source. In certain examples, the non-transitory computer readable storage medium includes an algorithm for determining a parameter of the photodetector based on one or more of the calculated signal amplitude and the comparison between the calculated signal amplitude and the light intensity of the light source. For example, the non-transitory computer readable storage medium may include an algorithm for determining a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, or a number of photoelectrons per unit power. The non-transitory computer readable storage medium may include an algorithm for determining a detector parameter over a range of operating voltages of the photodetector, such as when the parameter of the photodetector is determined over the entire operating voltage range of the photodetector.

[0016] In certain embodiments, aspects of the present disclosure also include multispectral particles (e.g., beads) having one or more fluorophores for performing one or more of the subject methods. Multispectral particles according to some embodiments include one or more, such as two or more, such as three or more, such as five or more fluorophores, including ten or more fluorophores. In some examples, the subject particles include single-peak multifluorophore beads, which provide bright photodetector signals across all light source wavelengths (e.g., across all LEDs or lasers of the system) and across the detection wavelengths of the photodetector. [Brief description of the drawings]

[0017] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, which include:

[0018] [Figure 1A] 1 illustrates measurements of varying light intensity from a light source over multiple discrete time intervals by a photodetector according to certain embodiments, showing a 50 step light intensity ramp over 2601 ms. [Figure 1B] 1B illustrates measurements of varying light intensity from a light source over multiple discrete time intervals by a photodetector according to certain embodiments, showing the first 5 steps (over 250 ms) of the 50-step light intensity ramp of FIG. 1A. [Diagram 2] 1 illustrates measurements of continuously varying light intensity in accordance with certain embodiments. [Figure 3A] 1 illustrates a flowchart for determining one or more parameters of a photodetector in accordance with certain embodiments. [Figure 3B] 4 illustrates a plot used to set an initial detector gain of a photodetector in accordance with certain embodiments. [Figure 4A] FIG. 1 illustrates a functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization, in accordance with certain embodiments. [Figure 4B] 1 illustrates a flow cytometer according to certain embodiments. [Diagram 5] FIG. 1 illustrates a functional block diagram of an example particle analyzer system in accordance with certain embodiments. [Figure 6A] 1 illustrates a schematic diagram of a particle sorter system in accordance with certain embodiments. [Figure 6B] 1 illustrates a schematic diagram of a particle sorter system in accordance with certain embodiments. [Figure 7] 1 illustrates a block diagram of a computing system in accordance with certain embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Aspects of the present disclosure include methods for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). The method according to certain embodiments includes illuminating a photodetector positioned in the particle analyzer with a light source (e.g., a continuous wave light source) at a first intensity for a first predetermined time interval, illuminating the photodetector with the light source at a second intensity for a second predetermined time interval, integrating a data signal from the photodetector over a period including the first and second predetermined time intervals, and determining one or more parameters of the photodetector based on the integrated data signal. Systems having a light source and a photodetector for performing the subject methods (e.g., a particle analyzer) are also described. A non-transitory computer-readable storage medium having stored thereon instructions for determining parameters of a photodetector according to the subject methods is also provided.

[0020] Before the present invention is described in more detail, it should be understood that the invention is not limited to the particular embodiments described, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing only particular embodiments, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0021] Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within this stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0022] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as the number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically stated number, the number that is close to or approximately the unstated number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically stated number.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described below.

[0024] All publications and patents cited herein are incorporated by reference herein to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0025] It should be noted that, as used in this specification and the appended claims, the articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like in connection with the recitation of claim elements, or for use of a "negative" limitation.

[0026] As will be apparent to those skilled in the art upon reading this disclosure, each of the separate embodiments described and illustrated herein has separate components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0027] Although the apparatus and methods have been or will be described with functional descriptions for grammatical fluidity, it is expressly understood that unless expressly recited under 35 U.S.C. 35 U.S.C. 112, the claims should not necessarily be construed as limited by construction of "means" or "step" limitations, but should be accorded the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of legal equivalents, and where the claims are expressly recited under 35 U.S.C. 35 U.S.C. 112, they should be accorded the full legal equivalents under 35 U.S.C. 35 U.S.C.

[0028] As summarized above, the present disclosure provides methods and systems for determining one or more parameters of a photodetector (e.g., a photodetector of a particle analyzer). In further describing embodiments of the present disclosure, a method is first described in more detail, which includes illuminating a photodetector with a first intensity for a first predetermined time interval and with a second intensity for a second predetermined time interval, and integrating a data signal from the photodetector over a time period including the first and second predetermined time intervals. Next, a system (e.g., a particle analyzer) having a light source and a photodetector for performing the subject method is described. A non-transitory computer-readable storage medium having instructions stored thereon for determining parameters of a photodetector according to the subject method is also provided.

[0029] Method for determining parameters of a photodetector - Patent application Aspects of the present disclosure include methods for determining parameters of a photodetector (e.g., a photodetector in a particle analyzer). In performing the subject methods, the photodetector is illuminated with a light source at a first intensity for a first predetermined time interval, followed by illuminating the photodetector with the light source at a second intensity for a second predetermined time interval. In certain embodiments, the light source is a continuous light source. The term "continuous light source" is used herein in its conventional sense to refer to a light source that provides an uninterrupted light flux and maintains illumination of the photodetector with little or no undesirable changes in light intensity. In some embodiments, the continuous light source emits non-pulsed or non-stroboscopic illumination. In certain embodiments, the continuous light source provides a substantially constant luminous intensity. For example, the continuous light source may provide an intensity of light emitted during a time interval of illumination that varies by 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, such as 0.5% or less, such as 0.1% or less, such as 0.01% or less, such as 0.001% or less, such as 0.0001% or less, such as 0.00001% or less, including cases where the intensity of light emitted during a time interval of illumination varies by 0.000001% or less. The intensity of the light output may be measured using any convenient protocol, including, but not limited to, scanning slit profilers, charge coupled devices (CCDs, e.g., intensified charge coupled devices, ICCDs), positioning sensors, power sensors (e.g., thermopile power sensors), optical power sensors, energy meters, digital laser photometers, laser diode detectors, among other types of optical detectors.

[0030] In a particular example, the light source is a pulsed light source. The term "pulsed light source" is used herein in its conventional sense to refer to a light source that provides a light beam at a predetermined time interval, such as by stroboscopic illumination. The pulse duration may vary depending on the type of light source, and may be 0.001 ns or more, such as 0.005 ns or more, such as 0.01 ns or more, such as 0.05 ns or more, such as 0.1 ns or more, such as 0.5 ns or more, such as 1 ns or more, such as 2 ns or more, such as 3 ns or more, such as 5 ns or more, such as 10 ns or more, such as 25 ns or more, such as 50 ns or more, such as 100 ns or more, such as 500 ns or more, such as 1000 ns or more, and includes pulse durations of 5 μs or more. For example, the pulse duration of the pulsed light source may be in the range of 0.00001 μs to 1000 μs, such as 0.00005 μs to 900 μs, for example 0.0001 μs to 800 μs, for example 0.0005 μs to 700 μs, for example 0.001 μs to 600 μs, for example 0.005 μs to 500 μs, for example 0.01 μs to 400 μs, for example 0.05 μs to 300 μs, for example 0.1 μs to 200 μs, and including pulse durations in the range of 1 μs to 100 μs.

[0031] The light source may be any convenient light source, including laser light sources and non-laser light sources. In certain embodiments, the light source is a non-laser light source, such as a narrowband light source that emits a specific wavelength or a narrow range of wavelengths. In some examples, the narrowband light source emits light having a narrow range of wavelengths, such as 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less, and includes light sources that emit light of a specific wavelength (i.e., monochromatic light). Any convenient narrowband light source protocol, such as a narrow wavelength LED, can be used.

[0032] 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 having a wide range of wavelengths, such as 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 500 nm or more. For example, one suitable broadband light source emits light having a wavelength between 200 nm and 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having a wavelength between 400 nm and 1000 nm. Any convenient broadband light source protocol may be employed, such as halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectra, ultra-bright light emitting diodes, semiconductor light emitting diodes, broad spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof, among other broadband light sources. In a particular embodiment, the light source for illuminating the flow stream through an opening in the particle sorting module during image capture includes an array of infrared LEDs.

[0033] In certain embodiments, the light source is a laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near infrared diode laser. In other embodiments, the laser may be a helium-neon (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 CO2 laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In other examples, the subject systems include dye lasers, such as stilbene, coumarin, or rhodamine lasers. In yet another example, the laser of interest includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet another example, the laser is a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a Tarim YAG laser, a ytterbium YAG laser, a Y2O3 laser, or a cerium-doped laser, and combinations thereof.

[0034] In some embodiments, the light source is a narrow band light source. In some examples, the light source is a light source that outputs a specific wavelength, including 200 nm to 1500 nm, such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm, and 400 nm to 800 nm. In certain embodiments, the continuous wave light source emits light having a wavelength of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm, or 850 nm.

[0035] The photodetector may be illuminated by the light source from any suitable distance from the flow stream, such as 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, and 100 mm or more. In addition, illumination of the photodetector may be at any suitable angle, such as an angle in the range of 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, such as 30° to 60°, such as an angle of 90°.

[0036] In an embodiment, the method includes illuminating the photodetector with a light source for two or more discrete time intervals. The term "discrete time interval" is used herein in its conventional sense to refer to illuminating the photodetector with a light source for a predetermined duration, followed by a period during which the intensity of the light source is altered (e.g., increased), followed by a subsequent discrete time interval or illumination. In some embodiments, the method includes illuminating the photodetector with discrete time intervals including 0.1 ms or more, such as 0.5 ms or more, such as 1.0 ms or more, such as 5 ms or more, such as 10 ms or more, such as 20 ms or more, such as 30 ms or more, such as 40 ms or more, such as 50 ms or more, such as 60 ms or more, such as 70 ms or more, such as 80 ms or more, such as 90 ms or more, and 100 ms or more. In certain embodiments, each predetermined time interval for illuminating the photodetector is of the same duration. For example, each predetermined time interval according to the subject method may be 50 ms. In other embodiments, each predetermined time interval is different. In certain embodiments, the method comprises illuminating the photodetector with a continuous wave light source at a plurality of intensities each over a plurality of discrete time intervals, such as three or more discrete time intervals, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as fifteen or more, such as twenty or more, such as twenty-five or more, such as fifty or more, such as seventy-five or more, and including one hundred or more discrete time intervals.

[0037] In some embodiments, each of the multiple time intervals is the same duration. In other embodiments, each of the multiple time intervals is a different duration. In still other embodiments, some of the time intervals may be the same duration and some of the time intervals may be different durations.

[0038] In some embodiments, the intensity of illumination by the light source is substantially constant over the duration of each predetermined time interval, for example the intensity of illumination varies by 10% or less, such as 9% or less, for example 8% or less, such as 7% or less, for example 6% or less, such as 5% or less, for example 4% or less, such as 3% or less, for example 2% or less, such as 1% or less, for example 0.5% or less, such as 0.1% or less, for example 0.01% or less, such as 0.001% or less, for example 0.0001% or less, such as 0.00001% or less, including cases where the intensity of illumination by the light source varies by 0.000001% or less over the duration of a predetermined time interval.

[0039] In some embodiments, the intensity of the light source is changed after each discrete illumination interval. In some embodiments, the intensity of illumination by the light source is increased. In other embodiments, the intensity of the light source is decreased. The intensity of the light used to illuminate the photodetector can be changed by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, including 100% or more, for each subsequent time interval. In certain examples, the intensity of the light is changed by 1.5 times or more, such as 2 times or more, such as 3 times or more, such as 4 times or more, including 5 times or more. In some embodiments, the method includes increasing the light intensity for each subsequent time interval by increasing the light intensity by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, including 100% or more, for each subsequent time interval. In other embodiments, the method comprises increasing the light intensity for each subsequent time interval by 1.5 times or more, such as 2 times or more, such as 3 times or more, including 4 times or more and 5 times or more.

[0040] In some examples, the method includes maintaining illumination of the light detector by the light source while the intensity is changing (e.g., while the light intensity is increasing). In some examples, the method includes increasing the intensity of the light from the light source over a period of time that includes at least a first predetermined time interval and a second predetermined time interval. In some examples, the intensity of the light from the light source increases linearly over a period of time that includes at least the first predetermined time interval and the second predetermined time interval. In other examples, the intensity of the light from the light source increases exponentially over a period of time that includes at least the first predetermined time interval and the second predetermined time interval.

[0041] In other examples, the method includes ceasing illumination of the light detector by the light source while the intensity of the light source is being changed (e.g., by turning the light source off or blocking the light source with a chopper, beam stopper, etc.). Any convenient protocol can be used to provide the intermittent illumination, such as an electronic switch for turning the light source on and off, such as a switch that is computer controlled and triggered based on a data signal (e.g., a received or input data signal), as described in more detail below. In some embodiments, the time interval for varying the intensity of the light source may be 0.001 ms or more, such as 0.005 ms or more, such as 0.01 ms or more, such as 0.05 ms or more, such as 0.1 ms or more, such as 0.5 ms or more, such as 1 ms or more, such as 2 ms or more, such as 3 ms or more, such as 4 ms or more, such as 5 ms or more, such as 6 ms or more, such as 7 ms or more, such as 8 ms or more, such as 9 ms or more, including 10 ms or more. For example, the period between each predetermined time interval for illuminating the photodetector with the light source may be from 0.001 ms to 25 ms, including for example 0.005 ms to 20 ms, for example 0.01 ms to 15 ms, including for example 0.05 ms to 10 ms, and 0.1 ms to 5 ms.

