Gain-matched amplifiers for optical detection.

Abstract

A method for adjusting the sensitivity of a photodiode in a light detection system is provided. [Solution] According to certain embodiments, a method includes detecting light with a light detection system having a photodiode and an amplifier, determining the responsivity of the photodiode across multiple wavelengths of light, and adjusting one or more parameters of the amplifier in response to the responsivity of the photodiode across the multiple wavelengths of light. A system (e.g., a particle analyzer) having a light source and a light detection system including a photodiode and an amplifier for practicing the subject methods is also described. A non-transitory computer-readable storage medium is also provided.

Description

[Background technology]

[0001] Light detection is often used to characterize components of a sample (e.g., a biological sample), for example, when the sample is used to diagnose a disease or condition. When a sample is illuminated, light can be scattered by the sample, transmitted through the sample, and emitted by the sample (e.g., by fluorescence). Differences in sample components, such as morphology, absorbance, and the presence of fluorescent labels, can cause differences in the light scattered, transmitted, or emitted by the sample. These differences can be used to characterize and identify the presence of components within the sample. To quantify these differences, light is collected and directed toward a detector surface.

[0002] One technique that utilizes light detection to characterize components within a sample is flow cytometry. Data generated from the detected light can be used to record the distribution of components and to sort for desired materials. Flow cytometers typically include a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid.

[0003] A flow cytometer transports particles (including cells) in a fluid sample as a cell stream into a flow cell while directing a sheath fluid into the flow cell. Within the flow cell, a liquid sheath forms around the cell stream, imparting a substantially uniform velocity to the cell stream. The flow cell hydrodynamically focuses the cells within the stream so that they pass through the center of a light source within the flow cell. Light from the light source can be detected as scattered light or by transmission spectroscopy, or it can be absorbed by one or more components in the sample and re-emitted as luminescence. Summary of the Invention

[0004] Aspects of the present disclosure include methods for adjusting the sensitivity of a photodiode in a light detection system. The method, according to certain embodiments, includes detecting light in a light detection system having a photodiode and an amplifier, determining the responsivity of the photodiode across multiple wavelengths of light, and adjusting one or more parameters of the amplifier in response to the responsivity of the photodiode across the multiple wavelengths of light. Systems (e.g., particle analyzers) having a light source and a light detection system including a photodiode and an amplifier for practicing the subject methods are also described. A non-transitory computer-readable storage medium is also provided.

[0005] In embodiments, the light is detected by a light detection system (e.g., in a particle analyzer) having a photodiode and an amplifier (e.g., a transimpedance amplifier) ​​in electrical communication with the photodiode. In some embodiments, the light detection system includes a plurality of photodiodes, including, for example, two or more photodiodes, for example, five or more photodiodes, for example, ten or more photodiodes, for example, twenty-five or more photodiodes, for example, fifty or more photodiodes, for example, one hundred or more photodiodes, and one thousand or more photodiodes. In certain embodiments, the light detection system includes an array of photodiodes. In practicing the subject methods, each photodiode detects light across multiple wavelengths of light, and the responsivity of the photodiode is determined across the multiple wavelengths. In some embodiments, the responsivity of each photodiode is determined across a spectrum of wavelengths of light, such as when the spectrum of light spans 200 or more wavelengths of light. In some cases, the responsivity of each photodiode is determined across a spectrum of light having wavelengths ranging from 200 nm to 1500 nm, e.g., from 400 nm to 1100 nm. In certain instances, the method includes plotting the responsivity of the photodiodes over a range of wavelengths of light to generate a responsivity curve for each photodiode in the light detection system.

[0006] In some embodiments, the method includes determining an average gain of the photodiode across multiple wavelengths. In some cases, the average gain of the photodiode is determined at each wavelength independently. In other cases, the average gain of the photodiode is determined across a range of wavelengths, e.g., a spectrum of wavelengths. A resistance of the amplifier is calculated based on the determined gain (e.g., the average gain of the amplifier across multiple wavelengths of light) and the determined responsivity of the photodiode. In some cases, the determined resistance is calculated to be the resistance of a feedback resistor used in the amplifier. In certain embodiments, the resistance of the amplifier is calculated to be R f ×R(λ)=G t where R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, and G t is the average gain of the photodiode over multiple wavelengths of light.

[0007] In some embodiments, the capacitance of each amplifier is adjusted based on the calculated resistance. In some cases, the capacitance is adjusted in a manner sufficient to produce a predetermined bandwidth for each photodiode. In particular cases, the capacitance of each amplifier is adjusted to a predetermined bandwidth according to the following formula: where BW is the bandwidth and R f is the resistance of the amplifier, and C f is the capacitance of the amplifier.

[0008]

number

[0009] In some embodiments, a method includes determining one or more parameters of a photodetector (e.g., a photodetector in a particle analyzer) by illuminating particles in a flowstream, where the particles include one or more fluorophores. In some cases, the particles are beads (e.g., polystyrene beads). In some cases, the method of determining the parameters of the photodetector includes illuminating a flowstream with particles including 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 flowstream with a photodetector having a light source, generating a data signal from the photodetector at the first illumination intensity, 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 cases, the method includes determining an average fluorescence intensity from the particles at the first illumination intensity and the second illumination intensity. In some cases, the method includes determining a variance of the average fluorescence intensity at the first illumination intensity and the second illumination intensity. In some cases, the method includes determining statistical photoelectrons (SPE) at the first illumination intensity and the second illumination intensity. In certain cases, the method further includes calculating a detector efficiency (Qdet) of the photodetector per fluorophore on the particle based on the statistical photoelectrons and the determined average fluorescence intensity of the fluorophore. In certain embodiments, the method includes determining a detector efficiency per detector channel of the photodetector. In some embodiments, the method further includes determining a background signal for each photodetector. In some embodiments, the method further includes determining electronic noise from each photodetector. In certain embodiments, the method further includes determining a detection limit of the photodetector. In some embodiments, the method further includes determining the photosensitivity of one or more photodetectors. In certain embodiments, determining the photosensitivity of the photodetector includes setting an initial detector gain for the photodetector.

[0010] Aspects of the present disclosure also include a system (e.g., a particle analyzer) having a light source and a light detection system including a photodiode and an amplifier. In some embodiments, the light detection system includes a plurality of photodiodes, including, for example, two or more photodiodes, for example, five or more photodiodes, for example, ten or more photodiodes, for example, twenty-five or more photodiodes, for example, fifty or more photodiodes, for example, one hundred or more photodiodes, and one thousand or more photodiodes. In some embodiments, the light detection system also includes a plurality of amplifiers, each amplifier in electrical communication with at least one photodiode, including, for example, two or more amplifiers, for example, five or more amplifiers, for example, ten or more amplifiers, for example, twenty-five or more amplifiers, for example, fifty or more amplifiers, for example, one hundred or more amplifiers, and one thousand or more amplifiers. In certain embodiments, the light detection system includes a photodetector array. In some cases, the light detection system includes a photodetector array having N photodiodes and an amplifier component having M amplifiers, where N is an integer between 4 and 10,000, and M is an integer between 4 and 10,000. In certain cases, the number of photodiodes in the array is the same as the number of amplifiers (i.e., N is equal to M). In other cases, the number of photodiodes in the array is greater than the number of amplifiers (i.e., N is greater than M). In still other cases, the number of photodiodes in the array is less than the number of amplifiers (i.e., N is less than M). In embodiments, the light detection system is configured to detect light across multiple wavelengths. In some embodiments, the photodiodes of the light detection system are configured to detect light across a spectrum of wavelengths of light, such as when the spectrum of light spans 200 or more wavelengths of light. In some cases, the photodiodes are configured to detect light across a spectrum of light having wavelengths ranging from 200 nm to 1500 nm, e.g., from 400 nm to 1100 nm.

[0011] In some embodiments, the system also includes a processor having a memory operably coupled to the processor, the memory including instructions that, when executed by the processor, cause the processor to determine a responsivity of the photodiode across multiple wavelengths of light from the light source and adjust one or more parameters of the amplifier in response to the responsivity of the photodiode across the multiple wavelengths of light. In some embodiments, the memory includes instructions to determine an average gain of the photodiode across the multiple wavelengths. In some cases, the memory includes instructions to determine the gain of the photodiode at each wavelength independently. In other cases, the memory includes instructions to determine the average gain of the photodiode across a range of wavelengths, such as across a spectrum of wavelengths.

[0012] In some embodiments, the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to calculate a resistance of one or more of the amplifiers. In these embodiments, the memory may include instructions to calculate the resistance of each amplifier based on a determined gain (e.g., the average gain of the amplifier across a spectrum of wavelengths) and a determined responsivity of each photodiode. In some cases, the memory includes instructions to calculate the resistance of a feedback resistor used in an amplifier in electrical communication with a photodiode. In particular embodiments, the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to calculate R f ×R(λ)=G t Calculate the amplifier resistance according to the formula, where R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, and G t is the average gain of the photodiode over multiple wavelengths of light.

[0013] In some embodiments, the memory includes instructions to adjust the capacitance of each amplifier based on the calculated resistance. In some cases, the memory includes instructions to adjust the capacitance to generate a predetermined bandwidth for each photodiode. In particular cases, the memory includes instructions to adjust the capacitance of each amplifier to a predetermined bandwidth according to the following formula: where BW is the bandwidth and R f is the resistance of the amplifier, and C f is the capacitance of the amplifier.

[0014]

number

[0015] Aspects of the present disclosure also include a non-transitory computer-readable storage medium for adjusting the sensitivity of a light detection system (e.g., in a particle analyzer). The non-transitory computer-readable storage medium according to certain embodiments includes an algorithm for detecting light in a light detection system including a photodiode and an amplifier, an algorithm for determining the responsivity of the photodiode across multiple wavelengths of light, and an algorithm for adjusting one or more parameters of the amplifier in response to the responsivity of the photodiode across the multiple wavelengths of light. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining the average gain of the photodiode across multiple wavelengths. In some cases, the non-transitory computer-readable storage medium includes an algorithm for determining the gain of the photodiode independently at each wavelength. In other cases, the non-transitory computer-readable storage medium includes an algorithm for determining the average gain of the photodiode across a range of wavelengths, such as across a spectrum of wavelengths.

[0016] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the resistance of one or more of the amplifiers. In these embodiments, the non-transitory computer-readable storage medium may include an algorithm for calculating the resistance of each amplifier based on a determined gain (e.g., the average gain of the amplifier across a spectrum of wavelengths) and a determined responsivity of each photodiode. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the resistance of a feedback resistor used in an amplifier in electrical communication with a photodiode. In certain embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating R f ×R(λ)=G t where R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, and G t is the average gain of the photodiode over multiple wavelengths of light.

[0017] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for adjusting the capacitance of each amplifier based on the calculated resistance. In some cases, the non-transitory computer-readable storage medium includes an algorithm for adjusting the capacitance to generate a predetermined bandwidth for each photodiode. In particular cases, the non-transitory computer-readable storage medium includes instructions for adjusting the capacitance of each amplifier to a predetermined bandwidth according to the following formula: where BW is the bandwidth and R f is the resistance of the amplifier, and C f is the capacitance of the amplifier.