[0042] The methods of the present disclosure also include detecting light with a photodetector, according to certain embodiments. Photodetectors for implementing the subject methods may be any convenient light detection protocol, including, but not limited to, light sensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or avalanche photodiodes (APDs), silicon photomultiplier tubes, and combinations thereof, among other photodetectors. In certain embodiments, the photodetector has a resolution of 0.01 cm. 2 ~10cm 2 , e.g. 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2 In another embodiment, the photodetector is a photomultiplier tube such as a photomultiplier tube having an active detection surface area of ​​0.01 cm 2 ~10cm 2 , e.g. 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2In some examples, the light is detected by an array of photodetectors, such as a photodetector array having two or more photodetectors, e.g., three or more photodetectors, e.g., five or more photodetectors, e.g., ten or more photodetectors, e.g., twenty-five or more photodetectors, e.g., fifty or more photodetectors, e.g., seventy-five or more photodetectors, e.g., one hundred or more photodetectors, e.g., five or more photodetectors, including a photodetector array having one thousand or more photodetectors. In a particular example, the light is detected by an array of avalanche photodiodes, such as a photodetector array having two or more avalanche photodiodes, for example three or more avalanche photodiodes, for example five or more avalanche photodiodes, for example ten or more avalanche photodiodes, for example twenty-five or more avalanche photodiodes, for example fifty or more avalanche photodiodes, for example seventy-five or more avalanche photodiodes, for example one hundred or more avalanche photodiodes, for example five hundred or more avalanche photodiodes, including a photodetector array having one thousand or more avalanche photodiodes.

[0043] In embodiments of the present disclosure, light may be measured by the photodetector at one or more wavelengths, such as at two or more wavelengths, for example at five or more different wavelengths, such as at ten or more different wavelengths, for example at twenty-five or more different wavelengths, such as at fifty or more different wavelengths, for example at one hundred or more different wavelengths, such as at two hundred or more different wavelengths, for example at three hundred or more different wavelengths, including measuring light from particles in the flow stream at four hundred or more different wavelengths.

[0044] In embodiments, the light can be measured continuously or at discrete intervals. In some examples, the subject detector is configured to measure the light continuously. In other examples, the subject detector is configured to measure the light at discrete intervals, including measuring the light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at some other interval.

[0045] Measurements of the light from the light source may be taken 1 or more times during each discrete time interval, including 2 or more times, such as 3 or more times, such as 5 or more times, and 10 or more times. In certain embodiments, the light from the light source is measured by the photodetector 2 or more times, and the data in certain instances is averaged.

[0046] 1A and 1B illustrate measurements of varying light intensity from a light source over multiple discrete time intervals by a photodetector according to certain embodiments. The photodetector is illuminated by the light source with a constant light intensity for each 50 ms time interval, followed by a 2 dB increase in light intensity over a 1 ms ramp-up time interval. FIG. 1A illustrates a 50-step light intensity ramp over 2601 ms. FIG. 1B illustrates the first 5 steps (over 250 ms) of the 50-step light intensity ramp of FIG. 1A. FIG. 2 illustrates measurements of continuously varying light intensity according to certain embodiments. The photodetector is illuminated with a light intensity that increases continuously over time, and the data signal is integrated over two or more predetermined time intervals to determine a parameter of the photodetector.

[0047] FIG. 3 shows a flow chart for determining one or more parameters of a photodetector according to a particular embodiment. In step 301, the photodetector is illuminated by a continuous wave light source at a first light intensity for a first predetermined discrete time interval. In step 302, the photodetector is illuminated by a light source at a second intensity for a second predetermined discrete time interval. In step 303, a data signal from the photodetector is integrated over a time period including at least the first time interval and the second time interval. Based on the integrated data signal, a signal amplitude from the photodetector is calculated in step 304. In step 305, the calculated signal amplitude is used to determine one or more parameters, for example, the calculated signal amplitude is compared to the intensity of light during each illumination interval. For example, a comparison of the calculated signal amplitude to the intensity of light during each illumination interval can be used to determine a minimum detection threshold 305a, a maximum detection threshold 305b, a detector sensitivity 305c, a detector dynamic range 305d, a detector signal-to-noise ratio 305e, or a number of photoelectrons per unit power 305f.

[0048] In an embodiment, the method includes integrating the data signal from the photodetector. In some embodiments, integrating the data signal from the photodetector includes integrating the data signal over 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more of the duration of each discrete interval of illumination, including integrating the data signal over 99% of the duration of each discrete interval of illumination. In some embodiments, the data signal from the photodetector is integrated over the entire duration of each discrete time interval of illumination according to the subject method.

[0049] In some embodiments, the method includes integrating the data signal from the photodetector over a period that includes at least each of the discrete time intervals of illumination at each of the different light intensities. For example, if the photodetector is illuminated by a continuous wave light source over 50 or more discrete intervals, the method includes integrating the data signal from the photodetector over a period that includes the duration of at least 50 discrete time intervals. In some embodiments, the method includes integrating the data signal from the photodetector over a period that includes a duration prior to illumination of the photodetector by the subject method, such as to measure a noise component for the photodetector signal. In these embodiments the method comprises integrating the data signal from the photodetector for 0.001 ms or more, such as 0.005 ms or more, for example 0.01 ms or more, such as 0.05 ms or more, for example 0.1 ms or more, such as 0.5 ms or more, for example 1 ms or more, such as 2 ms or more, for example 3 ms or more, such as 4 ms or more, for example 5 ms or more, such as 10 ms or more, for example 25 ms or more, such as 50 ms or more, for example 100 ms or more prior to illuminating the photodetector, including integrating for 250 ms or more prior to illuminating the photodetector. In other embodiments, the method comprises integrating the data signal from the photodetector after a final discrete time interval of illumination, including 0.005 ms or more, such as 0.01 ms or more, for example 0.05 ms or more, such as 0.1 ms or more, for example 0.5 ms or more, such as 1 ms or more, for example 2 ms or more, such as 3 ms or more, for example 4 ms or more, such as 5 ms or more, for example 10 ms or more, such as 25 ms or more, for example 50 ms or more, such as 100 ms or more, and 250 ms or more.

[0050] In certain embodiments, integrating the data signal from the photodetector includes calculating a signal amplitude over a period of time. In some examples, calculating the signal amplitude includes calculating a median signal amplitude. In certain examples, the method also includes comparing the calculated signal amplitude to the light intensity of the light source. In other examples, the method includes calculating an average signal amplitude. In some examples, the method also includes calculating a standard deviation of the signal amplitude. In other examples, the method includes calculating a variance and coefficient of variation (e.g., CV=standard deviation / mean) of the signal amplitude. Based on one or more of the calculated signal amplitude and a comparison of the calculated signal amplitude to the light intensity of the light source, parameters of the photodetector are calculated. For example, the method may include determining parameters for the photodetector such as a minimum detection threshold, a maximum detection threshold, a detector sensitivity (i.e., the ratio of detector output to detector input), a detector dynamic range (the range of detector signal from the minimum detection threshold to the maximum detection threshold), a detector signal-to-noise ratio, or a number of photoelectrons per unit output.

[0051] Each of the detector parameters may be determined over a range of the photodetector's operating voltage. In some embodiments, the parameters are determined based on signal amplitudes calculated over 10% or more of the photodetector's operating voltage, such as 15% or more, such as 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and the parameters are determined over 99% or more of the photodetector's operating voltage. In a particular example, each of the parameters may be determined over the entire photodetector's operating voltage range.

[0052] In certain examples, the parameters of the photodetector may be adjusted based on one or more of the calculated signal amplitude or a comparison between the calculated signal amplitude and the intensity of illumination during each discrete time interval. The term "adjust" is used herein in its conventional sense to refer to changing one or more functional parameters of the photodetector. For example, adjusting the photodetector may include increasing or decreasing the voltage gain of the photodetector. In certain embodiments, adjusting one or more parameters of the photodetector based on the calculated signal amplitude or a comparison between the calculated signal amplitude and the intensity of illumination during each discrete time interval of interest may be fully automated, such that the adjustments made require little or no human intervention or manual input by a user.

[0053] In some embodiments, the method includes determining one or more parameters of a photodetector (e.g., a photodetector in a particle analyzer) by illuminating particles in the flow stream, the particles including one or more fluorophores. In some examples, the particles are beads (e.g., polystyrene beads), as described in more detail below. In some examples, the subject method as described below provides for determining parameters of the photodetectors, including assigned relative fluorescence units (e.g., ABD units) for each photodetector, a robust coefficient of variation (rCV) for one or more of the photodetectors, maximum and minimum linearity for each photodetector, a relative change in rCV from baseline, a relative change in detector gain from baseline, and imaging specifications of the photodetectors, such as RF power or axial light loss.

[0054] In some examples, a method for determining a parameter of a photodetector includes illuminating a flow stream having particles comprising one or more fluorophores at a first intensity for a first predetermined time interval and at a second intensity for a second predetermined time interval, detecting light from the flow stream with a photodetector having a light source, generating a data signal from the photodetector at the first illumination intensity and generating a data signal from the photodetector at the second illumination intensity, and determining one or more parameters of the photodetector based on the data signals generated at the first intensity and the second intensity.

[0055] In some embodiments, the method includes determining a mean fluorescence intensity (M) from the particles at the first illumination intensity and the second illumination intensity. In some examples, the method includes determining the variance of the mean fluorescence intensity (V(M)) at the first illumination intensity and the second illumination intensity. In particular examples, the method includes determining a %rCV (robust coefficient of variation) of the photodetector. In particular embodiments, a linear fit of the variance is calculated according to:

[0056]

number

[0057] Here, Q led is given by 1 / c1, which is unit mean fluorescence intensity (M) (i.e., SPE / MFI). In certain embodiments, the variance is plotted and a linear fit of the variance is determined according to y=c1x+c0.

[0058] In embodiments, the mean fluorescence intensity and variance may be determined for a number of different illumination intensities, including for example two or more illumination intensities, such as three or more, for example four or more, such as five or more, for example six or more, such as seven or more, for example eight or more, such as nine or more, for example ten or more, and fifteen or more.

[0059] In some embodiments, the method includes determining a statistical photoelectron (SPE) at one or more of the illumination intensities, such as at least the first illumination intensity and the second illumination intensity. In a particular example, the method determines a detector efficiency (Q ) of a photodetector for each particle based on the statistical photoelectron and the determined average fluorescence intensity of the particles. det ) for determining detector efficiencies of one or more, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as twelve or more, such as six or more, such as twenty or more, such as twenty or more, such as thirty-six or more, such as forty-eight or more, such as seventy-two or more, detector channels of the photodetector, and determining detector efficiencies of 96 or more detector channels of the photodetector based on the statistical photoelectrons and the determined average fluorescence intensity of the particles. In a particular example, the method comprises determining detector efficiencies of all detector channels of the photodetector for each particle based on the statistical photoelectrons and the determined average fluorescence intensity of each particle. In a particular embodiment, the detector efficiency of the photodetector is determined according to:

[0060]

number

[0061] where SPE is the statistical photoelectron, MFI is the mean fluorescence intensity, and ABD is the allocation units per channel per particle lot.