[0018]

number

[0019] 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 fluorophores, for example, including two or more, e.g., three or more, e.g., five or more, and ten or more photodetectors. In some cases, the particle of interest includes a single-peak multifluorophore bead that provides a bright photodetector signal across all light source wavelengths (e.g., all LEDs or lasers in the system) and across the detection wavelengths of the photodetectors. [Brief explanation of the drawings]

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

[0021] [Figure 1] FIG. 2 illustrates an exemplary wavelength-dependent response curve of a photodiode in accordance with certain embodiments. [Figure 2] FIG. 1 illustrates an amplifier for adjusting the responsivity of a photodiode in accordance with certain embodiments. [Figure 3A] FIG. 10 is a flow diagram for adjusting the sensitivity of a photodiode in a light detection system in accordance with certain embodiments. [Figure 3B] FIG. 10 illustrates a plot used to set the initial detector gain of a photodetector in accordance with certain embodiments. [Figure 4A] FIG. 1 is a functional block diagram illustrating a particle analysis system for computation-based sample analysis and particle characterization in accordance with certain embodiments. [Figure 4B] FIG. 1 is a functional block diagram illustrating an example of a sorting control system in accordance with certain embodiments. [Figure 5] FIG. 1 is a functional block diagram illustrating an example of a particle analyzer control system in accordance with certain embodiments. [Figure 6A] FIG. 1 is a schematic diagram illustrating a particle sorter system in accordance with certain embodiments. [Figure 6B]FIG. 1 is a schematic diagram illustrating a particle sorter system in accordance with certain embodiments. [Figure 7] FIG. 1 is a block diagram illustrating a computing system in accordance with certain embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0022] Aspects of the present disclosure include methods for adjusting the sensitivity of a photodiode in a light detection system. The method, according to certain embodiments, includes detecting light in a light detection system having a photodiode and an amplifier, determining the responsivity of the photodiode across multiple wavelengths of light, and adjusting one or more parameters of the amplifier in response to the responsivity of the photodiode across the multiple wavelengths of light. Systems (e.g., particle analyzers) having a light source and a light detection system including a photodiode and an amplifier for practicing the subject methods are also described. A non-transitory computer-readable storage medium is also provided.

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

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

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

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

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

[0028] It should be noted that, as used in this specification and the appended claims, the 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. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for use of a "negative" limitation.

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

[0030] Although the apparatus and methods are described with functional descriptions for grammatical fluidity, it is expressly understood that unless expressly recited under 35 U.S.C. § 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 that if the claims are expressly recited under 35 U.S.C. § 112, they should be accorded the full legal equivalents under 35 U.S.C. § 112.

[0031] As summarized above, the present disclosure provides a method for adjusting the sensitivity of a photodiode in a light detection system. In further describing embodiments of the present disclosure, a method for detecting light in a light detection system having a photodiode and an amplifier, determining the responsivity of the photodiode across multiple wavelengths of light, and adjusting one or more parameters of the amplifier in response to the responsivity of the photodiode across multiple wavelengths of light is first described in more detail. Next, a system including a light source and a light detection system having a photodiode and an amplifier for practicing the subject method is described. A non-transitory computer-readable storage medium is also described.

[0032] Method for adjusting the sensitivity of a photodiode in a light detection system - Patent Application 20070122997 Aspects of the present disclosure include methods for adjusting the sensitivity of photodiodes in a light detection system. The term "sensitivity" is used herein in its conventional sense to refer to the ratio of detector output to detector input. In some embodiments, the sensitivity of a photodiode refers to the ratio of the current output from the photodiode to the current generated by the light detected by the photodiode. In embodiments, the subject methods increase the output (e.g., detector signal amplitude) from one or more photodiodes by 5% or more, including, for example, 10% or more, e.g., 25% or more, e.g., 50% or more, e.g., 75% or more, e.g., 90% or more, and 99% or more. In certain cases, the subject methods increase the detector output by 2x or more, including, for example, 3x or more, e.g., 4x or more, e.g., 5x or more, and 10x or more. In some embodiments, the subject methods increase the signal-to-noise ratio of one or more photodiodes by 5% or more, including, for example, 10% or more, for example, 25% or more, for example, 50% or more, for example, 75% or more, for example, 90% or more, and 99% or more. In certain cases, the subject methods increase the signal-to-noise ratio of one or more photodiodes by 2-fold or more, including, for example, 3-fold or more, for example, 4-fold or more, for example, 5-fold or more, and 10-fold or more. In certain embodiments, the disclosed methods are sufficient to extend the range of intensity detection and quantification by 2-fold or more, including, for example, 3-fold or more, for example, 5-fold or more, for example, 10-fold or more, for example, 25-fold or more, for example, 50-fold or more, and 100-fold or more.

[0033] In practicing the subject methods, light is detected by a photodiode in electrical communication with an amplifier in a light detection system (e.g., in a particle analyzer or flow cytometer, as described in more detail below). In certain embodiments, the light is from an illuminated sample in a flow stream. The detected light can be emitted light, transmitted light, scattered light, or a combination thereof. In embodiments, multiple wavelengths of light are detected by each photodiode. In some embodiments, the light detected by each photodiode includes 10 or more different wavelengths of light, including, for example, 15 or more, for example, 25 or more, for example, 50 or more, for example, 100 or more, for example, 200 or more, for example, 300 or more, for example, 400 or more, for example, 500 or more, for example, 1000 or more, for example, 1500 or more, for example, 2500 or more, and 5000 or more different wavelengths of light. In certain embodiments, the light detected by each photodiode comprises a spectrum of light, such as where the spectrum of light includes wavelengths ranging from 50 nm or more, including, for example, 100 nm or more, e.g., 200 nm or more, e.g., 300 nm or more, e.g., 400 nm or more, e.g., 500 nm or more, e.g., 600 nm or more, e.g., 700 nm or more, e.g., 800 nm or more, e.g., 900 nm or more, e.g., 1000 nm or more, and 1500 nm or more. For example, according to the subject methods, the light detected by each photodiode may range from 200 nm to 1500 nm, such as 400 nm to 1100 nm.

[0034] In embodiments, the light is detected with a light detection system including a photodiode and an amplifier in electrical communication with the photodiode. In some embodiments, the photodiode is an avalanche photodiode. In certain embodiments, the method includes detecting the light with a light detection system having a plurality of photodiodes, such as an array of photodiodes. For example, the detector may include four or more photodiodes, including, for example, 10 or more photodiodes, for example, 25 or more photodiodes, for example, 50 or more photodiodes, for example, 100 or more photodiodes, for example, 250 or more photodiodes, for example, 500 or more photodiodes, for example, 750 or more photodiodes, and 1000 or more photodiodes.

[0035] The photodiodes may be arranged in any geometric configuration as desired; configurations of interest include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, non-rectangular, decagonal, dodecagonal, circular, elliptical, and irregularly patterned configurations. The photodiodes within a photodiode array may be oriented relative to one another (as referenced in the XZ plane) at angles ranging from 10° to 180°, including, for example, 15° to 170°, for example, 20° to 160°, for example, 25° to 150°, for example, 30° to 120°, and 45° to 90°. The photodiodes may be of any suitable shape, including, for example, rectilinear shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curvilinear shapes such as circles and ellipses; and irregular shapes such as, for example, a parabolic base coupled to a planar top. In certain embodiments, the photodiode array has a rectangular active surface.

[0036] Each photodiode in the array may have an active surface with a width in the range of 5 μm to 250 μm, for example, 10 μm to 225 μm, for example, 15 μm to 200 μm, for example, 20 μm to 175 μm, for example, 25 μm to 150 μm, for example, 30 μm to 125 μm, and 50 μm to 100 μm, and a length in the range of 5 μm to 250 μm, for example, 10 μm to 225 μm, for example, 15 μm to 200 μm, for example, 20 μm to 175 μm, for example, 25 μm to 150 μm, for example, 30 μm to 125 μm, and 50 μm to 100 μm, and the surface area of ​​each photodiode in the array may be, for example, 50 μm 2 ~9000μm 2 , e.g., 75 μm 2 ~8000μm 2 , e.g., 100 μm 2 ~7000μm 2 , e.g., 150 μm 2 ~6000μm 2 , and 200 μm 2 ~5000μm 2 Including, 25μm 2 ~10,000 μm 2 The range is.

[0037] The size of the photodiode array can vary depending on the amount and intensity of light, the number of photodiodes, and the desired sensitivity, and can have a length ranging from 0.01 mm to 100 mm, including, for example, 0.05 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, for example, 2 mm to 50 mm, for example, 3 mm to 40 mm, for example, 4 mm to 30 mm, and 5 mm to 25 mm. The width of the photodiode array can also vary from 0.01 mm to 100 mm, including, for example, 0.05 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, for example, 2 mm to 50 mm, for example, 3 mm to 40 mm, for example, 4 mm to 30 mm, and 5 mm to 25 mm. Thus, the active surface of the photodetector array can be, for example, 0.5 mm. 2 ~5000mm 2 , e.g., 1 mm 2~1000m 2 , e.g., 5 mm 2 ~500mm 2 , and 10mm 2 ~100m 2 Including 0.1mm 2 ~10,000mm 2 The range may be:

[0038] The method may include measuring light continuously or at discrete intervals. In some cases, the photodiode of interest is configured to measure collected light continuously. In other cases, the light detection system is configured to measure at discrete intervals, such as every 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, and 1000 milliseconds, or some other interval.

[0039] In practicing the subject methods according to certain embodiments, each photodiode detects light across multiple wavelengths of light, and the responsivity of the photodiode is determined across the multiple wavelengths. Here, the term “responsivity” is used in its conventional sense to refer to the ratio of photocurrent generated by the photodiode as a function of the amount of incident light. In some embodiments, the responsivity of each photodiode is determined across 10 or more different wavelengths of light, including, for example, 15 or more, e.g., 25 or more, e.g., 50 or more, e.g., 100 or more, e.g., 200 or more, e.g., 300 or more, e.g., 400 or more, e.g., 500 or more, e.g., 1000 or more, e.g., 1500 or more, e.g., 2500 or more, and 5000 or more different wavelengths of light. In certain embodiments, the responsivity of each photodiode is determined across a spectrum of light, such as when the spectrum of light includes wavelengths ranging from 50 nm or greater, including, for example, 100 nm or greater, e.g., 200 nm or greater, e.g., 300 nm or greater, e.g., 400 nm or greater, e.g., 500 nm or greater, e.g., 600 nm or greater, e.g., 700 nm or greater, e.g., 800 nm or greater, e.g., 900 nm or greater, e.g., 1000 nm or greater, and 1500 nm or greater. For example, the responsivity of each photodiode according to the subject method may be determined across the spectrum of light in the range of 200 nm to 1500 nm, such as 400 nm to 1100 nm.

[0040] In certain instances, the method includes plotting the responsivity of the photodiodes over a range of wavelengths of light to generate a responsivity curve for each photodiode in the optical detection system. FIG. 1 illustrates an exemplary wavelength-dependent responsivity curve for a photodiode according to certain embodiments. In FIG. 1, the photodiode exhibits an increased responsivity from 450 nm to approximately 875 nm and a decreased responsivity from 875 nm to 1100 nm. As illustrated by the responsivity curve, the response of the photodiode detector is approximately 10 times greater at 875 nm than at 450 nm.