[0062] In certain embodiments, the method further comprises determining the background signal for one or more of the photodetectors.In some examples, the background signal is determined at one or more, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, for example, ten or more illumination intensities, including ten or more different illumination intensities.In some examples, the background signal is determined at all of the applied illumination intensities. The background signal can also be determined in one or more detector channels of the photodetector, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as twelve or more, such as six or more, such as twenty or more, such as twenty or more, such as thirty-six or more, such as forty-eight or more, such as seventy-two or more, including determining the background signal in 96 or more detector channels of the photodetector, and in certain examples, the background signal is determined in all of the detector channels of the photodetector. In some examples, the background signal is determined based on statistical photoelectron and detector efficiency of the photodetector. In certain examples, the background signal is determined according to the following:

[0063]

number

[0064] In some embodiments, the method further comprises determining electronic noise for one or more of the photodetectors. In some examples, the electronic noise of the photodetectors is determined at one or more, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more illumination intensities, including ten or more different illumination intensities. In some examples, the electronic noise of the photodetectors is determined at all of the applied illumination intensities. The electronic noise can also be determined in one or more detector channels of the photodetector, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as twelve or more, such as six or more, such as twenty or more, such as twenty or more, such as thirty-six or more, such as forty-eight or more, such as seventy-two or more, including determining the electronic noise in 96 or more detector channels of the photodetector, and in certain examples, the electronic noise is determined in all of the detector channels of the photodetector. In some examples, the electronic noise is determined based on statistical photoelectrons and detector efficiency of the photodetector. In certain examples, the electronic noise is determined according to the following:

[0065]

number

[0066] In some embodiments, the method further comprises determining a detection limit for one or more of the photodetectors. In some examples, the detection limit of the photodetector is determined for one or more, such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 12 or more, such as 16 or more, such as 20 or more, such as 24 or more, such as 36 or more, such as 48 or more, such as 72 or more detector channels of the photodetector, including determining the detection limit of the photodetector for 96 or more detector channels of the photodetector, and in certain examples, the detection limit is determined for all of the detector channels of the photodetector. In some examples, the detection limit of each photodetector is determined by 2+2SD=4(1+B SD) is used to determine the

[0067] In some embodiments, the method further includes determining a detector photosensitivity of one or more photodetectors. In certain embodiments, determining the detector photosensitivity of the photodetector includes setting an initial detector gain of the photodetector. In some examples, the method includes illuminating the photodetector with a light source (as described in detail above) at a plurality of different light intensities, generating data signals from the photodetector for the plurality of light intensities at one or more detector gains of the photodetector, and determining a minimum light illumination intensity that produces a data signal resolvable from a background data signal at each detector gain. In some examples, the method includes determining a minimum light illumination intensity that produces a data signal that is two standard deviations from the background data signal at each detector gain. In certain examples, the method includes setting the detector gain to a gain at which the minimum light illumination intensity that produces a data signal resolvable from the background data signal reaches a plateau when plotted as a function of light intensity. FIG. 3B illustrates a plot used to set the initial detector gain of the photodetector according to certain embodiments. As shown in Figure 3B, the detector gain of the photodetector is plotted as a function of light (e.g., LED) illumination intensity for two different fluorophores (e.g., fluorophores that are stably associated with particles, as described in more detail below). In setting the initial detector gain of the photodetector, the detector gain is determined when the lowest light illumination intensity that produces a data signal that is resolvable from the background data signal reaches a plateau, which in Figure 3B is approximately 575 volts.

[0068] System for determining parameters of a photodetector - Patent application Aspects of the present disclosure also include a system having a light source and a photodetector configured to detect light from the light source at different illumination intensities over a predetermined time interval. In some embodiments, the light source is a continuous wave light source. The term "continuous wave light source" is used herein in its conventional sense to refer to a light source that provides an uninterrupted light flux and maintains illumination of the photodetector with little or no undesirable changes in light intensity. In some embodiments, the continuous light source emits non-pulsed or non-stroboscopic illumination. In certain embodiments, the continuous light source provides a substantially constant light emission intensity. For example, the continuous light source may provide a light intensity emitted during a time interval of illumination that varies by 10% or less, such as 9% or less, for example 8% or less, such as 7% or less, for example 6% or less, such as 5% or less, such as 4% or less, for example 3% or less, such as 2% or less, for example 1% or less, such as 0.5% or less, such as 0.1% or less, for example 0.01% or less, such as 0.001% or less, such as 0.0001% or less, for example 0.00001% or less, including cases where the light intensity emitted during a time interval of illumination varies by 0.000001% or less.

[0069] In a particular example, the light source is a pulsed light source. The term "pulsed light source" is used herein in its conventional sense to refer to a light source that provides a light beam at a predetermined time interval, such as by stroboscopic illumination. The pulse duration may vary depending on the type of light source, and may be 0.001 ns or more, such as 0.005 ns or more, such as 0.01 ns or more, such as 0.05 ns or more, such as 0.1 ns or more, such as 0.5 ns or more, such as 1 ns or more, such as 2 ns or more, such as 3 ns or more, such as 5 ns or more, such as 10 ns or more, such as 25 ns or more, such as 50 ns or more, such as 100 ns or more, such as 500 ns or more, such as 1000 ns or more, and includes pulse durations of 5 μs or more. For example, the pulse duration of the pulsed light source may be in the range of 0.00001 μs to 1000 μs, such as 0.00005 μs to 900 μs, for example 0.0001 μs to 800 μs, for example 0.0005 μs to 700 μs, for example 0.001 μs to 600 μs, for example 0.005 μs to 500 μs, for example 0.01 μs to 400 μs, for example 0.05 μs to 300 μs, for example 0.1 μs to 200 μs, and including pulse durations in the range of 1 μs to 100 μs.

[0070] The continuous wave light source may be any convenient light source, including laser light sources and non-laser light sources. In certain embodiments, the light source is a non-laser light source, such as a narrow band light source that emits a specific wavelength or a narrow range of wavelengths. In some examples, the narrow band light source emits light having a narrow range of wavelengths, such as 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less, and includes light sources that emit light of a specific wavelength (i.e., monochromatic light). Any convenient narrow band light source protocol can be used, such as a narrow wavelength LED.

[0071] 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 having a wide range of wavelengths, such as 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 500 nm or more. For example, one suitable broadband light source emits light having a wavelength between 200 nm and 1500 nm. Another example of a suitable broadband light source includes a light source that emits light having a wavelength between 400 nm and 1000 nm. Any convenient broadband light source protocol may be employed, such as halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectra, ultra-bright light emitting diodes, semiconductor light emitting diodes, broad spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof, among other broadband light sources. In a particular embodiment, the light source for illuminating the flow stream through an opening in the particle sorting module during image capture includes an array of infrared LEDs.

[0072] In certain embodiments, the light source is a laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near infrared diode laser. In other embodiments, the laser may be a helium-neon (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 CO2 laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In other examples, the subject systems include dye lasers, such as stilbene, coumarin, or rhodamine lasers. In yet another example, the laser of interest includes a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet another example, the subject system includes a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a Tarim YAG laser, a Ytterbium YAG laser, a Y2O3 laser, or a cerium-doped laser, and combinations thereof.

[0073] In some embodiments, the light source is a narrowband light source. In some cases, the light source is a light source that outputs a specific wavelength, including 200 nm to 1500 nm, such as 250 nm to 1250 nm, such as 300 nm to 1000 nm, such as 350 nm to 900 nm, and 400 nm to 800 nm. In certain embodiments, the light source emits light having a wavelength of 365 nm, 385 nm, 405 nm, 460 nm, 490 nm, 525 nm, 550 nm, 580 nm, 635 nm, 660 nm, 740 nm, 770 nm, or 850 nm. In certain embodiments, the wavelength of the light output by the light source is matched with an optical tuning component used when illuminating a photodetector, such as a bandpass filter or a dichroic mirror. In some cases, the wavelength of the light output by the light source matches the spectral bandwidth of a bandpass filter in optical communication with the photodetector.

[0074] The light source may be positioned at any suitable distance from the light detector, for example the light source and the light detector are separated by a distance of 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, such as 25 mm or more, and 100 mm or more. Further, the light source may be positioned at any suitable angle relative to the light detector, for example an angle in the range of 10° to 90°, such as 15° to 85°, such as 20° to 80°, such as 25° to 75°, and including 30° to 60°, for example an angle of 90°.

[0075] The light source according to certain embodiments may also include one or more optical conditioning components. The term "optical conditioning" is used herein in its conventional sense to refer to any device capable of changing the spatial width of the illumination or other characteristics of a portion of the illumination from the light source, such as, for example, the illumination direction, wavelength, beam width, beam intensity, and focus. The optical conditioning protocol may be any convenient device that adjusts one or more characteristics of the light source, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof. In certain embodiments, the subject system includes one or more focusing lenses. The focusing lens may be, in one example, a reduction lens. In another example, the focusing lens is a magnification lens. In other embodiments, the subject system includes one or more mirrors. In yet other embodiments, the subject system includes an optical fiber.

[0076] When the optical conditioning component is configured to move, the optical conditioning component may be configured to move continuously or in discrete intervals. In some embodiments, the movement of the optical conditioning component is continuous. In other embodiments, the optical conditioning component can be moved in discrete intervals, including increments of, for example, 0.01 microns or more, such as 0.05 microns or more, such as 0.1 microns or more, such as 0.5 microns or more, such as 1 micron or more, such as 10 microns or more, such as 100 microns or more, such as 500 microns or more, such as 1 mm or more, such as 5 mm or more, such as 10 mm or more, and 25 mm or more.

[0077] Any displacement protocol may be employed to move the optical adjustment component structure, such as those employing stepper motors, servo motors, brushless electric motors, brushed DC motors, microstep drive motors, high resolution stepper motors, among other types of motors, coupled to a movable support stage, or directly coupled to a motor-actuated translation stage, a lead screw translation assembly, or a geared translation device.

[0078] In an embodiment, the continuous wave light source is configured to illuminate for two or more discrete time intervals, each time interval being a different illumination intensity. In some embodiments, the continuous wave light source is configured to illuminate at a specific intensity for a time interval including 0.1 ms or more, such as 0.5 ms or more, such as 1.0 ms or more, such as 5 ms or more, such as 10 ms or more, such as 20 ms or more, such as 30 ms or more, such as 40 ms or more, such as 50 ms or more, such as 60 ms or more, such as 70 ms or more, such as 80 ms or more, such as 90 ms or more, and 100 ms or more. For example, the continuous wave light source may be configured to illuminate at a specific light intensity for 50 ms.

[0079] In some embodiments, the light source is configured to maintain a substantially constant light intensity over the duration of each predetermined time interval, such as when the intensity of illumination changes by 10% or less, such as 9% or less, for example 8% or less, such as 7% or less, for example 6% or less, such as 5% or less, such as 4% or less, for example 3% or less, such as 2% or less, for example 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, including when the intensity of illumination by the light source changes by 0.000001% or less over the duration of the predetermined time interval.

[0080] In some examples, the light source is configured to increase its intensity over a period of time that includes at least a first and a second predetermined time interval. In some examples, the intensity of the light from the light source increases linearly over a period of time that includes at least the first and the second predetermined time interval. In other examples, the intensity of the light from the light source increases exponentially over a period of time that includes at least the first and the second predetermined time interval.

[0081] The photodetectors of the subject systems may be of any convenient light detection protocol, including, but not limited to, optical sensors or photodetectors such as active pixel sensors (APS), quadrant photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors or avalanche photodiodes, silicon photomultiplier tubes, and combinations thereof, among other photodetectors. In certain embodiments, the photodetectors have a resolution of 0.01 cm. 2 ~10cm 2 , e.g. 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2 In another embodiment, the photodetector is a photomultiplier tube such as a photomultiplier tube having an active detection surface area of ​​0.01 cm 2 ~10cm 2 , e.g. 0.05 cm 2 ~9cm 2 , e.g. 0.1 cm 2 ~8cm 2 , e.g. 0.5 cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2In some examples, the light is detected by an array of photodetectors, such as a photodetector array having two or more photodetectors, e.g., three or more photodetectors, e.g., five or more photodetectors, e.g., ten or more photodetectors, e.g., twenty-five or more photodetectors, e.g., fifty or more photodetectors, e.g., seventy-five or more photodetectors, e.g., one hundred or more photodetectors, e.g., five or more photodetectors, including a photodetector array having one thousand or more photodetectors. In a particular example, the light is detected by an array of avalanche photodiodes, such as a photodetector array having two or more avalanche photodiodes, for example three or more avalanche photodiodes, for example five or more avalanche photodiodes, for example ten or more avalanche photodiodes, for example twenty-five or more avalanche photodiodes, for example fifty or more avalanche photodiodes, for example seventy-five or more avalanche photodiodes, for example one hundred or more avalanche photodiodes, for example five hundred or more avalanche photodiodes, including a photodetector array having one thousand or more avalanche photodiodes.

[0082] In embodiments of the present disclosure, the photodetector may be configured to detect light at one or more wavelengths, such as at two or more wavelengths, such as at five or more different wavelengths, such as at ten or more different wavelengths, such as at twenty-five or more different wavelengths, such as at fifty or more different wavelengths, such as at one hundred or more different wavelengths, such as at two hundred or more different wavelengths, such as at three hundred or more different wavelengths, including measuring light from particles in the flow stream at four hundred or more different wavelengths.

[0083] In embodiments, the light detector may be configured to measure light continuously or at discrete intervals. In some examples, the subject detector is configured to measure light continuously. In other examples, the subject detector is configured to measure light at discrete intervals, including measuring light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at some other interval.

[0084] Measurements of the light from the light source may be taken 1 or more times during each discrete time interval, such as 2 or more times, such as 3 or more times, such as 5 or more times, and 10 or more times. In certain embodiments, the light from the light source is measured by the photodetector 2 or more times, and the data in certain instances is averaged.

[0085] In an embodiment, the system also includes a processor having a memory operably coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to integrate a data signal from the photodetector over a period of time that includes each of the discrete illumination intervals and determine one or more parameters of the photodetector based on the integrated data signal. In certain embodiments, the memory includes instructions that, when executed by the processor, cause the processor to calculate a median signal amplitude. In other examples, the memory includes instructions that, when executed by the processor, cause the processor to calculate a mean signal amplitude. In some examples, the memory includes instructions that, when executed by the processor, cause the processor to also calculate a standard deviation of the signal amplitude. In other examples, the memory includes instructions that, when executed by the processor, cause the processor to also calculate a variance and a coefficient of variation (e.g., CV=standard deviation / mean) of the signal amplitude. In certain embodiments, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to compare the calculated signal amplitude to the light intensity of the light source.

[0086] In some embodiments, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored thereon that, when executed by the processor, cause the processor to compare the calculated signal amplitude to the light intensity of the light source. Based on one or more of the calculated signal amplitude and the comparison between the calculated signal amplitude and the light intensity of the light source, the subject system includes a memory having instructions for calculating parameters of the photodetector. For example, the memory may include instructions for determining parameters for the photodetector, such as a minimum detection threshold, a maximum detection threshold, a detector sensitivity (i.e., the ratio of the detector output to the detector input), a detector dynamic range (the range of the detector signal from the minimum detection threshold to the maximum detection threshold), a detector signal-to-noise ratio, or a number of photoelectrons per unit output. In some embodiments, the memory includes instructions for determining the photodetector parameters over a range of operating voltages of the photodetector. In a particular embodiment, the memory includes instructions for calculating the signal amplitude of the photodetector over 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more of the operating voltage of the photodetector and determining the parameter over 99% or more of the operating voltage of the photodetector. In a particular example, the memory includes instructions for calculating the signal amplitude of the photodetector over the entire operating voltage range of the photodetector.

[0087] In some embodiments, the system 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 determine one or more parameters of the photodetectors, the memory including instructions for irradiating particles (e.g., multispectral beads as described below) in the flow stream, the particles including one or more fluorophores. In some examples, the memory includes instructions that, when executed by the processor, cause the processor to determine parameters of the photodetectors, including assigned relative fluorescence units (e.g., ABD units) for each photodetector, a robust coefficient of variation (rCV) for one or more of the photodetectors, maximum and minimum linearity for each photodetector, a relative change in rCV from baseline, a relative change in detector gain from baseline, and imaging specifications of the photodetectors such as RF power or axial light loss.

[0088] In some examples, the memory includes instructions for determining a parameter of the photodetector, the instructions including illuminating a flow stream having particles comprising one or more fluorophores at a first intensity for a first predetermined time interval and at a second intensity for a second predetermined time interval, detecting light from the flow stream with a photodetector having a light source, generating a data signal from the photodetector at the first illumination intensity and generating a data signal from the photodetector at the second illumination intensity, and determining one or more parameters of the photodetector based on the data signals generated at the first intensity and the second intensity.

[0089] In some embodiments, the memory includes instructions for determining a mean fluorescence intensity (M) from the particles at a first illumination intensity and a second illumination intensity. In some examples, the memory includes instructions for determining a variance of the mean fluorescence intensity (V(M)) at the first illumination intensity and the second illumination intensity. In particular examples, the memory includes instructions for determining a %rCV (robust coefficient of variation) of the photodetector. In particular embodiments, the memory includes instructions that, when executed by the processor, cause the process to calculate a linear fit of the variance according to:

[0090]

number

[0091] Here, Q led is given by 1 / c1, which is unit mean fluorescence intensity (M) (i.e., SPE / MFI). In certain embodiments, the memory includes instructions for plotting the variance to determine a linear fit of the variance according to y=c1x+c0.

[0092] In embodiments the mean fluorescence intensity and variance may be determined for a number of different illumination intensities, such as two or more illumination intensities, such as three or more, for example four or more, such as five or more, for example six or more, such as seven or more, for example eight or more, such as nine or more, for example ten or more, and fifteen or more different illumination intensities.

[0093] In some embodiments, the memory includes instructions for determining a statistical photoelectron (SPE) at one or more of the illumination intensities, such as at least the first illumination intensity and the second illumination intensity. In a particular example, the memory determines a detector efficiency (Q) of the photodetector for each particle based on the statistical photoelectron and the determined average fluorescence intensity of the particles. det) for calculating the average fluorescence intensity of the particles. In certain embodiments, the memory includes instructions for determining detector efficiencies of one or more, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as twelve or more, such as sixteen or more, such as twenty or more, such as twenty or more, such as thirty-six or more, such as forty-eight or more, such as seventy-two or more, detector channels of the photodetector and determining detector efficiencies of 96 or more detector channels of the photodetector based on the statistical photoelectrons and the determined average fluorescence intensity of the particles. In certain examples, the memory includes instructions for determining detector efficiencies of all detector channels of the photodetector for each particle based on the statistical photoelectrons and the determined average fluorescence intensity of each particle. In certain embodiments, the memory includes instructions that, when executed by the processor, cause the processor to determine a detector efficiency determined according to:

[0094]

number

[0095] where SPE is the statistical photoelectron, MFI is the mean fluorescence intensity, and ABD is the allocation units per channel per particle lot.

[0096] In certain embodiments, the memory includes instructions for determining background signals for one or more of the photodetectors. In some examples, the background signal is determined at one or more, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more illumination intensities, including ten or more different illumination intensities. In some examples, the memory includes instructions for determining background signals at all of the applied illumination intensities. In some embodiments, the memory includes instructions for determining background signals at one or more, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as twelve or more, such as sixteen or more, such as twenty or more, such as twenty four or more, such as thirty six or more, such as forty eight or more, such as seventy two or more detector channels of the photodetector, and the memory includes instructions for determining background signals at ninety six or more detector channels of the photodetector. In particular examples, the memory includes instructions for determining a background signal in all of the detector channels of the photodetector. In some examples, the memory includes instructions that, when executed by the processor, cause the processor to determine the background signal based on statistical photoelectrons and detector efficiency of the photodetector. In particular examples, the memory includes instructions for determining the background signal according to:

[0097]

number

[0098] In some embodiments, the memory includes instructions for determining electronic noise for one or more of the photodetectors. In some examples, the electronic noise of the photodetector is determined at one or more, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more illumination intensities, including ten or more different illumination intensities. In some examples, the electronic noise of the photodetector is determined at all of the applied illumination intensities. The electronic noise can similarly be determined at one or more, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as twelve or more, such as sixteen or more, such as twenty or more, such as twenty four or more, such as thirty six or more, such as forty eight or more, such as seventy two or more detector channels of the photodetector, including determining electronic noise at ninety six or more detector channels of the photodetector. In particular examples, the memory includes instructions for determining electronic noise in all of the detector channels of the photodetector. In some examples, the memory includes instructions for determining electronic noise based on statistical photoelectrons and detector efficiency of the photodetector. In particular examples, the memory includes instructions for determining electronic noise according to:

[0099]

number

[0100] In some embodiments, the memory includes instructions for determining the detection limit of one or more of the photodetectors. In some examples, the memory includes instructions for determining the detection limit of the photodetectors in one or more, such as two or more, such as three or more, such as four or more, such as five or more, such as six or more, such as seven or more, such as eight or more, such as nine or more, such as ten or more, such as twelve or more, such as sixteen or more, such as twenty or more, such as twenty four or more, such as thirty six or more, such as forty eight or more, such as seventy two or more detector channels of the photodetectors, and the memory includes instructions for determining the detection limit of the photodetectors in ninety six or more detector channels of the photodetectors. In a particular example, the memory includes instructions for determining the detection limit in all of the detector channels of the photodetectors. In some examples, the memory includes instructions for determining the detection limit of the photodetectors in a range of 2+2SD=4(1+B SD ) for determining the detection limit of each photodetector according to

[0101] In some embodiments, the memory includes instructions for determining a detector photosensitivity of one or more photodetectors. In certain embodiments, the memory includes instructions for setting an initial detector gain for the photodetector. In some examples, the memory includes instructions for illuminating the photodetector with a light source (as described in detail above) at a plurality of different light intensities, instructions for generating data signals from the photodetector for the plurality of light intensities at one or more detector gains of the photodetector, and instructions for determining a minimum light illumination intensity that produces a data signal resolvable from a background data signal at each detector gain. In some examples, the memory includes instructions for determining a minimum light illumination intensity that produces a data signal that is two standard deviations from the background data signal at each detector gain. In certain examples, the memory includes instructions for setting the detector gain to a gain at which the minimum light illumination intensity that produces a data signal resolvable from the background data signal reaches a plateau when plotted as a function of light intensity.

[0102] In certain embodiments, the photodetector is a photodetector positioned in a particle analyzer, such as a particle sorter. In certain embodiments, the subject system is a flow cytometry system that includes a photodetector as part of a light detection system for detecting light emitted by a sample in a flow stream. Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt 1): 17-28; Linden, et al., Semin Thromb Hemost. 2004 Oct; 30(5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222(4): 335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are incorporated herein by reference.In particular examples, flow cytometry systems of interest include BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortess™ X-20 flow cytometer, and BD Biosciences FACSCalibur™ flow cytometer, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, and BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, and BD and the Biosciences FACSMelody™ cell sorter.

[0103] In some embodiments, the subject particle sorting systems may be configured in accordance with U.S. Pat. Nos. 10,006,852, 9,952,076, 9,933,341, 9,784,661, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, 7, and flow cytometry systems such as those described in Nos. 544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, and 5,602,039, the disclosures of which are incorporated herein by reference in their entireties.

[0104] In certain embodiments, the subject systems are flow cytometry systems having an excitation module that uses radio frequency multiplexing excitation to generate multiple frequency shifted beams of light. In certain examples, the subject systems are flow cytometry systems such as those described in U.S. Patent Nos. 9,423,353 and 9,784,661, and U.S. Patent Application Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0105] In some embodiments, the subject system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting determination module having a plurality of sorting determination units, such as that described in U.S. Provisional Patent Application No. 62 / 803,264, filed February 8, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a method for sorting components of a sample includes sorting particles (e.g., cells in a biological sample) using a particle sorting module having deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0106] In some embodiments, the subject system includes a particle analysis system that can be used to analyze and characterize particles with or without physical sorting of the particles into a collection vessel. FIG. 4A shows a functional block diagram of an example of a particle analysis system. In some embodiments, the particle analysis system 401 is a flow system. The particle analysis system 401 shown in FIG. 4A can be configured to perform the methods described herein in whole or in part. The particle analysis system 401 includes a fluidics system 402. The fluidics system 402 can include or be coupled to a sample tube 405 and a moving fluid column within the sample tube through which the particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0107] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection station 408 generally refers to a monitoring area 407 of the common sample path. Detection, in some implementations, may include detecting one or more other characteristics of the light or particle 403 as it passes through the monitoring area 407. In FIG. 4A, one detection station 408 is shown with one monitoring area 407. Some implementations of the particle analysis system 401 may include multiple detection stations. Additionally, some detection stations may monitor more than one area.

[0108] Each signal is assigned a signal value to form a data point for each particle. As explained above, this data may be referred to as event data. The data points may be multi-dimensional data points that include values ​​of each characteristic measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0109] The particle analysis system 401 may also include a control system 306. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operatively associated with the fluidics system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the Poisson distribution and the number of data points collected by the detection system 404 during the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. The control system 406 may further compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0110] 4B shows a system 400 for flow cytometry in accordance with an exemplary embodiment of the invention. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. Flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more longpass ("LP") filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0111] The pump lasers 415a-415c emit light in the form of laser beams. The wavelengths of the laser beams emitted from the pump lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively, in the exemplary system of FIG. 4B. The laser beams are first directed through one or more of the beam splitters 445a and 445b. The beam splitter 445a transmits 488 nm light and reflects 633 nm light. The beam splitter 445b transmits UV light (light having a wavelength in the range of 10-400 nm) and reflects 488 nm and 633 nm light.

[0112] The laser beam is then directed to a focusing lens 420, which focuses the beam onto the portion of the fluid stream where the sample particles are located in a flow chamber 425. The flow chamber is part of a fluidics system that directs the particles, typically one at a time, in a stream to the focused laser beam for inspection. The flow chamber may comprise a flow cell in a benchtop cytometer or a nozzle tip in a stream-in-air cytometer.

[0113] Light from the laser beam interacts with particles in the sample by diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at a variety of different wavelengths depending on the particle's properties, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or in the particle. The fluorescent emissions, as well as the diffracted, refracted, reflected, and scattered light, may be routed through one or more of beam splitters 445a-g, bandpass filters 450a-e, longpass filters 455a-b, and fluorescence collection lens 440 to one or more of a forward scatter detector 430, a side scatter detector 435, and one or more fluorescence detectors 460a-f.

[0114] Fluorescence collection lens 440 collects light emitted from particle-laser beam interactions and routes the light toward one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on either side of the center of the spectral band, i.e., from 500 nm to 520 nm. Shortpass filters transmit wavelengths of light below a particular wavelength. Longpass filters, such as longpass filters 455a-455b, transmit wavelengths of light above a specified wavelength of light. For example, longpass filter 455a is a 670 nm longpass filter and transmits light above 670 nm. Filters are often selected to optimize the specificity of the detector for a particular fluorochrome. The filters can be configured so that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0115] Beam splitters direct different wavelengths of light in different directions. Beam splitters can be characterized by filter properties such as short pass and long pass. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 445g transmits wavelengths of light less than or equal to 620 nm and reflects wavelengths of light longer than 620 nm in different directions. In one embodiment, beam splitters 445a-445g can include optical mirrors such as dichroic mirrors.