[0041] In some embodiments, the method includes using the responsivity of the photodiode across multiple wavelengths to determine an average gain of the photodiode across multiple wavelengths. In some cases, the average gain of the photodiode is determined independently at each wavelength. For example, if the responsivity of the photodiode is determined across a spectrum of wavelengths (e.g., 200 nm to 1500 nm, such as 400 nm to 1100 nm), the method includes determining the average gain of the photodiode every 1 nm across the spectrum of wavelengths, including, for example, every 2 nm, every 5 nm, e.g., every 10 nm, and every 25 nm across the spectrum of wavelengths or some other interval. In other embodiments, the average gain of the photodiode is determined across a range of wavelengths, including, for example, over a range of 1 nm or more, e.g., over a range of 2 nm or more, e.g., over a range of 5 nm or more, e.g., over a range of 10 nm or more, e.g., over a range of 25 nm or more, e.g., over a range of 50 nm or more, e.g., over a range of 100 nm or more, e.g., over a range of 250 nm or more, e.g., over a range of 500 nm or more, and over 1000 nm or more.

[0042] The resistance of the amplifier is calculated based on the determined gain (e.g., the average gain of the amplifier over multiple wavelengths of light) and the determined responsivity of the photodiode. In some cases, the determined resistance is calculated to be the resistance of a feedback resistor used in the amplifier. In certain embodiments, the resistance of the amplifier is R f ×R(λ)=G t where R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, and G t is the average gain of the photodiode over multiple wavelengths of light.

[0043] In some embodiments, the capacitance of each amplifier is adjusted based on the calculated resistance. In certain embodiments, the capacitance is adjusted by 5% or more, including, for example, 10% or more, for example, 20% or more, for example, 30% or more, for example, 40% or more, for example, 50% or more, for example, 60% or more, for example, 70% or more, for example, 80% or more, for example, 90% or more, and 95% or more. In some cases, the capacitance of the amplifier is adjusted to compensate for the wavelength-dependent responsivity of the photodiode.

[0044] In some cases, the capacitance of the amplifier is increased to compensate for the wavelength-dependent responsivity of the photodiode. In other cases, the capacitance of the amplifier is decreased to compensate for the wavelength-dependent responsivity of the photodiode. In certain cases, the capacitance of the amplifier is adjusted to produce a wavelength responsivity of the photodiode that is substantially the same across multiple wavelengths (e.g., across a spectrum of wavelengths). In other cases, the capacitance of the amplifier is adjusted to produce a desired responsivity of the photodiode at one or more predetermined detection wavelengths.

[0045] 2 shows an amplifier for adjusting the responsivity of a photodiode according to certain embodiments. Amplifier 201 is in electrical communication with photodiode 202 through capacitor 203 and feedback resistor 204. The resistance of feedback resistor 204 is determined based on the responsivity of the photodiode at each wavelength and the average gain of the photodiode across multiple wavelengths of light.

[0046] In some cases, the capacitance is adjusted in a manner sufficient to produce a predetermined bandwidth for each photodiode. In particular cases, the capacitance of each amplifier is adjusted to a predetermined bandwidth according to the following formula, where BW is the bandwidth and R f is the resistance of the amplifier, and C f is the capacitance of the amplifier.

[0047]

number

[0048] Depending on the average gain of the amplifier, the predetermined bandwidth may be, for example, 10 kHz or greater, including predetermined bandwidths of 25 kHz or greater, for example, 50 kHz or greater, for example, 100 kHz or greater, for example, 150 kHz or greater, for example, 200 kHz or greater, for example, 250 kHz or greater, for example, 500 kHz or greater, and 1000 kHz or greater. 6 In one example, in volts / amps, the predetermined bandwidth may be, for example, 275 kHz or greater, such as 300 kHz or greater, such as 325 kHz or greater, such as 350 kHz or greater, such as 375 kHz or greater, such as 400 kHz or greater, such as 425 kHz or greater, such as 450 kHz or greater, such as 475 kHz or greater, and 250 kHz or greater, including predetermined bandwidths of 500 kHz or greater.

[0049] FIG. 3A shows a flow diagram for adjusting the sensitivity of photodiodes in a light detection system according to certain embodiments. In step 301, light from a light source is detected by a photodiode in electrical communication with an amplifier. In step 302, the responsivity of the photodiode is determined across multiple wavelengths, such as across a spectrum of wavelengths. In certain embodiments, determining the responsivity of the photodiode includes generating a wavelength-dependent responsivity curve comparing the responsivity of the photodiode as a function of the wavelength of the light. In step 303, the average gain of the amplifier is determined across multiple wavelengths (e.g., across a spectrum of wavelengths), and the resistance of the amplifier is calculated based on the determined responsivity of the photodiode and the average gain of the amplifier. The capacitance of each amplifier is adjusted in step 304 based on the calculated resistance. In certain cases, the capacitance of the amplifier is adjusted to produce a wavelength responsivity of the photodiode that is substantially the same across multiple wavelengths (e.g., across a spectrum of wavelengths). In other cases, the capacitance of the amplifier is adjusted to produce a desired responsivity of the photodiode at one or more predetermined detection wavelengths.

[0050] In other embodiments, the diodes of the subject methods are light sources, such as broadband light sources emitting light having a wide range of wavelengths, e.g., spanning 50 nm or more, e.g., 100 nm or more, e.g., 150 nm or more, e.g., 200 nm or more, e.g., 250 nm or more, e.g., 300 nm or more, e.g., 350 nm or more, e.g., 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 emitting light having a wavelength between 400 nm and 1000 nm. When the light source is a broadband light source, broadband light source protocols of interest may include, but are not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, continuous spectrum broadband LEDs, superluminescent diodes, semiconductor light-emitting diodes, wide-spectrum LED white light sources, and multi-LED integrated white light sources, among other broadband light sources or any combination thereof.

[0051] In other embodiments, the light source is a narrowband light source that emits a specific wavelength or narrow range of wavelengths, such as a light source that emits light in a narrow wavelength range, such as a range of 40 nm or less, e.g., 30 nm or less, e.g., 25 nm or less, e.g., 20 nm or less, e.g., 15 nm or less, e.g., 10 nm or less, e.g., 5 nm or less, e.g., 2 nm or less, and a light source that emits a specific wavelength of light (i.e., monochromatic light), such as a range of 50 nm or less. When the method includes irradiating with a narrowband light source, narrowband light source protocols of interest may include, but are not limited to, a narrow wavelength LED, a laser diode, or a broadband light source coupled to one or more optical bandpass filters, a diffraction grating, a monochromator, or any combination thereof.

[0052] In certain embodiments, the light source comprises one or more lasers, such as a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, a CO laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In other cases, the method comprises irradiating the flow stream with a dye laser, such as a stilbene, coumarin, or rhodamine laser. In still other cases, the light source comprises 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 still other cases, the light source comprises 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 slim YAG laser, a ytterbium YAG laser, a Y2O3 laser, or a cerium-doped laser, and combinations thereof.

[0053] The light source may include any combination of light sources. The light source may be configured to emit light at wavelengths in the range of 200 nm to 1500 nm, including, for example, 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. For example, the light source may be a broadband light source that emits light at wavelengths in the range of 200 nm to 900 nm. In other cases, the light source includes multiple narrowband light sources that emit light at specific wavelengths in the range of 200 nm to 900 nm. For example, the light source may be multiple narrowband LEDs (1 nm to 25 nm), each independently emitting light having a wavelength in the range of 200 nm to 900 nm. In other embodiments, the narrowband light source includes one or more lasers (e.g., a laser array) that emit light at specific wavelengths in the range of 200 nm to 700 nm, such as a laser array having a gas laser, excimer laser, dye laser, metal vapor laser, and solid-state laser, as described above.

[0054] When two or more light sources are employed, the light sources can be configured to irradiate simultaneously, sequentially, or a combination thereof. In one example, the light sources are configured to irradiate simultaneously. In other embodiments, the light sources are configured to irradiate sequentially. When two or more light sources are employed to irradiate sequentially, the duration of irradiation by each light source can be independently 0.001 microseconds or more, including, for example, 0.01 microseconds or more, such as 0.1 microseconds or more, such as 1 microsecond or more, such as 5 microseconds or more, such as 10 microseconds or more, such as 30 microseconds or more, and 60 microseconds or more. For example, the light source (e.g., a laser) can be configured to irradiate for a duration ranging from 0.001 microseconds to 100 microseconds, including, for example, 0.01 microseconds to 75 microseconds, such as 0.1 microseconds to 50 microseconds, such as 1 microsecond to 25 microseconds, and 5 microseconds to 10 microseconds. The duration of irradiation by each light source can be the same or different.

[0055] The period between illumination by each light source can also vary, as needed, separated by a delay of 0.001 microseconds or more, including, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 15 microseconds or more, for example, 30 microseconds or more, and 60 microseconds or more. For example, the period between illumination by each light source can range from 0.001 microseconds to 60 microseconds, including, for example, 0.01 microseconds to 50 microseconds, for example, 0.1 microseconds to 35 microseconds, for example, 1 microsecond to 25 microseconds, and 5 microseconds to 10 microseconds. In certain embodiments, the period between illumination by each light source is 10 microseconds. The delay between illumination by each light source can be the same or different.

[0056] The illumination can be continuous or at discrete intervals. In some cases, the light source illuminates continuously. In other cases, the light source illuminates at discrete intervals, including every 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, and 1000 milliseconds, or some other interval.

[0057] In certain embodiments, the light source is an optical beam generator configured to generate two or more frequency-shifted beams of light. In some cases, the optical beam generator includes a laser and a radio frequency generator configured to apply a radio frequency drive signal to an acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous wave laser. For example, lasers in optical beam generators of interest include gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO2 lasers, argon fluorine (ArF) excimer lasers, krypton fluorine (KrF) excimer lasers, xenon chlorine (XeCl) excimer lasers, or xenon fluorine (XeF) excimer lasers, or combinations thereof; dye lasers such as stilbene, coumarin, or rhodamine lasers; helium cadmium (HeCd) lasers; helium mercury (HeHg) lasers; helium selenium (HeS) lasers; e) Lasers may be metal vapor lasers such as helium silver (HeAg) lasers, strontium lasers, neon copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof, solid state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers, or cerium doped lasers, and combinations thereof.

[0058] The acousto-optical device can be any convenient acousto-optical protocol configured to detect frequency-shifted laser light using an applied acoustic wave. In certain embodiments, the acousto-optical device is an acousto-optical deflector. The acousto-optical device in the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied radio frequency drive signal. The radio frequency drive signal can be applied to the acousto-optical device with any suitable radio frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0059] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams in the output laser beam, e.g., three or more radio frequency drive signals, e.g., four or more radio frequency drive signals, e.g., five or more radio frequency drive signals, e.g., six or more radio frequency drive signals, e.g., seven or more radio frequency drive signals, e.g., eight or more radio frequency drive signals, e.g., nine or more radio frequency drive signals, e.g., ten or more radio frequency drive signals, e.g., fifteen or more radio frequency drive signals, e.g., twenty-five or more radio frequency drive signals, e.g., fifty or more radio frequency drive signals, and including being configured to apply one hundred or more radio frequency drive signals.