[0116] The forward scatter detector 430 is positioned slightly off-axis from the direct beam through the flow cell and is configured to detect diffracted light, i.e., excitation light traveling mostly in a forward direction through or around the particle. The intensity of light detected by the forward scatter detector 430 depends on the overall size of the particle. The forward scatter detector 430 may include a photodiode. The side scatter detector 435 is configured to detect refracted and reflected light from the surface and internal structure of the particle, which tends to increase as the complexity of the particle's structure increases. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected by the forward scatter detector 430, the side scatter detector 435 and the fluorescence detector can be converted to electronic signals (voltages) by the detectors. This data can provide information about the sample.

[0117] Those skilled in the art will recognize that flow cytometers according to embodiments of the present invention are not limited to the flow cytometer shown in Figure 4B, but may include any flow cytometer known in the art. For example, a flow cytometer can have any number of lasers, beam splitters, filters, and detectors at various wavelengths and in a variety of different configurations.

[0118] During operation, the operation of the cytometer is controlled by the controller / processor 490, and measurement data from the detectors may be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 190 may be coupled to the detectors to receive output signals therefrom, and may also be coupled to electrical and electromechanical components of the flow cytometer 400 to control lasers, fluid flow parameters, and the like. Input / output (I / O) functionality 497 may also be provided within the system. The memory 495, controller / processor 490, and I / O 497 may be provided entirely as an integral part of the flow cytometer 410. In such an embodiment, a display may also form part of the I / O functionality 497 for presenting experimental data to a user of the cytometer 400. Alternatively, the memory 495 and the controller / processor 490 and some or all of the I / O functionality may be part of one or more external devices, such as a general purpose computer. In some embodiments, some or all of the memory 495 and the controller / processor 490 may be in wireless or wired communication with the cytometer 410. The controller / processor 490, in conjunction with memory 495 and I / O 497, can be configured to perform a variety of functions associated with the preparation and analysis of experiments in a flow cytometer.

[0119] The system illustrated in FIG. 4B includes six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as “filter windows” for a given detector) as defined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used for a flow cytometer experiment emit light in their own characteristic wavelength bands. The particular fluorescent labels used in the experiment and their associated fluorescent emission bands may be selected to generally coincide with the filter windows of the detectors. However, when more detectors are provided and more labels are utilized, perfect correspondence between filter windows and fluorescent emission spectra is not possible. Generally, the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of one particular detector, but a portion of the emission spectrum of that label will also overlap with the filter window of one or more other detectors. This may be referred to as spillover. The I / O 497 may be configured to receive data for a flow cytometer experiment having a panel of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of the multiple markers. The I / O 497 may also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experimental data, such as label spectral characteristics and flow cytometer configuration data, may also be stored in memory 495. The controller / processor 490 may be configured to evaluate the assignment of one or more of the labels to markers.

[0120] 5 shows a functional block diagram of an example of a particle analyzer control system for analyzing and displaying biological events, such as an analysis controller 500. The analysis controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0121] The particle analyzer or sorting system 502 may be configured to acquire biological event data. For example, a flow cytometer may generate flow cytometry event data. The particle analyzer 502 may be configured to provide the biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel.

[0122] The analysis controller 500 can be configured to receive biological event data from a particle analyzer or sorting system 502. The biological event data received from the particle analyzer or sorting system 502 can include flow cytometry event data. The analysis controller 500 can be configured to provide a graphical display including an initial plot of the biological event data on a display device 506. The analysis controller 500 can be further configured to render, for example, a region of interest overlaid on the initial plot as a gate around a population of biological event data shown by the display device 506. In some embodiments, the gate can be a logical combination of one or more graphical regions of interest depicted on a single parameter histogram or bivariate plot. In some embodiments, the display can be used to display particle parameters or saturation detector data.

[0123] Analysis controller 500 may further be configured to display the biological event data within the gate on display device 506 differently from other events in the biological event data outside the gate. For example, analysis controller 500 may be configured to render the color of the biological event data contained within the gate differently from the color of the biological event data outside the gate. Display device 506 may be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0124] The analysis controller 500 can be configured to receive a gate selection signal identifying a gate from a first input device. For example, the first input device can be implemented as a mouse 510. The mouse 510 can initiate a gate selection signal to the analysis controller 500 that identifies a gate to be displayed on or operated through the display device 506 (e.g., by clicking on or in the desired gate once a cursor is positioned there). In some implementations, the first device can be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices can include multiple input functions. In such implementations, each input function can be considered an input device. For example, as shown in FIG. 5, the mouse 510 can include a right mouse button and a left mouse button, each of which can generate a trigger event.

[0125] The trigger event can cause the analysis controller 500 to change the way the data is displayed, which portions of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selection of a subject population for particle sorting.

[0126] In some embodiments, the analysis controller 500 can be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 can be further configured to automatically modify the plot visualization to facilitate the gating process. The modification can be based on a particular distribution of the biological event data received by the analysis controller 500.

[0127] The analysis controller 500 can be connected to a storage device 504. The storage device 504 can be configured to receive and store biological event data from the analysis controller 500. The storage device 504 can also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 can be further configured to enable retrieval of biological event data, such as flow cytometry event data, by the analysis controller 500.

[0128] The display device 506 can be configured to receive display data from the analysis controller 500. The display data can include a plot of the biological event data and a gate that outlines an area of ​​the plot. The display device 506 can be further configured to modify the information presented according to input received from the analysis controller 500 in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0129] In some implementations, the analysis controller 500 can generate a user interface for receiving the exemplary events for sorting. For example, the user interface can include a control for receiving the exemplary events or exemplary images. The exemplary events or images or exemplary gates can be provided prior to collection of event data for the sample or based on an initial set of events for a portion of the sample.

[0130] In some embodiments, the subject system includes a particle sorter system. FIG. 6A is a schematic diagram of a particle sorter system 600 (e.g., particle analyzer or sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorter system 600 is a cell sorter system. As shown in FIG. 6A, a droplet forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which can be coupled to, can include, or can be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are aligned to cross a monitoring area 611 (e.g., where laser streams intersect) illuminated by an illumination source 612 (e.g., a laser). Vibration of droplet forming transducer 602 causes a moving fluid column 608 to break up into multiple droplets 610 , some of which contain particles 609 .

[0131] In operation, the detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitoring area 611. The detection station 614 powers a timing circuit 628, which in turn powers a flash charge circuit 630. At a droplet break-off point, signaled by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 608 such that the droplet of interest carries a charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect the droplet into a vessel, such as a collection tube or a multi-well or microwell sample plate, where a well or microwell can be associated with a particular droplet of interest. As shown in FIG. 6A, the droplets can be collected in a drain reservoir 638.

[0132] The detection system 616 (e.g., a droplet boundary detector) functions to automatically determine the phase of the droplet drive signal as a particle of interest passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Pat. No. 7,679,039, which is incorporated by reference herein in its entirety. The detection system 616 allows the instrument to accurately calculate the location of each detected particle in the droplet. The detection system 616 can provide an amplitude signal 620 and / or a phase signal 618, which are provided (via an amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 can be included in the control system.

[0133] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled with a memory configured to store the detected events and the sorting decisions based thereon. The sorting decisions can be included in the particle's event data. In some implementations, the detection system 616 and detection station 614 can be implemented as a single detection unit or can be communicatively coupled such that event measurements can be collected by one of the detection system 616 or detection station 614 and provided to a non-collection element.

[0134] FIG. 6B is a schematic diagram of a particle sorter system according to one embodiment presented herein. The particle sorter system 600 shown in FIG. 6B includes deflection plates 652 and 654. An electric charge can be applied via a stream charging wire in the barb. This generates a stream of droplets 610 containing particles 609 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The information about the particles is analyzed, such as by sorting electronics or other detection systems (not shown in FIG. 6B). The deflection plates 652 and 654 can be independently controlled to attract or repel the charged droplets to direct the droplets toward a desired collection vessel (e.g., one of 672, 674, 676, or 678). As shown in FIG. 6B, the deflection plates 652 and 654 can be controlled to direct the particles along a first path 662 toward vessel 674 or along a second path 668 toward vessel 678. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflector may allow the particle to continue along flow path 664. Such uncharged droplets may be diverted into a waste container, such as via aspirator 670.

[0135] Sorting electronics may be included to initiate measurement collection, receive the fluorescent signal of the particles, and determine how to adjust the deflection plates to cause sorting of the particles. Exemplary implementations of the embodiment shown in Figure 6B include the BD FACSAria™ line of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0136] Computer Control System Aspects of the present disclosure further include a computer control system, the system further comprising one or more computers for full or partial automation. In some embodiments, the system includes a computer having a computer readable storage medium having a computer program stored thereon, the computer program, when loaded into the computer, includes instructions for illuminating the light detector with a light source at a first intensity for a first predetermined time interval, illuminating the light detector with a light source at a second intensity for a second predetermined time interval, integrating a data signal from the light detector over a period including the first and second predetermined time intervals, and determining one or more parameters of the light detector based on the integrated data signal. In some embodiments, the system includes a computer having a computer readable storage medium having a computer program stored thereon, the computer program, when loaded into the computer, includes instructions for increasing the intensity of the light from the light source over a period including at least the first and second predetermined time intervals. In some examples, the computer program includes instructions for linearly increasing the intensity of the light from the light source. In some examples, the computer program includes instructions for exponentially increasing the intensity of the light from the light source.

[0137] In some embodiments, the computer program includes instructions for calculating a signal amplitude from the integrated data signal and comparing the calculated signal amplitude to the intensity of illumination by the light source. In certain embodiments, the computer program includes instructions for determining one or more of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and a number of photoelectrons per unit output of an illuminated photodetector. In certain examples, the computer program includes instructions for determining a parameter of the photodetector over an operating voltage range of the photodetector, e.g., over the entire operating voltage range of the photodetector.

[0138] In an embodiment, the system includes an input module, a processing module, and an output module. The subject systems may include both hardware and software components, where the hardware components may take the form of one or more platforms, e.g., servers, such that the functional elements of the system, i.e., elements of the system that perform particular tasks (e.g., managing the input and output of information, processing information, etc.), may be implemented by executing software applications on and across one or more computer platforms represented by the system.

[0139] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, and the like. The processing module includes a processor that accesses a memory having instructions stored therein to perform the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are available or that become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor coordinating and executing the functions of various computer programs, which may be written in a variety of programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow a user to manually align the light source with the flow stream based on the first and second optical signals, hi some embodiments, the processor includes analog electronics that provide feedback control, such as, for example, negative feedback control.

[0140] The system memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-write compact disk, flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices, including a compact disk drive, tape drive, removable hard disk drive, or disk drive. Such types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk, magnetic tape, removable hard disk, or magnetic disk, respectively. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or in program storage devices used in conjunction with the memory storage devices.

[0141] In some embodiments, a computer program product is described comprising a computer usable medium having control logic (computer software program including program code) stored therein. The control logic, when executed by a processor, causes the computer to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example using hardware state machines. Implementations of hardware state machines to perform the functions described herein will be apparent to those skilled in the relevant art.

[0142] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic or optical disk, or tape, or RAM, or any other suitable device, either fixed or portable). The processor may include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming may be provided remotely to the processor over a communication channel, or may be pre-stored in a computer program product, such as a memory or some other portable or fixed computer-readable storage medium, using any of those devices together with the memory. For example, a magnetic or optical disk may carry the programming and can be read by a disk writer / reader. The system of the present invention also includes programming, for example in the form of a computer program product, an algorithm for use in implementing the above-described method. The programming according to the present invention may be recorded on a computer-readable medium, for example any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as magnetic disks, hard disk storage media, and magnetic tape, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROM, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0143] The processor may also have access to a communication channel for communicating with a user at a remote location, meaning that the user does not have direct contact with the system, but rather relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), a telephone network, a satellite network, or any other suitable communication channel, including a cellular phone (i.e., a smartphone).

[0144] In some embodiments, a system according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0145] In one embodiment, the communications interface is configured to include one or more communications ports, e.g., a physical port or interface such as a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communications between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment) configured for similar complementary data communications.

[0146] In one embodiment, the communications interface is configured for infrared communications, Bluetooth® communications, or any other suitable wireless communications protocol to enable the subject system to communicate with other devices, such as a computer terminal and / or network, a communications-enabled mobile phone, a personal digital assistant, or any other communications device that a user may use in conjunction with.

[0147] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing the Internet Protocol (IP) via a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0148] In one embodiment, the subject system is configured to communicate wirelessly with the server device via a communications interface using common standards such as, for example, 802.11 or Bluetooth RF protocols, or IrDA infrared protocol. The server device may be another portable device, such as a smartphone, a personal digital assistant (PDA) or a notebook computer, or a larger device, such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touch screen.

[0149] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored within the subject system, e.g., within the optional data storage unit, with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0150] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of picture elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be accomplished in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network, in accordance with known techniques. The presentation of data by the output manager may be implemented in accordance with a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the subject system are typically of a class of computers commonly referred to as servers, but may be any type of known or future developed computer platform. However, they may be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cabling or other communication systems, including wireless systems, either networked or not. They may be co-located or physically separated.In some cases, depending on the type and / or configuration of the computer platform selected, various operating systems may be employed on any of the computer platforms. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, etc.

[0151] FIG. 7 illustrates a general architecture of an exemplary computing device 700 according to certain embodiments. The general architecture of the computing device 700 illustrated in FIG. 7 includes a configuration of computer hardware and software components. The computing device 700 may include more (or less) elements than those illustrated in FIG. 7. However, not all of these typical conventional elements need to be illustrated to provide an enabling disclosure. As illustrated, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which may communicate with each other via a communication bus. The network interface 720 may provide connectivity to one or more networks or computing systems. Thus, the processing unit 710 may receive information and instructions from other computing systems or services via a network. The processing unit 710 may also communicate with a memory 770 and further provide output information for an optional display 750 via the input / output device interface 740. The input / output device interface 740 can also accept input from optional input devices 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game pad, accelerometer, gyroscope, or other input device.

[0152] Memory 770 may include computer program instructions (grouped as modules or components in some embodiments) that processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions used by processing unit 710 in the general management and operation of computing device 700. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0153] Computer-readable storage medium Aspects of the present disclosure further include a non-transitory computer-readable storage medium having instructions for carrying out the subject methods. The computer-readable storage medium may be employed on one or more computers for full or partial automation of a system for carrying out the methods described herein. In certain embodiments, instructions according to the methods described herein may be encoded on a computer-readable medium in the form of "programming", and the term "computer-readable medium" as used herein refers to any non-transitory storage medium involved in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include magnetic disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid-state disks, and network-attached storage (NAS), whether such devices are internal or external to the computer. A file containing information may be "stored" on a computer-readable medium, where "storing" means recording information so that it can be accessed and retrieved at a later date by a computer. The computer-implemented methods described herein may be performed using programming that may be written in one or more of any number of computer programming languages. Such languages ​​include, for example, Java (Sun Microsystems, Inc., Santa Clara, Calif.), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as any number of others.

[0154] In some embodiments, a subject computer readable storage medium includes a computer program stored thereon, the computer program including instructions that, when loaded into a computer, have an algorithm for illuminating the photodetector with a continuous wave light source at a first intensity for a first predetermined time interval, an algorithm for illuminating the photodetector with the light source at a second intensity for a second predetermined time interval, an algorithm for integrating a data signal from the photodetector over a period including the first predetermined time interval and the second predetermined time interval, and an algorithm for determining one or more parameters of the photodetector based on the integrated data signal.

[0155] In certain examples, the non-transitory computer readable storage medium includes an algorithm for illuminating a photodetector with multiple light intensities over multiple time intervals. In these examples, the non-transitory computer readable storage medium includes an algorithm for integrating a data signal from the photodetector over a time period that includes the multiple illumination time intervals.

[0156] 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 certain examples, the non-transitory computer readable storage medium includes an algorithm for comparing the calculated signal amplitude to a light intensity of the light source. In certain examples, the non-transitory computer readable storage medium includes an algorithm for determining a parameter of the photodetector based on one or more of the calculated signal amplitude and a comparison between the calculated signal amplitude and the light intensity of the light source. For example, the non-transitory computer readable storage medium may include an algorithm for determining a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, or a number of photoelectrons per unit output. The non-transitory computer readable storage medium may include an algorithm for determining a detector parameter over a range of operating voltages of the photodetector, such as when the parameter of the photodetector is determined over the entire operating voltage range of the photodetector.

[0157] The computer readable storage medium may be employed on one or more computer systems having a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, and the like. The processing module includes a processor that accesses a memory having stored instructions to perform the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are available or become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor coordinating and executing the functions of various computer programs, which may be written in a variety of programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

[0158] Multispectral fluorescent particles As summarized above, aspects of the present disclosure also include particles (e.g., beads) having one or more fluorophores for carrying out certain methods described herein. Particles of interest according to certain embodiments may include single-peak multi-fluorophore beads that provide bright photodetector signals across all light source wavelengths (e.g., across all LEDs or lasers of the system) and across the detection wavelengths of the photodetector.

[0159] In embodiments, the subject particles are formulated (e.g., in a fluid composition) to flow in a flow stream illuminated by a light source as described above. Each particle may have one or more different types of fluorophores, for example, 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more, or 25 or more, or 30 or more, or 35 or more, or 40 or more, or 45 or more, 50 or more different types of fluorophores. For example, each particle may include 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 different types of fluorophores.

[0160] In embodiments, each fluorophore is stably associated with the particle. Stably associated means that the fluorophore does not easily dissociate from the particle and contact with liquid medium, e.g., aqueous medium. In some embodiments, one or more of the fluorophores are covalently bound to the particle. In other embodiments, one or more of the fluorophores are physically associated with the particle (i.e., non-covalently bound). In other embodiments, one or more of the fluorophores are covalently bound to the particle and one or more of the fluorophores are physically associated with the particle.

[0161] In some embodiments, each particle includes two or more different types of fluorophores. Any two fluorophores are considered to be different if one or more of the molecular formula, excitation maximum, and emission maximum are different from each other. Thus, different or distinct fluorophores may differ from each other in terms of chemical composition or in terms of one or more properties of the fluorophores. For example, different fluorophores may differ from each other in at least one of excitation maximum and emission maximum. In some cases, different fluorophores differ from each other in their excitation maxima. In some cases, different fluorophores differ from each other in their emission maxima. In some cases, different fluorophores differ from each other in both their excitation maxima and emission maxima. Thus, in embodiments including a first and a second fluorophore, the first and second fluorophores may differ from each other in at least one of excitation maximum and emission maximum. For example, the first and second fluorophores may differ from each other in excitation maximum, emission maximum, or both excitation and emission maxima. A given set of fluorophores can be considered distinct if they differ from one another with respect to their excitation or emission maxima, where the magnitude of such difference is, in some cases, 5 nm or more, 10 nm or more, including 15 nm or more, and in some cases the magnitude of the difference is in the range of 5 to 400 nm, such as 10 to 200 nm, including 15 to 100 nm, such as 25 to 50 nm.

[0162] Fluorophores of interest according to certain embodiments have excitation maxima in the range of 100 nm to 800 nm, such as 150 nm to 750 nm, for example 200 nm to 700 nm, such as 250 nm to 650 nm, for example 300 nm to 600 nm, and including 400 nm to 500 nm. Fluorophores of interest according to certain embodiments have emission maxima in the range of 400 nm to 1000 nm, such as 450 nm to 950 nm, for example 500 nm to 900 nm, for example 550 nm to 850 nm, and including 600 nm to 800 nm. In a particular example, the fluorophore is a luminescent dye, such as a fluorescent dye having a peak emission wavelength of 200 nm or more, such as 250 nm or more, for example 300 nm or more, such as 350 nm or more, for example 400 nm or more, such as 450 nm or more, for example 500 nm or more, such as 550 nm or more, for example 600 nm or more, such as 650 nm or more, for example 700 nm or more, such as 750 nm or more, for example 800 nm or more, such as 850 nm or more, for example 900 nm or more, such as 950 nm or more, for example 1000 nm or more, and including 1050 nm or more. For example, the fluorophore may be a fluorescent dye having a peak emission wavelength in the range of 200 nm to 1200 nm, such as 300 nm to 1100 nm, for example 400 nm to 1000 nm, such as 500 nm to 900 nm, including a fluorescent dye having a peak emission wavelength of 600 nm to 800 nm. In certain embodiments, the subject multispectral particles provide stable excitation by lasers emitting at or near wavelengths of 349 nm (UV laser), 488 nm (blue laser), 532 nm (Nd:YAG solid state laser), 640 nm (red laser), and 405 nm (violet laser). In certain examples, the subject multispectral particles provide stable excitation by light sources across the entire spectral detection band, such as from 350 nm to 850 nm.

[0163] Fluorophores of interest include, but are not limited to, bodipy dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurydine dyes, safranine dyes, indamines, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronine dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, squaraines, bodipy, squarin-loccitane, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine, or tetrapyrrole, and combinations thereof. In certain embodiments, the conjugate may comprise two or more dyes, for example, two or more dyes selected from bodipy dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurydine dyes, safranine dyes, indamines, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronine dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, squaraines, bodipy, squarin-loccitane, naphthalenes, coumarins, oxadiazoles, anthracenes, pyrenes, acridines, arylmethines, or tetrapyrroles, and combinations thereof.

[0164] In certain embodiments, fluorophores of interest may include, but are not limited to, fluorescein isothiocyanate (FITC), phycoerythrin (PE) dyes, peridinin chlorophyll protein-cyanine dyes (e.g., PerCP-Cy5.5), phycoerythrin-cyanine (PE-Cy) dyes (PE-Cy7), allophycocyanin (APC) dyes (e.g., APC-R700), allophycocyanin-cyanine dyes (e.g., APC-Cy7), coumarin dyes (e.g., V450 or V500). In certain examples, the fluorophore may include one or more of 1,4-bis-(o-methylstyryl)-benzene (bis-MSB 1,4-bis[2-(2-methylphenyl)ethenyl]-benzene), C510 dye, C6 dye, Nile Red dye, T614 dye (e.g., N-[7-(methanesulfonamido)-4-oxo-6-phenoxycyclomen-3-yl]formaldehyde), LDS821 dye ((2-(6-(p-dimethylaminophenyl)-2,4-neopentylene-1,3,5-hexatrienyl)-3-ethylbenzothiazolium perchlorate), mFluor dye (e.g., mFluor Red dye such as mFluor 780NS).

[0165] The particles may be of any convenient shape for illumination by a light source as described above. In some examples, the particles are solid supports shaped or configured into disks, spheres, ovals, cubes, blocks, cones, etc., as well as irregular shapes. The mass of the particles may vary, and in some examples ranges from 0.01 mg to 20 mg, such as 0.05 mg to 19.5 mg, such as 0.1 mg to 19 mg, such as 0.5 mg to 18.5 mg, such as 1 mg to 18 mg, such as 1.5 mg to 17.5 mg, such as 2 mg to 15 mg, including 3 mg to 10 mg. The particles may be sized to a size of 0.01 mm or less. 2 More than, for example, 0.05 mm 2 More than, for example, 0.1 mm 2 More than, for example, 0.5 mm 2 More than, for example, 1 mm 2 More than, for example, 1.5 mm 2 More than, for example, 2 mm 2 More than, for example, 2.5 mm 2More than, for example, 3 mm 2 More than 3.5mm, for example 2 More than 4mm, for example 2 Above, for example, 4.5 mm 2 or more, and 5mm 2 and the like, may have a surface area determined using, for example, a Vertex system or equivalent.

[0166] The size of the particles may vary as desired, in some examples the particles have a longest dimension in the range of 0.01 mm to 10 mm, such as 0.05 mm to 9.5 mm, for example 0.1 mm to 9 mm, such as 0.5 mm to 8.5 mm, for example 1 mm to 8 mm, for example 1.5 mm to 7.5 mm, for example 2 mm to 7 mm, for example 2.5 mm to 6.5 mm, for example 3 mm to 6 mm. In particular examples the particles have a shortest dimension in the range of 0.01 mm to 5 mm, such as 0.05 mm to 4.5 mm, for example 0.1 mm to 4 mm, for example 0.5 mm to 3.5 mm, and including 1 mm to 3 mm.

[0167] In certain instances, the subject particles are porous, e.g., the particles have a porosity in the range including 5μ to 100μ, such as 10μ to 90μ, such as 15μ to 85μ, such as 20μ to 80μ, such as 25μ to 75μ, and 30μ to 70μ, e.g., 50μ, as determined, for example, using a Capillary Flow Porometer or equivalent.

[0168] The particles can be formed from any convenient material. Of interest in some embodiments are particles (e.g., beads) with low or no autofluorescence. Suitable materials include, but are not limited to, glass materials (e.g., silicates), ceramic materials (e.g., calcium phosphates), metallic materials, and polymeric materials (e.g., polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, etc.). In some examples, the particles are formed from a solid support, such as a porous matrix as described in U.S. Patent Application Publication No. 9,797,899, the disclosure of which is incorporated herein by reference. Thus, the surface area of ​​the particles can be any suitable macroporous or microporous substrate, suitable macroporous and microporous substrates include, but are not limited to, ceramic matrices, frits such as fritted glass, polymeric matrices, and metal-organic polymeric matrices. In some embodiments, the porous matrix is ​​a frit. The term "frit" is used herein in its conventional sense to refer to a porous composition formed from a sintered granular solid, such as glass. The frits may have varying chemical compositions depending on the type of sintered granules used to prepare the frit, where frits that may be used include, but are not limited to, frits composed of aluminosilicates, boron trioxide, borophosphosilicate glass, borosilicate glass, glaze, cobalt glass, cranberry glass, fluorophosphate glass, fluorosilicate glass, fused quartz, germanium dioxide, metal and sulfide embedded borosilicates, lead glass, phosphate glass, phosphorus pentoxide glass, phosphosilicate glass, potassium silicate, soda lime glass, sodium hexametaphosphate glass, sodium silicate, tellurite glass, uranium glass, vitrites, and combinations thereof. In some embodiments, the porous matrix is ​​a glass frit, such as a borosilicate, aluminosilicate, fluorosilicate, potassium silicate, or borophosphosilicate glass frit.