[0060] In some cases, to generate an intensity profile of the angularly deflected laser beam within the output laser beam, the controller is configured to apply a radio frequency drive signal having a varying amplitude, including, for example, from about 0.001 V to about 500 V, for example, from about 0.005 V to about 400 V, for example, from about 0.01 V to about 300 V, for example, from about 0.05 V to about 200 V, for example, from about 0.1 V to about 100 V, for example, from about 0.5 V to about 75 V, for example, from about 1 V to about 50 V, for example, from about 2 V to about 40 V, for example, from 3 V to about 30 V, and from about 5 V to about 25 V. Each applied radio frequency drive signal, in some embodiments, has a frequency of, for example, about 0.005 MHz to about 400 MHz, for example, about 0.01 MHz to about 300 MHz, and has a frequency of, for example, about 0.001 MHz to about 500 MHz, including, for example, about 0.05 MHz to about 200 MHz, for example, about 0.1 MHz to about 100 MHz, for example, about 0.5 MHz to about 90 MHz, for example, about 1 MHz to about 75 MHz, for example, about 2 MHz to about 70 MHz, for example, about 3 MHz to about 65 MHz, for example, about 4 MHz to about 60 MHz, and about 5 MHz to about 50 MHz.

[0061] In some embodiments, the method includes determining one or more parameters of a photodetector (e.g., a photodetector in a particle analyzer), such as by illuminating particles in a flowstream, where the particles include one or more fluorophores. In some cases, the particles are beads (e.g., polystyrene beads), as described in more detail below. In some cases, the subject methods described below provide for determining parameters of the photodetectors, including relative fluorescence units (e.g., ABD units) assigned to 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.

[0062] In some cases, a method of determining a parameter of a photodetector includes illuminating a flowstream with 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 flowstream with a photodetector having a light source, generating a data signal from the photodetector at the first illumination intensity, 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.

[0063] In some embodiments, the method includes determining the mean fluorescence intensity (M) from the particles at a first illumination intensity and a second illumination intensity. In some cases, the method includes determining the variance of the mean fluorescence intensity (V(M)) at the first illumination intensity and the second illumination intensity. In certain cases, the method includes determining the rCV% (robust coefficient of variation) of the photodetector. In certain embodiments, the linear fit of the variance is calculated according to the following formula, where Q led is given by 1 / c1, the statistical photoelectrons per unit mean fluorescence intensity (M) (i.e., SPE / MFI).

[0064]

number

[0065] In certain embodiments, the variance is plotted to determine the linear fit of the variance according to y=c1x+c0.

[0066] In embodiments, the mean fluorescence intensity and variance may be determined for a plurality of different illumination intensities, such as two or more illumination intensities of light, including, for example, three or more, such as four or more, for example, five or more, such as six or more, for example, seven or more, such as eight or more, for example, nine or more, such as ten or more, and fifteen or more different wavelengths of light.

[0067] In some embodiments, the method includes determining statistical photoelectrons (SPE) at one or more of the illumination intensities, such as at least a first illumination intensity and a second illumination intensity. In certain cases, the method further includes calculating a detector efficiency (Qdet) of the photodetector for each particle based on the statistical photoelectrons and the determined mean fluorescence intensity of the particles. In certain embodiments, the method includes determining detector efficiencies for one or more detector channels of the photodetector, 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, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty four or more, for example, thirty six or more, for example, forty eight or more, for example, seventy two or more, and including determining detector efficiencies for 96 or more detector channels of the photodetector based on the statistical photoelectrons and the determined mean fluorescence intensity of the particles. In certain instances, the method includes determining a detector efficiency for all detector channels of the photodetector for each particle based on the statistical photoelectrons and the determined mean fluorescence intensity of each particle. In certain embodiments, the detector efficiency for the photodetector is determined according to the following formula, where SPE is the statistical photoelectrons, MFI is the mean fluorescence intensity, and ABD is the assigned units per channel per particle lot.

[0068]

number

[0069] In certain embodiments, the method further comprises determining a background signal for one or more of the photodetectors. In some cases, the background signal is determined at one or more illumination intensities, including, 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, and ten or more different illumination intensities. In some cases, 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, including determining the background signal in 96 or more detector channels of the photodetector based on the determined average fluorescence intensity of statistical photoelectrons and particles, 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, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty four or more, for example, thirty six or more, for example, forty eight or more, for example, seventy two or more, and in certain cases, the background signal is determined in all detector channels of the photodetector. In some cases, the background signal is determined based on the detector efficiency of statistical photoelectrons and photodetectors. In certain cases, the background signal is determined in B SD =B SD,MFI ×Q led and the following formula:

[0070]

number

[0071] In some embodiments, the method further includes determining electronic noise for one or more of the photodetectors. In some cases, the electronic noise of the photodetectors is determined at one or more illumination intensities, including, 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, and ten or more different illumination intensities. In some cases, the electronic noise of the photodetectors is determined at all of the applied illumination intensities. Electronic noise can also be determined in one or more detector channels of a photodetector, including determining electronic noise in, 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, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty four or more, for example, thirty six or more, for example, forty eight or more, for example, seventy two or more, and 96 or more of a photodetector based on the determined average fluorescence intensity of statistical photoelectrons and particles, and in certain cases, electronic noise is determined in all of the detector channels of a photodetector. In some cases, electronic noise is determined based on the detector efficiency of statistical photoelectrons and photodetectors. In certain cases, electronic noise is determined based on the EN SD =EN SD,MFI ×Q led and the following formula:

[0072]

number

[0073] In some embodiments, the method further includes determining a detection limit of one or more of the photodetectors. In some cases, the detection limit of the photodetector can be determined within one or more detector channels of the photodetector, including determining the detection limits of, 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, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty-four or more, for example, thirty-six or more, for example, forty-eight or more, for example, seventy-two or more, and 96 or more photodetectors within the photodetector; in certain cases, the detection limit of the photodetector is determined across all of the detector channels of the photodetector. In some cases, the detection limit of each photodetector is determined as 2 + 2SD = 4(1 + B SD ) is judged according to

[0074] In some embodiments, the method further includes determining the optical sensitivity of one or more photodetectors. In certain embodiments, determining the optical sensitivity of the photodetector includes setting an initial detector gain of the photodetector. In some cases, 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, at each detector gain, the lowest light illumination intensity that produces a data signal resolvable from a background data signal. In some cases, the method includes determining, at each detector gain, the lowest light illumination intensity that produces a data signal that is two standard deviations from the background data signal. In certain cases, the method includes setting the detector gain to the gain of the lowest light illumination intensity that produces a data signal resolvable from a background data signal plateau when plotted as a function of light intensity. Figure 3B shows 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 stably associated with particles, as described in more detail below). In setting the initial detector gain for the photodetector, the lowest light illumination intensity that produces a data signal resolvable from the background data signal plateau is determined, which in Figure 3B is approximately 575 volts.

[0075] System for adjusting the sensitivity of a photodiode As summarized above, aspects of the present disclosure also include a system (e.g., a particle analyzer) having a light source and a light detection system including a photodiode and an amplifier. As noted above, the term "sensitivity" is used herein to refer to the ratio of the detector output to the detector input. In some embodiments, the sensitivity of a photodiode refers to the ratio of the current output from the photodiode to the current generated by light detected by the photodiode. A system according to certain embodiments includes a light source, a light detection system (e.g., positioned in a housing of the particle analyzer) having a photodiode and an amplifier, and a processor with a memory operably coupled to the processor, the memory including instructions stored in the processor that, when executed by the processor, cause the processor to determine a responsivity of the photodiode across multiple wavelengths of light from the light source and adjust one or more parameters of the amplifier in response to the responsivity of the photodiode across the multiple wavelengths of light.

[0076] In embodiments, the light source may be any suitable broadband or narrowband light source. Depending on the components within the sample (e.g., cells, beads, non-cellular particles, etc.), the light source may be configured to emit wavelengths of light ranging from 200 nm to 1500 nm, including, for example, 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, and 400 nm to 800 nm. For example, the light source may include a broadband light source emitting light having a wavelength between 200 nm and 900 nm. In other cases, the light source may include a narrowband light source emitting wavelengths in the range of 200 nm to 900 nm. For example, the light source may be a narrowband LED (1 nm to 25 nm) emitting light having a wavelength in the range of 200 nm to 900 nm. In certain embodiments, the light source is a laser. In some cases, the subject systems include 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 cases, the subject systems include a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, lasers of interest include metal vapor lasers, 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 still other instances, the subject systems include solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, slim YAG lasers, ytterbium YAG lasers, Y2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0077] In other embodiments, the light source is a non-laser light source such as a lamp including, but not limited to, a halogen lamp, a deuterium arc lamp, a xenon arc lamp, a light emitting diode such as a broadband LED with a continuous spectrum, a superluminescent diode, a semiconductor light emitting diode, a broad spectrum LED white light source, a multi-LED integration, etc. In some cases, the non-laser light source is a stabilized fiber-coupled broadband light source, a white light source, or any combination thereof, among other light sources.

[0078] The light source may be positioned at any suitable distance from the sample (e.g., a flow stream in a flow cytometer), such as at a distance of 0.001 mm or more from the flow stream, including, for example, at a distance of 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, and 100 mm or more. In addition, the light source illuminates the sample at any suitable angle (e.g., relative to the perpendicular axis of the flow stream), such as at an angle ranging from 10° to 90°, for example, at a 90° angle, including, for example, from 15° to 85°, for example, from 20° to 80°, for example, from 25° to 75°, and from 30° to 60°.

[0079] The light source can be configured to illuminate the sample continuously or at discrete intervals. In some cases, the system includes a light source configured to continuously illuminate the sample, such as with a continuous wave laser that continuously illuminates the flow stream at an interrogation point within the flow cytometer. In other cases, the system of interest includes a light source configured to illuminate the sample at discrete intervals, including every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 milliseconds, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or some other interval. When the light source is configured to illuminate the sample at discrete intervals, the system may include one or more additional components to provide intermittent illumination of the sample with the light source. For example, the subject systems of these embodiments may include one or more laser beam choppers, manual or computer-controlled beam stops for blocking and exposing the sample to the light source.

[0080] In some embodiments, the light source is a laser. Lasers of interest may include pulsed or continuous wave lasers. For example, the laser may be a gas laser such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO2 laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof; a dye laser such as a stilbene, coumarin, or rhodamine laser; a helium cadmium (HeCd) laser, a helium mercury (HeHg) laser, a helium selenium (HeSe) laser, a helium silver (HeAg) laser, a strontium laser, or a neon copper laser. The laser may be a metal vapor laser such as a (NeCu) laser, a copper laser, or a gold laser, and combinations thereof; 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 thulium YAG laser, a ytterbium YAG laser, a Y2O3 laser, or a cerium-doped laser, and combinations thereof; a semiconductor diode laser; an optically pumped semiconductor laser (OPSL), or a frequency doubled or tripled implementation of any of the above lasers.