[0169] In some embodiments, the particles are formed from porous organic polymers. Porous organic polymers of interest will vary depending on the sample volume, components in the sample, and assay reagents present, and may include, but are not limited to, porous polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), ethylvinylacetate (EVA), polycarbonate, polycarbonate alloys, polyurethane, polyethersulfone, copolymers, and combinations thereof. For example, porous polymers of interest include homopolymers, heteropolymers, and copolymers composed of monomer units such as styrene, monoalkylene arylene monomers (e.g., ethylstyrene, α-methylstyrene, vinyltoluene, and vinylethylbenzene), (meth)acrylic acid esters such as methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, isodecyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, cyclohexyl (meth)acrylate, and benzyl (meth)acrylate, chlorine-containing monomers such as vinyl chloride, vinylidene chloride, and chloromethylstyrene, acrylonitrile compounds such as acrylonitrile and methacrylonitrile, and vinyl acetate, vinyl propionate, n-octadecylacrylamide, ethylene, propylene, and butane, and combinations thereof.

[0170] In some embodiments, the particles are formed from a metal organic polymer matrix, for example, an organic polymer matrix having a framework structure containing a metal such as aluminum, barium, antimony, calcium, chromium, copper, erbium, germanium, iron, lead, lithium, phosphorus, potassium, silicon, tantalum, tin, titanium, vanadium, zinc, or zirconium. In some embodiments, the porous organic matrix is ​​an organosiloxane polymer, including, but not limited to, polymers of methyltrimethoxysilane, dimethyldimethoxysilane, tetraethoxysilane, methacryloxypropyltrimethoxysilane, bis(triethoxysilyl)ethane, bis(triethoxysilyl)butane, bis(triethoxysilyl)pentane, bis(triethoxysilyl)hexane, bis(triethoxysilyl)heptane, bis(triethoxysilyl)octane, and combinations thereof.

[0171] kit Kits are also provided that include one or more components of the subject systems. Kits according to certain embodiments include one or more continuous wave light sources, such as narrowband light emitting diodes, and a photodetector (e.g., a photomultiplier tube), where analysis of one or more parameters of the photodetector is desired. The kits may also include optical conditioning components, such as lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof.

[0172] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for carrying out the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, such as a sheet or sheets of paper on which the information is printed, in the kit packaging, in a package insert, etc. Yet another form in which these instructions may be present is a computer readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Yet another form in which these instructions may be present is a website address that may be used via the Internet to access the information at a remote site.

[0173] Utilities The subject methods, systems, and computer systems are used in a variety of applications where it is desirable to calibrate or optimize a photodetector, such as a particle analyzer. The subject methods and systems also find application for photodetectors used to analyze and sort particle components in samples in fluid media, such as biological samples. The present disclosure also finds use in flow cytometry, where it is desirable to provide a flow cytometer with improved cell sorting accuracy, increased particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and improved particle deflection during cell sorting. In embodiments, the present disclosure reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In certain embodiments, the subject methods and systems provide fully automated protocols such that adjustments of the flow cytometer during use require little, if any, human input.

[0174] Aspects (including embodiments) of the subject matter described herein may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the description, certain non-limiting aspects of the present disclosure, numbered 1-115, are provided below as appendices. As will be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding aspects or any of the aspects following the individually numbered aspect. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0175] 1. A method for determining a parameter of a photodetector in a particle analyzer, comprising: illuminating a photodetector positioned within the particle analyzer with a light source at a first intensity for a first predetermined time interval; illuminating the photodetector with a light source at a second intensity for a second predetermined time interval; integrating the data signal from the photodetector over a period of time that includes a first predetermined time interval and a second predetermined time interval; determining one or more parameters of the photodetector based on the integrated data signal; A method comprising: 2. The method of claim 1, wherein the particle analyzer is a flow cytometer. 3. The method of claim 1 or 2, wherein the light detector is positioned within the particle analyzer to detect light from particles in the flow stream. 4. The method of any one of claims 1 to 3, wherein the light source is a continuous wave light source. 5. The method according to any one of claims 1 to 3, wherein the light source is a pulsed light source.

[0176] 6. The method of any one of claims 1 to 5, wherein the light source is a light emitting diode. 7. The method of any one of claims 1 to 6, wherein the light source is a narrowband light source. 8. The method of claim 7, wherein the light source emits light having wavelengths ranging from 20 nm or less. 9. The method of any one of claims 1-8, wherein the method includes illuminating the photodetector with a second intensity greater than the first intensity. 10. The method of any one of claims 1 to 9, wherein the first predetermined time interval and the second predetermined time interval have the same duration.

[0177] 11. The method of any one of claims 1 to 10, wherein the method includes continuously illuminating the photodetector over a period of time that includes a first predetermined time interval and a second predetermined time interval. 12. The method of any one of claims 1-11, wherein the method includes increasing the intensity of light from the light source from a first intensity to a second intensity for a third predetermined time interval. 13. The method of any one of claims 1 to 11, wherein the method includes increasing an intensity of light from the light source over a period of time that includes a first predetermined time interval and a second predetermined time interval. 14. The method of claim 13, wherein the intensity of light from the light source increases linearly over a period of time that includes a first predetermined time interval and a second predetermined time interval. 15. The method of claim 13, wherein the intensity of the light from the light source increases exponentially over a period of time that includes a first predetermined time interval and a second predetermined time interval.

[0178] 16. The method of any one of claims 1 to 15, wherein integrating the data signal from the photodetector includes calculating a signal amplitude over a period of time. 17. The method of claim 16, wherein the method includes calculating a median signal amplitude over a period of time. 18. The method of claim 16 or 17, wherein the method includes comparing the calculated signal amplitude to the light intensity of the light source. 19. The method of any one of claims 1 to 18, wherein the method includes determining one or more parameters of the photodetector selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and a number of photoelectrons per unit output. 20. Method illuminating a photodetector with a light source at a plurality of intensities for a plurality of predetermined time intervals; integrating the data signal from the photodetector over a period of time that includes a plurality of predetermined time intervals; determining one or more parameters of the photodetector based on the integrated data signal; 20. The method according to any one of claims 1 to 19, comprising:

[0179] 21. The method of any one of claims 1 to 20, wherein the parameters of the photodetector are determined over a range of operating voltages of the photodetector. 22. The method of claim 21, wherein the parameters of the photodetector are determined over the entire operating voltage range of the photodetector. 23. The method of any one of claims 1 to 22, further comprising calculating an optimal detector gain for the optical detector based on the determined parameters.

[0180] 24. Illuminating the photodetector with a light source at a first intensity for a first predetermined time interval; illuminating the photodetector with a light source at a second intensity for a second predetermined time interval; integrating the data signal from the photodetector over a period of time that includes a first predetermined time interval and a second predetermined time interval; determining one or more parameters of the photodetector based on the integrated data signal; A method comprising: 25. The method of claim 24, wherein the light source is a continuous wave light source. 26. The method of claim 24, wherein the light source is a pulsed light source. 27. The method of any one of claims 24 to 26, wherein the light source is a light-emitting diode. 28. The method of any one of appendices 24 to 27, wherein the light source is a narrowband light source.

[0181] 29. The method of claim 28, wherein the light source emits light having wavelengths ranging from 20 nm or less. 30. The method of any one of claims 24 to 29, wherein the method includes illuminating the photodetector with a second intensity greater than the first intensity. 31. The method of any one of claims 24 to 30, wherein the first predetermined time interval and the second predetermined time interval have the same duration. 32. The method of any one of claims 24 to 31, wherein the method includes continuously illuminating the photodetector over a period of time that includes a first predetermined time interval and a second predetermined time interval. 33. The method of any one of claims 24 to 32, wherein the method includes increasing the intensity of light from the light source from a first intensity to a second intensity for a third predetermined time interval.

[0182] 34. The method of any one of claims 24 to 32, wherein the method includes increasing an intensity of light from the light source over a period of time that includes a first predetermined time interval and a second predetermined time interval. 35. The method of claim 34, wherein the intensity of light from the light source increases linearly over a period of time that includes a first predetermined time interval and a second predetermined time interval. 36. The method of claim 34, wherein the intensity of light from the light source increases exponentially over a period of time that includes a first predetermined time interval and a second predetermined time interval. 37. The method of any one of claims 24 to 36, wherein integrating the data signal from the photodetector includes calculating a signal amplitude over a period of time. 38. The method of claim 37, wherein the method includes calculating a median signal amplitude over a period of time.

[0183] 39. The method of claim 37 or 38, wherein the method includes comparing the calculated signal amplitude with the light intensity of the light source. 40. The method of any one of appendixes 24 to 39, wherein the method includes determining one or more parameters of the photodetector selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and a number of photoelectrons per unit output. 41. The method is illuminating a photodetector with a light source at a plurality of intensities for a plurality of predetermined time intervals; integrating the data signal from the photodetector over a period of time that includes a plurality of predetermined time intervals; determining one or more parameters of the photodetector based on the integrated data signal; 41. The method according to any one of claims 24 to 40, comprising: 42. The method of any one of appendixes 24 to 41, wherein the parameters of the photodetector are determined over a range of operating voltages of the photodetector. 43. The method of claim 42, wherein the parameters of the photodetector are determined over the entire operating voltage range of the photodetector.

[0184] 44. The method of any one of claims 24 to 43, further comprising calculating an optimal detector gain for the optical detector based on the determined parameters. 45. The method of any one of claims 24 to 44, wherein the optical detector is positioned within a flow cytometer. 46. ​​The method of claim 45, wherein the flow cytometer includes a flow cell for propagating particles in a flow stream.

[0185] 47. A particle analyzer comprising: A light source; an optical detection system positioned within a housing of the particle analyzer, the optical detection system comprising: Detecting light from the light source at a first intensity for a first predetermined time interval; detecting light from the light source at a second intensity for a second predetermined time interval; a light detection system having a light detector configured to: a processor having a memory operatively coupled to the processor; Equipped with The memory includes instructions stored thereon that, when executed by the processor, cause the processor to: integrating the data signal from the photodetector over a period of time that includes a first predetermined time interval and a second predetermined time interval; determining one or more parameters of the photodetector based on the integrated data signal; A particle analyzer that performs the above. 48. The particle analyzer of claim 47, wherein the particle analyzer is incorporated into a flow cytometer. 49. The particle analyzer of claim 47 or 48, wherein the light source is a continuous wave light source. 50. The particle analyzer of claim 47 or 48, wherein the light source is a pulsed light source. 51. A particle analyzer according to any one of 47 to 50, wherein the light source is a light emitting diode.

[0186] 52. The particle analyzer of any one of notes 47 to 51, wherein the light source is a narrow bandwidth light source. 53. The particle analyzer of 52, wherein the light source emits light including wavelengths ranging from 20 nm or less. 54. The particle analyzer of any one of notes 47 to 53, wherein the second intensity is greater than the first intensity. 55. The particle analyzer of any one of notes 47 to 54, wherein the first predetermined time interval and the second predetermined time interval have the same duration. 56. A particle analyzer according to any one of notes 47 to 55, wherein the light source is configured to continuously illuminate the light detector over a period of time that includes a first predetermined time interval and a second predetermined time interval.

[0187] 57. The particle analyzer of claim 56, wherein 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 increase an intensity of light from the light source from a first intensity to a second intensity over a third predetermined time interval. 58. A particle analyzer as described in any one of appendices 47 to 56, wherein 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 increase an intensity of light from the light source over a period of time that includes a first predetermined time interval and a second predetermined time interval. 59. The particle analyzer of claim 58, wherein the intensity of light from the light source increases linearly over a period of time that includes a first predetermined time interval and a second predetermined time interval. 60. The particle analyzer of claim 58, wherein the intensity of light from the light source increases exponentially over a period of time that includes a first predetermined time interval and a second predetermined time interval. 61. A particle analyzer as described in any one of appendixes 47 to 60, wherein 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 calculate signal amplitude over a period of time.