[0081] In certain embodiments, the light source is an optical beam generator configured to generate two or more frequency-shifted beams of light. In some cases, the optical beam generator includes a laser and a radio frequency generator configured to apply a radio frequency drive signal to an acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous wave laser. For example, lasers in optical beam generators of interest include gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO2 lasers, argon fluorine (ArF) excimer lasers, krypton fluorine (KrF) excimer lasers, xenon chlorine (XeCl) excimer lasers, or xenon fluorine (XeF) excimer lasers, or combinations thereof; dye lasers such as stilbene, coumarin, or rhodamine lasers; helium cadmium (HeCd) lasers; helium mercury (HeHg) lasers; helium selenium (HeS) lasers; e) Lasers may be metal vapor lasers such as helium silver (HeAg) lasers, strontium lasers, neon copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof, solid state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Y2O3 lasers, or cerium doped lasers, and combinations thereof.

[0082] The acousto-optical device can be any convenient acousto-optical protocol configured to detect frequency-shifted laser light using an applied acoustic wave. In certain embodiments, the acousto-optical device is an acousto-optical deflector. The acousto-optical device in the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied radio frequency drive signal. The radio frequency drive signal can be applied to the acousto-optical device with any suitable radio frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0083] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams in the output laser beam, e.g., three or more radio frequency drive signals, e.g., four or more radio frequency drive signals, e.g., five or more radio frequency drive signals, e.g., six or more radio frequency drive signals, e.g., seven or more radio frequency drive signals, e.g., eight or more radio frequency drive signals, e.g., nine or more radio frequency drive signals, e.g., ten or more radio frequency drive signals, e.g., fifteen or more radio frequency drive signals, e.g., twenty-five or more radio frequency drive signals, e.g., fifty or more radio frequency drive signals, and including being configured to apply one hundred or more radio frequency drive signals.

[0084] In some cases, to generate an intensity profile of the angularly deflected laser beam within the output laser beam, the controller is configured to apply a radio frequency drive signal having a varying amplitude, including, for example, from about 0.001 V to about 500 V, for example, from about 0.005 V to about 400 V, for example, from about 0.01 V to about 300 V, for example, from about 0.05 V to about 200 V, for example, from about 0.1 V to about 100 V, for example, from about 0.5 V to about 75 V, for example, from about 1 V to about 50 V, for example, from about 2 V to about 40 V, for example, from 3 V to about 30 V, and from about 5 V to about 25 V. Each applied radio frequency drive signal, in some embodiments, has a frequency of, for example, about 0.005 MHz to about 400 MHz, for example, about 0.01 MHz to about 300 MHz, and has a frequency of, for example, about 0.001 MHz to about 500 MHz, including, for example, about 0.05 MHz to about 200 MHz, for example, about 0.1 MHz to about 100 MHz, for example, about 0.5 MHz to about 90 MHz, for example, about 1 MHz to about 75 MHz, for example, about 2 MHz to about 70 MHz, for example, about 3 MHz to about 65 MHz, for example, about 4 MHz to about 60 MHz, and about 5 MHz to about 50 MHz.

[0085] In certain embodiments, the controller has a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to generate an output laser beam having an angularly deflected laser beam with a desired intensity profile. For example, the memory may include instructions to generate two or more angularly deflected laser beams, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more, having the same intensity, or the memory may include instructions to generate one hundred or more angularly deflected laser beams having the same intensity. In other embodiments, the memory may include instructions to generate two or more angularly deflected laser beams, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more, having different intensities, or the memory may include instructions to generate one hundred or more angularly deflected laser beams having different intensities.

[0086] In certain embodiments, the controller has a memory operatively coupled to the processor, the memory including instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam having an increasing intensity from the edge to the center of the output laser beam along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam can be in the range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., about 2% to about 85%, e.g., about 3% to about 80%, e.g., about 4% to about 75%, e.g., about 5% to about 70%, e.g., about 6% to about 65%, e.g., about 7% to about 60%, e.g., about 8% to about 55%, and including about 10% to about 50% of the intensity of the angularly deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a memory operatively coupled to the processor, the memory including instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam having an increasing intensity from the edge to the center of the output laser beam along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam can be in the range of 0.1% to about 99%, e.g., 0.5% to about 95%, e.g., 1% to about 90%, e.g., about 2% to about 85%, e.g., about 3% to about 80%, e.g., about 4% to about 75%, e.g., about 5% to about 70%, e.g., about 6% to about 65%, e.g., about 7% to about 60%, e.g., about 8% to about 55%, and including about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In yet another embodiment, the controller has a memory operably coupled to the processor, the memory including instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam having an intensity profile with a Gaussian distribution along a horizontal axis.In yet another embodiment, the controller has a memory operably coupled to the processor, the memory including instructions stored therein that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0087] In embodiments, the optical beam generator of interest can be configured to generate angularly polarized laser beams within the spatially separated output laser beam. Depending on the applied radio frequency drive signal and the desired illumination profile of the output laser beam, the angularly polarized laser beams can be separated by 0.001 μm or more, including, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more, for example, 500 μm or more, for example, 1000 μm or more, and 5000 μm or more. In some embodiments, the system is configured to generate angularly polarized laser beams within the output laser beam that overlap with adjacent angularly polarized laser beams along the horizontal axis of the output laser beam, for example. The overlap between adjacent angularly deflected laser beams (e.g., beam spot overlap) can be 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, and 100 μm or more, including 0.001 μm or more.

[0088] In certain cases, the light beam generator configured to generate two or more beams of frequency-shifted light includes a laser excitation module as described in U.S. Patent Nos. 9,423,353, 9,784,661, and 10,006,852, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0089] In embodiments, the system includes a light detection system having one or more photodiodes. In some embodiments, the photodiode is an avalanche photodiode. In certain embodiments, the light detection system includes a plurality of photodiodes, such as an array of photodiodes. For example, the detector may include four or more photodiodes, including, for example, 10 or more photodiodes, for example, 25 or more photodiodes, for example, 50 or more photodiodes, for example, 100 or more photodiodes, for example, 250 or more photodiodes, for example, 500 or more photodiodes, for example, 750 or more photodiodes, and 1000 or more photodiodes.

[0090] The photodiodes may be arranged in any geometric configuration as desired; configurations of interest include, but are not limited to, square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, non-rectangular, decagonal, dodecagonal, circular, elliptical, and irregularly patterned configurations. The photodiodes within a photodiode array may be oriented relative to one another (as referenced in the XZ plane) at angles ranging from 10° to 180°, including, for example, 15° to 170°, for example, 20° to 160°, for example, 25° to 150°, for example, 30° to 120°, and 45° to 90°. The photodiodes may be of any suitable shape, including, for example, rectilinear shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curvilinear shapes such as circles and ellipses; and irregular shapes such as, for example, a parabolic base coupled to a planar top. In certain embodiments, the photodiode array has a rectangular active surface.

[0091] Each photodiode in the array may have an active surface with a width in the range of 5 μm to 250 μm, for example, 10 μm to 225 μm, for example, 15 μm to 200 μm, for example, 20 μm to 175 μm, for example, 25 μm to 150 μm, for example, 30 μm to 125 μm, and 50 μm to 100 μm, and a length in the range of 5 μm to 250 μm, for example, 10 μm to 225 μm, for example, 15 μm to 200 μm, for example, 20 μm to 175 μm, for example, 25 μm to 150 μm, for example, 30 μm to 125 μm, and 50 μm to 100 μm, and the surface area of ​​each photodiode in the array may be, for example, 50 μm 2 ~9000μm 2 , e.g., 75 μm 2 ~8000μm 2 , e.g., 100 μm 2 ~7000μm 2 , e.g., 150 μm 2 ~6000μm 2 , and 200 μm 2 ~5000μm 2 Including, 25μm 2 ~10,000 μm 2 The range is.

[0092] The size of the photodiode array can vary depending on the amount and intensity of light, the number of photodiodes, and the desired sensitivity, and can have a length ranging from 0.01 mm to 100 mm, including, for example, 0.05 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, for example, 2 mm to 50 mm, for example, 3 mm to 40 mm, for example, 4 mm to 30 mm, and 5 mm to 25 mm. The width of the photodiode array can also vary from 0.01 mm to 100 mm, including, for example, 0.05 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, for example, 2 mm to 50 mm, for example, 3 mm to 40 mm, for example, 4 mm to 30 mm, and 5 mm to 25 mm. Thus, the active surface of the photodetector array can be, for example, 0.5 mm. 2 ~5000mm 2 , e.g., 1 mm 2~1000m 2 , e.g., 5 mm 2 ~500mm 2 , and 10mm 2 ~100m 2 Including 0.1mm 2 ~10,000mm 2 The range may be:

[0093] Photodiodes of interest are configured to measure collected light at one or more wavelengths, including, for example, two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths of light, e.g., fifteen or more, e.g., twenty-five or more, e.g., fifty or more, e.g., one hundred or more, e.g., two hundred or more, e.g., three hundred or more, e.g., four hundred or more, e.g., five hundred or more, e.g., one thousand or more, e.g., one fifth hundred or more, e.g., two fifths or more, and five thousand or more different wavelengths of light. In certain embodiments, the photodiodes are configured to measure the spectrum of light, such as when the spectrum of light includes wavelengths spanning 50 nm or more, including, for example, 100 nm or more, e.g., 200 nm or more, e.g., 300 nm or more, e.g., 400 nm or more, e.g., 500 or more, e.g., 600 nm or more, e.g., 700 nm or more, e.g., 800 nm or more, e.g., 900 nm or more, e.g., 1000 nm or more, and 1500 nm or more. For example, the photodiode may be configured to measure light in the range of 200 nm to 1500 nm, such as 400 nm to 1100 nm.

[0094] The light detection system is configured to measure light continuously or at discrete intervals. In some cases, the photodiode of interest is configured to continuously measure collected light. In other cases, the light detection system is configured to measure at discrete intervals, such as measuring light every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or some other interval.

[0095] As summarized above, the light detection system also includes an amplifier component. In embodiments, the amplifier component is configured to amplify the output signal from the photodiode in response to the detected light. In some embodiments, the amplifier component includes a current-to-voltage converter, such as a transimpedance amplifier. In other embodiments, the amplifier component includes an operational amplifier circuit, such as a summing amplifier. In embodiments, the output current from the photodiode is converted to a voltage, in certain cases combined with a summing amplifier, and propagated to a processor to output a data signal.

[0096] Depending on the number of photodiodes employed in the light detection system, the amplifier component may include two or more amplifiers, including, for example, three or more amplifiers, for example, four or more amplifiers, for example, five or more amplifiers, for example, six or more amplifiers, for example, seven or more amplifiers, for example, eight or more amplifiers, for example, nine or more amplifiers, for example, ten or more amplifiers, for example, fifteen or more amplifiers, for example, twenty-five or more amplifiers, for example, fifty or more amplifiers, for example, one hundred or more amplifiers, for example, two ... In certain embodiments, the amplifier component comprises two or more transimpedance amplifiers, for example including three or more transimpedance amplifiers, for example four or more transimpedance amplifiers, for example five or more transimpedance amplifiers, for example six or more transimpedance amplifiers, for example seven or more transimpedance amplifiers, for example eight or more transimpedance amplifiers, for example nine or more transimpedance amplifiers, such as ten or more transimpedance amplifiers, for example fifteen or more transimpedance amplifiers, for example twenty-five or more transimpedance amplifiers, such as fifty or more transimpedance amplifiers, for example one hundred or more transimpedance amplifiers, for example two hundred and fifty or more transimpedance amplifiers, such as five hundred or more transimpedance amplifiers, for example seven hundred or more transimpedance amplifiers, and one thousand or more transimpedance amplifiers. In other embodiments, the amplifier component comprises two or more summing amplifiers, including, for example, three or more summing amplifiers, for example four or more summing amplifiers, for example five or more summing amplifiers, for example six or more summing amplifiers, such as seven or more summing amplifiers, for example eight or more summing amplifiers, for example nine or more summing amplifiers, such as ten or more summing amplifiers, for example fifteen or more summing amplifiers, for example twenty-five or more summing amplifiers, such as fifty or more summing amplifiers, for example one hundred or more summing amplifiers, for example two hundred and fifty or more summing amplifiers, such as five hundred or more summing amplifiers, for example seven hundred or more summing amplifiers, and one thousand or more summing amplifiers.

[0097] In some embodiments, the photodetection system includes a number of amplifiers equal to the number of photodiodes. For example, the photodetection system may include N photodiodes and N amplifiers, where N is an integer between 2 and 1024, for example, where N is between 4 and 512, including between 8 and 256, and between 16 and 128. In certain cases, N is 4 (i.e., the photodetection system includes four photodiodes and four amplifiers). In other cases, N is 8. In still other cases, N is 16. In still other cases, N is 32. In other embodiments, the photodetection system may include N photodiodes and 2N amplifiers, where N is an integer between 2 and 1024, for example, where N is between 4 and 512, including between 8 and 256, and between 16 and 128. For example, the photodetection system may include a transimpedance amplifier and a summing amplifier for each photodiode.

[0098] An amplifier component (e.g., a transimpedance amplifier) ​​is in electrical communication with the plurality of photodiodes. In some embodiments, the amplifier component is in direct electrical communication with (i.e., immediately downstream from) the plurality of photodiodes. In other embodiments, the amplifier component is in electrical communication with the plurality of photodiodes via an electronic switch component. In particular embodiments, the photodetection system of interest includes a first amplifier component electrically positioned between the photodiodes and the electronic switch component, and a second amplifier component electrically downstream from the electronic switch component. In some cases, the first amplifier component includes a plurality of transimpedance amplifiers configured to receive output signals from the photodiodes, and the second amplifier component includes a plurality of summing amplifiers configured to receive output signals from the electronic switch.

[0099] Each amplifier in the amplifier component can be configured to receive electronic signals from one or more of the photodiodes. For example, if the photodiodes include N photodiodes, each amplifier in the amplifier component can be configured to receive signals from N photodiodes or a portion thereof, such as N / 2 photodiodes, N / 4 photodiodes, N / 8 photodiodes, N / 16 photodiodes, N / 32 photodiodes, or some other portion. In one example, the photodetection system includes 64 photodiodes, and each amplifier is configured to receive signals from all 64 different photodiodes (i.e., configured to receive signals from N photodiodes). In another example, the photodetection system includes 64 photodiodes, and each amplifier is configured to receive signals from 32 different photodiodes (i.e., configured to receive signals from N / 2 photodiodes). In yet another example, the photodetection system includes 64 photodiodes, and each amplifier is configured to receive signals from 16 different photodiodes (i.e., configured to receive signals from N / 4 photodiodes). In these embodiments, the electronic switch component can multiplex or demultiplex the electronic signals from the photodiodes and transmit either multiplexed or non-multiplexed electronic signals from the photodiodes.

[0100] In embodiments, the system is configured to adjust the sensitivity of photodiodes in a light detection system. In some embodiments, the system includes a processor system, also including a processor having a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to determine the responsivity of the photodiodes across multiple wavelengths of light from a light source. Here, the term "responsivity" is used in its conventional sense to refer to the ratio of photocurrent generated by the photodiode as a function of the amount of incident light. In some embodiments, the memory includes instructions to determine the responsivity of each photodiode across 10 or more different wavelengths of light, including, for example, 15 or more, for example, 25 or more, for example, 50 or more, for example, 100 or more, for example, 200 or more, for example, 300 or more, for example, 400 or more, for example, 500 or more, for example, 1000 or more, for example, 1500 or more, for example, 2500 or more, and 5000 or more different wavelengths of light. In certain embodiments, the memory includes instructions for determining the responsivity across a spectrum of light, such as including wavelengths spanning 50 nm or more, including 100 nm or more, e.g., 200 nm or more, e.g., 300 nm or more, e.g., 400 nm or more, e.g., 500 nm or more, e.g., 600 nm or more, e.g., 700 nm or more, e.g., 800 nm or more, e.g., 900 nm or more, e.g., 1000 nm or more, and 1500 nm or more. For example, the memory includes instructions for determining the responsivity across a spectrum of light ranging from 200 nm to 1500 nm, such as 400 nm to 1100 nm. In certain cases, the system includes a processor system and a processor having memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to plot the responsivity of the photodiodes across a range of wavelengths of light to generate a wavelength-response curve for each photodiode in the light detection system. In certain embodiments, the memory includes instructions for determining the responsivity of a photodiode to a predetermined wavelength using a wavelength responsivity curve.

[0101] In some embodiments, the system includes a processor system and also includes a processor having a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to use the responsivity of the photodiode across the plurality of wavelengths to determine an average gain of the photodiode across the plurality of wavelengths. In some cases, the memory includes instructions for independently determining the average gain of the photodiode at each wavelength. For example, if the responsivity of the photodiode is determined across a spectrum of wavelengths (e.g., 200 nm to 1500 nm, such as 400 nm to 1100 nm), the memory may include instructions for determining the average gain of the photodiode every 1 nm across the spectrum of wavelengths, including, for example, every 2 nm, every 5 nm, e.g., every 10 nm, and every 25 nm across the spectrum of wavelengths or some other interval. In other cases, the memory includes instructions to determine the average gain of the photodiode over a range of wavelengths, including, for example, over a range of 1 nm or more, for example, over a range of 2 nm or more, for example, over a range of 5 nm or more, for example, over a range of 10 nm or more, for example, over a range of 25 nm or more, for example, over a range of 50 nm or more, for example, over a range of 100 nm or more, for example, over a range of 250 nm or more, for example, over a range of 500 nm or more, and over 1000 nm or more.

[0102] In some embodiments, the system includes a processor system and also includes a processor having a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to calculate a resistance of the amplifier based on a determined gain (e.g., an average gain of the amplifier over multiple wavelengths of light) and a determined responsivity of the photodiode. In some cases, the memory includes instructions to calculate the resistance of a feedback resistor used in the amplifier. In particular embodiments, the memory includes instructions to calculate R f ×R(λ)=G t wherein R fis the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, and G t is the average gain of the photodiode over multiple wavelengths of light.

[0103] In some embodiments, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacitance of each amplifier based on the calculated resistance. In particular embodiments, the memory includes instructions to adjust the capacitance by 5% or more, including, for example, 10% or more, for example, 20% or more, for example, 30% or more, for example, 40% or more, for example, 50% or more, for example, 60% or more, for example, 70% or more, for example, 80% or more, for example, 90% or more, and 95% or more.

[0104] In some cases, the memory includes instructions to adjust the capacitance of the amplifier to compensate for the wavelength-dependent responsivity of the photodiode. In some cases, the capacitance of the amplifier is increased to compensate for the wavelength-dependent responsivity of the photodiode. In other cases, the capacitance of the amplifier is decreased to compensate for the wavelength-dependent responsivity of the photodiode. In particular cases, the memory includes instructions to adjust the capacitance of the amplifier to produce a wavelength responsivity of the photodiode that is substantially the same across multiple wavelengths (e.g., across a spectrum of wavelengths). In other cases, the memory includes instructions to adjust the capacitance of the amplifier to produce a desired responsivity of the photodiode at one or more predetermined detection wavelengths.

[0105] In some cases, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacitance in a manner sufficient to produce a predetermined bandwidth per photodiode. In particular cases, the memory includes instructions to adjust the capacitance of each amplifier to the predetermined bandwidth according to the following formula: f is the resistance of the amplifier, and C fis the capacitance of the amplifier.

[0106]

number

[0107] In some embodiments, the system includes a processor having a memory operatively coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacity of the amplifier to achieve a predetermined bandwidth of 10 kHz or greater, e.g., 25 kHz or greater, e.g., 50 kHz or greater, e.g., 100 kHz or greater, e.g., 150 kHz or greater, e.g., 200 kHz or greater, e.g., 250 kHz or greater, e.g., 500 kHz or greater, and 1000 kHz or greater. For example, when the average gain of the amplifier is 10 6 In one embodiment, the memory is volts / amps, and includes instructions to adjust the capacitance of the amplifier to achieve a predetermined bandwidth, which may be 250 kHz or greater, including predetermined bandwidths of 275 kHz or greater, such as 300 kHz or greater, such as 325 kHz or greater, such as 350 kHz or greater, such as 375 kHz or greater, such as 400 kHz or greater, such as 425 kHz or greater, such as 450 kHz or greater, such as 475 kHz or greater, and 500 kHz or greater.

[0108] In some embodiments, the system includes a processor having a memory operably coupled to the processor, the memory including instructions stored in the memory that, when executed by the processor, cause the processor to determine one or more parameters of the photodetectors, the memory including instructions for illuminating particles (e.g., multispectral beads as described below) comprising one or more fluorophores in the flow stream. In some cases, the memory includes instructions that, when executed by the processor, cause the processor to determine parameters of the photodetectors, including relative fluorescence units (e.g., ABD units) assigned to 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.

[0109] In some cases, the memory includes instructions for determining a parameter of a photodetector; illuminating a flowstream with 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 flowstream with a photodetector having a light source; generating a data signal from the photodetector at the first illumination intensity; 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.

[0110] 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 cases, 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 cases, the memory includes instructions for determining an rCV% (robust coefficient of variation) of the photodetector. In particular embodiments, the memory includes instructions that, when executed by the processor, cause the processor to calculate a linear fit of the variance according to the formula:led is given by 1 / c1, the statistical photoelectrons per unit mean fluorescence intensity (M) (i.e., SPE / MFI).

[0111]

number

[0112] In certain embodiments, the memory includes instructions for plotting the variance to determine a linear fit of the variance according to y=c1x+c0.

[0113] In embodiments, the mean fluorescence intensity and variance may be determined for a plurality of different illumination intensities, such as two or more illumination intensities of light, including, for example, three or more, such as four or more, for example, five or more, such as six or more, for example, seven or more, such as eight or more, for example, nine or more, such as ten or more, and fifteen or more different wavelengths of light.

[0114] In some embodiments, the memory includes instructions for determining statistical photoelectrons (SPEs) at one or more of the illumination intensities, such as at least a first illumination intensity and a second illumination intensity. In certain cases, the memory includes instructions for calculating a detector efficiency (Qdet) of the photodetector for each particle based on the statistical photoelectrons and the determined mean fluorescence intensity of the particles. In certain embodiments, the memory includes instructions for determining detector efficiencies for one or more detector channels of the photodetector, including determining detector efficiencies for, 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, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty four or more, for example, thirty six or more, for example, for forty eight or more, for example, seventy two or more, and 96 or more detector channels of the photodetector based on the determined mean fluorescence intensities of the statistical photoelectrons and the particles. In certain cases, the memory includes instructions for determining a detector efficiency for all detector channels of the photodetector per particle based on the statistical photoelectrons and the determined mean fluorescence intensity of each particle. In certain embodiments, the memory includes instructions that, when executed by the processor, cause the processor to determine the determined detector efficiency according to the following formula: where SPE is the statistical photoelectrons, MFI is the mean fluorescence intensity, and ABD is the assigned units per channel per particle lot.