[0188] 62. The particle analyzer of claim 61, wherein 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 calculate a median signal amplitude over a period of time. 63. A particle analyzer as described in appended claim 61 or 62, wherein the processor has a memory operatively coupled to the processor, the memory including instructions stored thereon, the instructions, when executed by the processor, causing the processor to compare the calculated signal amplitude with the light intensity of the light source. 64. A particle analyzer according to any one of appendices 47 to 63, wherein one or more parameters of the optical detector are selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and a number of photoelectrons per unit output. 65. A particle analyzer according to any one of claims 47 to 64, wherein the light source is configured to illuminate the photodetector at multiple intensities and for multiple predetermined time intervals. 66. A processor has a memory operatively coupled to the processor, the memory including instructions stored thereon, the instructions, when executed by the processor, causing the processor to: integrating the data signal from the photodetector over a period of time that includes a plurality of predetermined time intervals; determining one or more parameters of the photodetector based on the integrated data signal; 66. The particle analyzer of claim 65,

[0189] 67. A particle analyzer as described in any one of appendixes 47 to 66, wherein 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 determine parameters of the photodetector over a range of operating voltages of the photodetector. 68. The particle analyzer of claim 67, wherein 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 determine parameters of the photodetector over an operating voltage range of the photodetector. 69. A particle analyzer as described in any one of appendixes 47 to 68, wherein 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 calculate an optimal detector gain for the optical detector based on the determined parameters.

[0190] 70. A system comprising: A light source; 1. An optical detection system comprising: Detecting light from the light source at a first intensity for a first predetermined time interval; detecting light from the light source at a second intensity for a second predetermined time interval; a light detection system having a light detector configured to: a processor having a memory operatively coupled to the processor; Equipped with The memory includes instructions stored thereon that, when executed by the processor, cause the processor to: integrating the data signal from the photodetector over a period of time that includes a first predetermined time interval and a second predetermined time interval; determining one or more parameters of the photodetector based on the integrated data signal; A system that allows the user to: 71. The system of claim 70, wherein the light source is a continuous wave light source. 72. The system of claim 70, wherein the light source is a pulsed light source. 73. The system of any one of notes 70 to 72, wherein the light source is a light-emitting diode. 74. The system of any one of notes 70 to 73, wherein the light source is a narrowband light source.

[0191] 75. The system of claim 70, wherein the light source emits light having wavelengths ranging from 20 nm or less. 76. The system of any one of notes 70 to 75, wherein the second intensity is greater than the first intensity. 77. The system of any one of notes 70 to 76, wherein the first predetermined time interval and the second predetermined time interval have the same duration. 78. A system according to any one of notes 70 to 77, wherein the light source is configured to continuously illuminate the light detector over a period of time including a first predetermined time interval and a second predetermined time interval. 79. The system of claim 78, wherein 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 increase an intensity of light from the light source from a first intensity to a second intensity over a third predetermined time interval.

[0192] 80. The system of any one of notes 70-78, wherein 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 increase an intensity of light from the light source over a period of time that includes a first predetermined time interval and a second predetermined time interval. 81. The system of claim 80, wherein the intensity of light from the light source increases linearly over a period of time that includes a first predetermined time interval and a second predetermined time interval. 82. The system of claim 80, wherein the intensity of light from the light source increases exponentially over a period of time that includes a first predetermined time interval and a second predetermined time interval. 83. The system of any one of appendixes 70-82, wherein 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 calculate a signal amplitude over a period of time. 84. The system of claim 83, wherein 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 calculate a median signal amplitude over a period of time.

[0193] 85. The system of claim 83 or 84, wherein the processor has a memory operatively coupled to the processor, the memory including instructions stored thereon, the instructions, when executed by the processor, causing the processor to compare the calculated signal amplitude with the light intensity of the light source. 86. A system described in any one of appendixes 83 to 85, wherein one or more parameters of the photodetector are selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and a number of photoelectrons per unit output. 87. The system of any one of claims 85 to 86, wherein the light source is configured to illuminate the photodetector at multiple intensities and for multiple predetermined time intervals. 88. A processor has a memory operatively coupled to the processor, the memory including instructions stored thereon, the instructions, when executed by the processor, causing the processor to: integrating the data signal from the photodetector over a period of time that includes a plurality of predetermined time intervals; determining one or more parameters of the photodetector based on the integrated data signal; 88. The system of claim 87, 89. The system of any one of notes 70 to 88, wherein 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 determine parameters of the photodetector over a range of operating voltages of the photodetector.

[0194] 90. The system of claim 89, wherein 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 determine parameters of the photodetector over an operating voltage range of the photodetector. 91. The system of any one of appendixes 70-90, wherein 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 calculate an optimal detector gain for the optical detector based on the determined parameters. 92. The system of any one of appendices 70 to 91, wherein the system is a flow cytometer. 93. The system of claim 92, wherein the flow cytometer includes a flow cell for propagating particles in a flow stream. 94. The system of claim 93, wherein the light detector is positioned to detect light from particles in the flow stream.

[0195] 95. A non-transitory computer-readable storage medium, comprising: and instructions stored thereon, the instructions comprising: an algorithm for illuminating the photodetector with a light source at a first intensity for a first predetermined time interval; an algorithm for illuminating the photodetector with the light source at a second intensity for a second predetermined time interval; an algorithm for integrating a data signal from the photodetector over a period of time that includes a first predetermined time interval and a second predetermined time interval; an algorithm for determining one or more parameters of the photodetector based on the integrated data signal; A non-transitory computer readable storage medium comprising: 96. The non-transitory computer-readable storage medium of claim 95, wherein the light source is a continuous wave light source. 97. The non-transitory computer-readable storage medium of claim 95, wherein the light source is a pulsed light source. 98. The non-transitory computer-readable storage medium of any one of appendixes 95 to 97, wherein the light source is a light-emitting diode. 99. The non-transitory computer-readable storage medium of any one of claims 95 to 98, wherein the light source is a narrowband light source.

[0196] 100. The non-transitory computer-readable storage medium of claim 99, wherein the light source emits light having wavelengths ranging from 20 nm or less. 101. The non-transitory computer-readable storage medium of any one of claims 95 to 100, wherein the non-transitory computer-readable storage medium includes an algorithm for illuminating the photodetector with a second intensity that is greater than the first intensity. 102. The non-transitory computer-readable storage medium of any one of appendices 95 to 101, wherein the first predetermined time interval and the second predetermined time interval have the same duration. 103. The non-transitory computer-readable storage medium of any one of appendices 95 to 102, wherein the non-transitory computer-readable storage medium includes an algorithm for continuously illuminating a photodetector over a period of time that includes a first predetermined time interval and a second predetermined time interval. 104. The non-transitory computer-readable storage medium of claim 103, wherein the non-transitory computer-readable storage medium includes an algorithm for increasing an intensity of light from the light source from a first intensity to a second intensity over a third predetermined time interval.

[0197] 105. The non-transitory computer-readable storage medium of claim 103, wherein the non-transitory computer-readable storage medium includes an algorithm for increasing an intensity of light from the light source over a period of time that includes a first predetermined time interval and a second predetermined time interval. 106. The non-transitory computer-readable storage medium of claim 105, wherein the intensity of light from the light source increases linearly over a period of time that includes a first predetermined time interval and a second predetermined time interval. 107. The non-transitory computer-readable storage medium of claim 105, wherein the intensity of light from the light source increases exponentially over a period of time that includes a first predetermined time interval and a second predetermined time interval. 108. The non-transitory computer-readable storage medium of any one of appendixes 95 to 107, wherein the non-transitory computer-readable storage medium includes an algorithm for calculating a signal amplitude over a period of time. 109. The non-transitory computer-readable storage medium of claim 108, wherein the non-transitory computer-readable storage medium includes an algorithm for calculating median signal amplitude over a period of time.

[0198] 110. The non-transitory computer-readable storage medium of claim 108 or 109, wherein the non-transitory computer-readable storage medium includes an algorithm for comparing the calculated signal amplitude to the light intensity of the light source. 111. The non-transitory computer-readable storage medium of any one of appendices 95 to 110, comprising an algorithm for determining one or more parameters of a photodetector selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and a number of photoelectrons per unit output. 112. A non-transitory computer-readable storage medium comprising: an algorithm for illuminating the photodetector with a light source at a plurality of intensities for a plurality of predetermined time intervals; an algorithm for integrating the data signal from the photodetector over a period of time that includes a plurality of predetermined time intervals; an algorithm for determining one or more parameters of the photodetector based on the integrated data signal; 113. A non-transitory computer-readable storage medium according to any one of claims 95 to 111, comprising: 113. The non-transitory computer-readable storage medium of any one of Clauses 95 to 112, wherein the non-transitory computer-readable storage medium includes an algorithm for determining parameters of a photodetector over a range of operating voltages of the photodetector. 114. The non-transitory computer-readable storage medium of claim 113, wherein the non-transitory computer-readable storage medium includes an algorithm for determining a parameter of a photodetector over an operating voltage range of the photodetector. 115. The non-transitory computer-readable storage medium of any one of appendixes 95 to 114, wherein the non-transitory computer-readable storage medium includes an algorithm for calculating an optimal detector gain for the photodetector based on the determined parameters.

[0199] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of this invention, certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

[0200] Thus, the above merely illustrates the principles of the invention. It is understood that those skilled in the art can devise various arrangements that embody the principles of the invention and are within its spirit and scope, although not expressly described or shown herein. Furthermore, all examples and conditional language described herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts that the inventors contribute to furthering the art, and should not be construed as being limited to such specifically described examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, regardless of structure, i.e., any elements developed to perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly set forth in the claims.

[0201] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly defined as being invoked for a limitation in a claim only if the precise phrase "means for" or the precise phrase "step for" appears at the beginning of such limitation in the claim, and if such precise phrase is not used in the limitation in the claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0202] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Patent Application No. 63 / 012,765, filed April 20, 2020, the disclosure of which is incorporated herein by reference.

Claims

1. 1. A method for determining a parameter of a photodetector in a particle analyzer, comprising: illuminating a photodetector positioned within the particle analyzer with a light source at a first intensity for a first predetermined time interval; illuminating the photodetector with the light source at a second intensity for a second predetermined time interval; integrating a data signal from the photodetector over a period of time that includes the first predetermined time interval and the second predetermined time interval; determining one or more parameters of the photodetector based on the integrated data signal; A method comprising:

2. The method of claim 1 , wherein the light source is a continuous wave light source.

3. The method according to claim 1 or 2, wherein the light source is a light emitting diode.

4. The method of any one of claims 1 to 3, wherein the method comprises illuminating the photodetector with a second intensity greater than the first intensity.

5. A method according to any preceding claim, wherein the method comprises increasing the intensity of light from the light source from the first intensity to the second intensity over a third predetermined time interval.

6. 5. The method of claim 1, wherein the method comprises increasing an intensity of light from the light source over the period of time that includes the first predetermined time interval and the second predetermined time interval.

7. 7. The method of claim 1, wherein integrating the data signal from the photodetector comprises calculating a signal amplitude over the period of time and comparing the calculated signal amplitude to the light intensity of the light source.

8. 8. The method of any one of claims 1 to 7, wherein the method comprises determining one or more parameters of the photodetector selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal to noise ratio, and a number of photoelectrons per unit power.

9. The method comprises: determining a parameter of the photodetector over a range of operating voltages of the photodetector; calculating an optimal detector gain for the photodetector based on the determined parameters; The method according to any one of claims 1 to 8, comprising:

10. 1. A particle analyzer comprising: A light source; a light detection system positioned within a housing of the particle analyzer, the light detection system having a light detector configured to detect light from the light source at a first intensity for a first predetermined time interval and to detect light from the light source at a second intensity for a second predetermined time interval; a processor having a memory operatively coupled thereto; It is equipped with The memory includes instructions stored thereon that, when executed by the processor, cause the processor to: integrating a data signal from the photodetector over a period of time that includes the first predetermined time interval and the second predetermined time interval; determining one or more parameters of the photodetector based on the integrated data signal. Particle analyzer.

11. The particle analyzer of claim 10 , wherein the light source is a continuous wave light source.

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

13. 13. The particle analyzer of any one of claims 10 to 12, wherein the processor has a memory operatively coupled thereto, the memory including instructions stored thereon which, when executed by the processor, cause the processor to increase an intensity of light from the light source over the period of time that includes the first predetermined time interval and the second predetermined time interval.

14. The processor has a memory operatively coupled thereto, the memory including instructions stored thereon that, when executed by the processor, cause the processor to: a) calculating the signal amplitude over said period of time; Determining one or more parameters of the photodetector selected from the group consisting of a minimum detection threshold, a maximum detection threshold, a detector sensitivity, a detector dynamic range, a detector signal-to-noise ratio, and a number of photoelectrons per unit power; or b) calculating the signal amplitude over said period of time; determining a parameter of the photodetector over a range of operating voltages of the photodetector; Calculating an optimal detector gain for the photodetector based on the determined parameters. A particle analyzer according to any one of claims 10 to 13, further comprising:

15. A non-transitory computer-readable storage medium having instructions stored thereon, the instructions comprising: an algorithm for illuminating the photodetector with a light source at a first intensity for a first predetermined time interval; an algorithm for illuminating the photodetector with the light source at a second intensity for a second predetermined time interval; an algorithm for integrating a data signal from the photodetector over a period of time that includes the first predetermined time interval and the second predetermined time interval; an algorithm for determining one or more parameters of the photodetector based on the integrated data signal; and A non-transitory computer readable storage medium comprising:

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