[0115]

number

[0116] In certain embodiments, the memory includes instructions for determining a background signal for one or more of the photodetectors. In some cases, the background signal is determined at one or more illumination intensities, including, 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, and ten or more different illumination intensities. In some cases, the memory includes instructions for determining the background signal at all of the applied illumination intensities. In some embodiments, the memory includes instructions for determining detector efficiency in one or more detector channels of the photodetector, 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, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty-four or more, for example, thirty-six or more, for example, forty-eight or more, for example, seventy-two or more, and including cases where the memory includes instructions for determining background signals in 96 or more detector channels of the photodetector. In certain cases, the memory includes instructions for determining background signals in all of the detector channels of the photodetector. In some cases, 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 certain cases, the memory includes instructions for determining background signals in all of the detector channels of the photodetector. SD =B SD,MFI ×Q led and instructions for determining the background signal according to the formula:

[0117]

number

[0118] In some embodiments, the memory includes instructions for determining electronic noise for one or more of the photodetectors. In some cases, the electronic noise of the photodetectors is determined at one or more illumination intensities, including, 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, and ten or more different illumination intensities. In some cases, the electronic noise of the photodetectors is determined at all of the applied illumination intensities. Electronic noise can similarly be determined within one or more detector channels of a photodetector, including determining electronic noise within, 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, for example, twelve or more, for example, sixteen or more, for example, twenty or more, for example, twenty-four or more, for example, thirty-six or more, for example, forty-eight or more, for example, seventy-two or more, and ninety-six or more detector channels in a photodetector. In certain cases, the memory includes instructions for determining electronic noise in all of the detector channels of the photodetector. In some cases, the memory includes instructions for determining electronic noise based on statistical photoelectron and detector efficiency of the photodetector. In certain cases, the memory includes instructions for determining electronic noise based on EN SD =EN SD,MFI ×Q led and instructions for determining electronic noise according to the formula:

[0119]

number

[0120] In some embodiments, the memory includes instructions for determining the detection limit of one or more of the photodetectors. In some cases, the memory includes instructions for determining the detection limit of the photodetectors in one or more detector channels of the photodetector, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., twelve or more, e.g., sixteen or more, e.g., twenty or more, e.g., twenty-four or more, e.g., thirty-six or more, e.g., forty-eight or more, e.g., seventy-two or more, and including cases where the memory includes instructions for determining the detection limit of the photodetectors in 96 or more detector channels of the photodetector. In particular cases, the memory includes instructions for determining the detection limit in all of the detector channels of the photodetector. In some cases, the memory includes instructions for determining the detection limit in all of the detector channels of the photodetector, e.g., 2 + 2SD = 4(1 + B SD ) to determine the detection limit of each photodetector.

[0121] In some embodiments, the memory includes instructions for determining the optical sensitivity of one or more photodetectors. In particular embodiments, the memory includes instructions for setting an initial detector gain for the photodetector. In some cases, the memory includes instructions for 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, at each detector gain, a lowest light illumination intensity that produces a data signal resolvable from a background data signal. In some cases, the memory includes instructions for determining, at each detector gain, a lowest light illumination intensity that produces a data signal that is two standard deviations from the background data signal. In particular cases, the memory includes instructions for setting the detector gain to the gain of the lowest light illumination intensity that produces a data signal resolvable from the background data signal when plotted as a function of light intensity.

[0122] In certain embodiments, the light detection system having one or more photodiodes and amplifier components described above is part of or positioned in a particle analyzer, such as a particle sorter. In certain embodiments, the subject system is a flow cytometry system that includes a photodiode and amplifier components as part of the light detection system described above for detecting light emitted by a sample in a flow stream. Flow cytometry systems are described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.) Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt1):17-28; Linden, et al. Semin 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, including but not limited to:In particular instances, flow cytometry systems of interest include the BD Biosciences FACSCanto™ II flow cytometer, the BD Accuri™ flow cytometer, the BD Biosciences FACSCelesta™ flow cytometer, the BD Biosciences FACSLyric™ flow cytometer, the BD Biosciences FACSVerse™ flow cytometer, the BD Biosciences FACSymphony™ flow cytometer, the BD Biosciences LSRFortessa™ flow cytometer, the BD Biosciences LSRFortess™ X-20 flow cytometer, and the BD Biosciences FACSCalibur™ flow cytometer, the BD Biosciences FACSCount™ cell sorter, the BD Biosciences FACSLyric™ cell sorter, and the BD Biosciences Via™ cell sorter, the BD Biosciences Influx™ cell sorter, the BD Biosciences Jazz™ cell sorter, and the BD Biosciences Aria™ cell sorter, and BD Biosciences FACSMelody™ cell sorter.

[0123] In some embodiments, the subject particle analyzer system may be configured with any of the following particle analyzers: Nos. 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, and 5,602,039, the disclosures of which are incorporated herein by reference in their entireties.

[0124] In certain embodiments, the subject system is a flow cytometry system having an excitation module that uses radio frequency multiplexing excitation to generate multiple frequency-shifted beams of light. In particular cases, the subject system is a flow cytometry system such as those described in U.S. Patent Nos. 9,423,353, 9,784,661, and U.S. Patent Publication Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0125] 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 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 multiple sorting determination units, such as that described in U.S. Patent Publication No. 62 / 803,264, filed February 8, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a method of sorting components of a sample includes sorting particles (e.g., cells in a biological sample) using a particle sorting module having a deflector plate, such as that described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0126] In some embodiments, the system of interest includes a particle analysis system that can be used to analyze and characterize particles, whether or not the particles are physically sorted into a collection vessel. FIG. 4A shows a functional block diagram of an example 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, in whole or in part, methods described herein, for example. 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 particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0127] 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. A detection station 408 generally refers to a monitoring area 407 of the common sample path. Detection, in some implementations, can include detecting light or one or more other properties of the 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 can include multiple detection stations. Additionally, some detection stations can monitor more than one area.

[0128] 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 multidimensional data points that include values ​​for each property measured for the particle. The detection system 404 is configured to collect a series of such data points at a first time interval.

[0129] 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 a 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 additionally compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0130] 4B shows a system 400 for flow cytometry according to an exemplary embodiment of the invention. System 400 includes a flow cytometer 410, a controller / processor 490, and 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 filters ("LP") 455a-455b, and one or more fluorescence detectors 460a-460f.

[0131] Pump lasers 115a-c emit light in the form of laser beams. The wavelengths of the laser beams emitted from pump lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively, in the example system of FIG. 4B. The laser beams are first directed through one or more of beam splitters 445a and 445b. Beam splitter 445a transmits light at 488 nm and reflects light at 633 nm. Beam splitter 445b transmits UV light (light with wavelengths in the range of 10-400 nm) and reflects light at 488 nm and 633 nm.

[0132] 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 fluid dynamic system that directs particles in the stream (typically one at a time) into the focused laser beam for inspection. The flow chamber can comprise a flow cell in a benchtop cytometer or a nozzle tip in a stream-in-air cytometer.

[0133] Light from the laser beam interacts with particles in the sample by diffraction, refraction, reflection, scattering, and absorption, with re-emission at a variety of different wavelengths depending on particle properties such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. Fluorescence emission, as well as diffracted, refracted, reflected, and scattered light, can 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 forward scatter detector 430, side scatter detector 435, and one or more fluorescence detectors 460a-f.

[0134] The 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 each side of the center of the spectral band, or from 500 nm to 520 nm. Shortpass filters transmit wavelengths of light below a specified wavelength. Longpass filters, such as longpass filters 455a-455b, transmit wavelengths of light above a specified wavelength. For example, longpass filter 455a, a 670 nm longpass filter, transmits light above 670 nm. Filters are often selected to optimize the detector's specificity 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.

[0135] Beam splitters direct light of different wavelengths in different directions. Beam splitters can be characterized by filter properties such as short-pass and long-pass. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 445g transmits light with wavelengths of 620 nm or less and reflects light with wavelengths of 620 nm or greater in different directions. In one embodiment, beam splitters 445a-445g can comprise optical mirrors, such as dichroic mirrors.

[0136] 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, or excitation light traveling mostly forward through or around the particle. The intensity of light detected by the forward scatter detector depends on the particle's overall size. The forward scatter detector may include a photodiode. The side scatter detector 435 is configured to detect refracted and reflected light from the particle's surface and internal structure, which tends to increase as particle complexity 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, side scatter detector 435, and fluorescence detector can be converted to electronic signals (voltage) by the detectors. This data can provide information about the sample.

[0137] Those skilled in the art will recognize that a flow cytometer according to one embodiment of the present invention is not limited to the flow cytometer shown in Figure 4B, but can 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.

[0138] During operation, cytometer operation is controlled by controller / processor 490, and measurement data from the detectors may be stored in memory 495 and processed by controller / processor 490. While not explicitly shown, controller / processor 490 may be coupled to the detectors to receive output signals therefrom, and may also be coupled to the electrical and electromechanical components of flow cytometer 400 to control lasers, fluid flow parameters, etc. Input / output (I / O) 497 may also be provided within the system. Memory 495, controller / processor 490, and I / O 497 may be provided entirely as an integral part of flow cytometer 410. In such embodiments, a display may also form part of I / O 497 for presenting experimental data to a user of cytometer 400. Alternatively, some or all of memory 495 and controller / processor 490 and I / O 497 may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of memory 495 and controller / processor 490 may be in wireless or wired communication with cytometer 410. The controller / processor 490 in conjunction with memory 495 and I / O 497 can be configured to perform a variety of functions related to the preparation and analysis of flow cytometer experiments.

[0139] The system shown in FIG. 4B includes six different detectors that detect fluorescent light 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 in a flow cytometer experiment emit in their own characteristic wavelength bands. The particular fluorescent labels used in the experiment and their associated fluorescent emission bands may be selected to roughly match the filter windows of the detectors. However, as more detectors are provided and more labels are utilized, perfect correspondence between filter windows and fluorescent emission spectra is not possible. While the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of one particular detector, it is generally true that some of the emission spectrum of that label also overlaps with the filter windows of one or more other detectors. This may be referred to as spillover. The I / O 497 can be configured to receive data for a flow cytometer experiment with a panel of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of the multiple markers. I / O 497 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission 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, can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more assignments of labels to markers.

[0140] 5 shows a functional block diagram of one embodiment 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.

[0141] The particle analyzer or sorting system 502 is configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide the biological event data to the analysis controller 500. A data communication channel can be included between the particle analyzer or sorting system 502 and the analysis controller 500. The biological event data can be provided to the analysis controller 500 via the data communication channel.

[0142] The analysis controller or sorting system 500 can be configured to receive biological event data from the particle analyzer 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 a first plot of the biological event data on a display device 506. The analysis controller 500 can be further configured to render a region of interest, for example, as a gate around a population of the biological event data shown by the display device 506, overlaid on the first plot. 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.

[0143] Analysis controller 500 can be further configured to display the biological event data within the gate differently from other events in the biological event data outside the gate on display device 506. For example, analysis controller 500 can be configured to render the color of the biological event data contained within the gate distinct from the color of the biological event data outside the gate. Display device 506 can be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0144] 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 manipulated via the display device 506 (e.g., by clicking on or within the desired gate when 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 touchscreen, 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.

[0145] The trigger event can cause the analysis controller 500 to change the manner in which the data is displayed, what portions of the data are actually displayed on the display device 506, and / or provide input for further processing, such as selection of a population of interest for particle sorting.

[0146] 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 visualization of the plot 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.

[0147] 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.

[0148] 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 presented information according to input received from the analysis controller 500 in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0149] In some implementations, the analysis controller 500 can generate a user interface for receiving example events for filtering. For example, the user interface can include controls for receiving example events or example images. The example events or example images, or example gates, can be provided prior to collection of event data for the sample or based on an initial event set for a portion of the sample.

[0150] In some embodiments, the system of interest 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 into a moving fluid column 608 (e.g., a stream) containing particles 609. Within the moving fluid column 608, the particles 609 (e.g., cells) are aligned single file across a monitoring area 611 (e.g., a laser stream intersection) and illuminated by an illumination source 612 (e.g., a laser). Vibration of droplet-forming transducer 602 causes moving fluid column 608 to break up into multiple droplets 610 , some of which contain particles 609 .

[0151] During operation, the detection station 614 (e.g., an event detector) identifies when a particle of interest (or cell of interest) crosses the monitoring area 611. The detection station 614 feeds a timing circuit 628, which in turn feeds a flash charge circuit 630. At the droplet break-off point, signaled by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 608, thus causing the droplet of interest to carry a charge. The droplet of interest can contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown), which can deflect the droplets into a container such as a collection tube or a multi-well or microwell sample plate in which wells or microwells can be associated with droplets of particular interest. However, as shown in FIG. 6A, the droplets can be collected in a drain container 638.

[0152] Detection system 616 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal as a particle of interest passes through monitoring area 611. An exemplary droplet boundary detector is described in U.S. Pat. No. 7,679,039, which is incorporated herein by reference in its entirety. Detection system 616 enables the instrument to accurately calculate the position of each detected particle within the droplet. Detection system 616 provides amplitude signal 620 and / or phase signal 618, which can then be provided (via amplifier 622) to amplitude control circuit 626 and / or frequency control circuit 624. Amplitude control circuit 626 and / or frequency control circuit 624 then control droplet forming transducer 602. Amplitude control circuit 626 and / or frequency control circuit 624 can be included within a control system.

[0153] In some implementations, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled to a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can be included in the event data for the particle. 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 either the detection system 616 or the detection station 614 and provided to a non-collection element.

[0154] 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 of charging wires within the barbs, creating a stream of droplets 610 containing particles 610 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 and direct them 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 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 sort 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.

[0155] Sorting electronics can be included to initiate the collection of measurements, receive fluorescent signals about the particles, and determine how to adjust the deflection plates to cause particle sorting. Exemplary implementations of the embodiment shown in Figure 6B include the BD FACSAria® line of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0156] Computer Control System Aspects of the present disclosure further include a computer control system, the system further including one or more computers for full or partial automation. In some embodiments, the system includes a computer having a computer-readable storage medium having a computer program stored on the system, the computer program, when loaded into the computer, including instructions for detecting light with a photodiode in electrical communication with the amplifier, determining a responsivity of the photodiode across multiple wavelengths of light, and adjusting one or more parameters of the amplifier in response to the responsivity of the photodiode across the multiple wavelengths of light. In some embodiments, the computer program includes instructions for determining the responsivity of the photodiode across a spectrum of wavelengths of light. In some embodiments, the computer program includes instructions for determining an average gain of the photodiode across the multiple wavelengths. In some cases, the computer program includes instructions for calculating a resistance of the amplifier based on the determined responsivity and average gain of the photodiode across the multiple wavelengths of light. In certain cases, the computer program includes instructions for calculating R f ×R(λ)=G t wherein R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, and G t is the average gain of the photodiode over multiple wavelengths of light.

[0157] In some embodiments, the computer program includes instructions to adjust the capacitance of each amplifier based on the calculated resistance. In some cases, the computer program includes instructions to adjust the capacitance of the amplifier to compensate for the wavelength-dependent responsivity of the photodiode. In some cases, the capacitance of the amplifier is increased to compensate for the wavelength-dependent responsivity of the photodiode. In other cases, the capacitance of the amplifier is decreased to compensate for the wavelength-dependent responsivity of the photodiode. In particular cases, the computer program includes instructions to adjust the capacitance of the amplifier to produce a wavelength responsivity of the photodiode that is substantially the same across multiple wavelengths (e.g., across a spectrum of wavelengths). In other cases, the computer program includes instructions to adjust the capacitance of the amplifier to produce a desired responsivity of the photodiode at one or more predetermined detection wavelengths.

[0158] In some cases, the computer program includes instructions to adjust the capacitance in a manner sufficient to produce a predetermined bandwidth for each photodiode. In particular cases, the computer program includes instructions to adjust the capacitance of each amplifier to a predetermined bandwidth according to the following formula: where BW is the bandwidth and R f is the resistance of the amplifier, and C f is the capacitance of the amplifier.

[0159]

number

[0160] In an embodiment, the system includes an input module, a processing module, and an output module. The subject systems may include both hardware and software components, and the hardware components may take the form of one or more platforms, e.g., servers, such that functional elements, i.e., those elements of the system that perform specific tasks of the system (such as managing the input and output of information, processing information, etc.), may be performed by the execution of software applications on and across one or more computer platforms represented in the system.

[0161] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses 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 that become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor's coordination and execution of functions of various computer programs, which may be written in a variety of programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. 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, e.g., negative feedback control.

[0162] 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, a flash memory device, or other memory storage device. The memory storage device can be any of a variety of known or future devices, including a compact disk drive, a tape drive, a removable hard disk drive, or a 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, a removable hard disk, or a floppy disk, respectively. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also called computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0163] In some embodiments, a computer program product is described comprising a computer-usable medium having control logic (a computer software program including program code) stored therein. The control logic, when executed by a processor, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementation of hardware state machines to perform the functions described herein will be apparent to one skilled in the relevant art.

[0164] 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 to the processor remotely via a communications channel, or may be pre-stored in a computer program product, such as memory or some other portable or fixed computer-readable storage medium, using any of these devices together with the memory. For example, a magnetic or optical disk may carry the programming and be readable by a disk writer / reader. The system of the present invention also includes programming in the form of a computer program product, e.g., algorithms for use in implementing the above-described methods. The programming according to the present invention may be recorded on a computer-readable medium, e.g., any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as CD-ROMs; electrical storage media such as RAM and ROM; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0165] 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 mobile phone (i.e., a smartphone).

[0166] In some embodiments, a system according to the present disclosure may be configured to include a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communications interface may be configured for wired or wireless communications, including, but not limited to, radio frequency (RF) communications (e.g., radio frequency identification (RFID), ZigBee communications protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocol, and cellular communications such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0167] In one embodiment, the communications interface is configured to include one or more communications ports, e.g., physical ports or interfaces 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.

[0168] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, allowing the subject system to communicate with other devices, such as a computer terminal and / or network, a communication-enabled mobile phone, a personal digital assistant, or any other communication device that a user may use in conjunction with the system.

[0169] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing 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.

[0170] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communications interface using a common standard, such as, for example, 802.11 or Bluetooth® RF protocols, or the IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touchscreen.

[0171] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored within the subject system, e.g., in the optional data storage unit, with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0172] 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 pixels. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user and for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network, in accordance with known techniques. Presentation of data by the output manager may be performed in accordance with various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may 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 also 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, various operating systems may be employed on any of the computer platforms, depending on the type and / or configuration of the computer platform selected. 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.

[0173] FIG. 7 illustrates the general architecture of an exemplary computing device 800 according to certain embodiments. The general architecture of computing device 700 illustrated in FIG. 7 includes an arrangement of computer hardware and software components. Computing device 700 may include more (or fewer) elements than those illustrated in FIG. 7 . However, it is not necessary to show all of these typically conventional elements to provide a useful disclosure. As illustrated, computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750, and input devices 760, all of which may communicate with each other via a communications bus. Network interface 820 may provide connectivity to one or more networks or computing systems. Thus, processing unit 710 may receive information and instructions from other computing systems or services via a network. Processing unit 710 also communicates with memory 770 and may further provide output information to optional display 750 via input / output device interface 740. The input / output device interface 740 may also accept input from optional input devices 760 such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0174] Memory 770 may include computer program instructions (which in some embodiments may be grouped as modules or components) that processing unit 710 executes to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by processing unit 710 in the general management and operation of computing device 700. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0175] Non-transitory computer-readable storage medium for adjusting sensitivity of photodiodes in a light detection system Aspects of the present disclosure further include non-transitory computer-readable storage media having instructions for implementing the subject methods. The computer-readable storage medium may be employed by one or more computers for fully or partially automating systems for implementing 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 floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tape, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid-state disks, and network-attached storage (NAS). Files containing information may be "stored" on a computer-readable medium, where "storing" refers to recording information such that it can be accessed and retrieved at a later date by a computer. The computer-implemented methods described herein may be implemented using programming that can be written in one or more of any number of computer programming languages. Such languages ​​include, for example, Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as many others.

[0176] In some embodiments, a computer-readable storage medium of interest includes a computer program stored on the computer-readable storage medium, the computer program, when loaded into a computer, including an algorithm for detecting light with a photodiode in electrical communication with the amplifier, an algorithm for determining the responsivity of the photodiode across multiple wavelengths of light, and an algorithm for adjusting one or more parameters of the amplifier in response to the responsivity of the photodiode across the multiple wavelengths of light.

[0177] In some embodiments, a computer-readable storage medium of interest includes a computer program stored on the computer-readable storage medium, the computer program including instructions having an algorithm for, when loaded into a computer, determining the responsivity of a photodiode across a spectrum of wavelengths of light. In some embodiments, the computer program includes an algorithm for determining the average gain of the photodiode across multiple wavelengths. In some cases, the computer program includes an algorithm for calculating the resistance of an amplifier based on the determined responsivity and average gain of the photodiode across multiple wavelengths of light. In particular cases, the computer program includes an algorithm for calculating the resistance of an amplifier based on the determined responsivity and average gain of the photodiode across multiple wavelengths of light. f ×R(λ)=G t where R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, and G t is the average gain of the photodiode over multiple wavelengths of light.

[0178] In some embodiments, the computer program includes an algorithm for adjusting the capacitance of each amplifier based on the calculated resistance. In some cases, the computer program includes an algorithm for adjusting the capacitance of the amplifier to compensate for the wavelength-dependent responsivity of the photodiode. In some cases, the computer program includes an algorithm for increasing the capacitance of the amplifier to compensate for the wavelength-dependent responsivity of the photodiode. In other cases, the computer program includes an algorithm for decreasing the capacitance to compensate for the wavelength-dependent responsivity of the photodiode. In particular cases, the computer program includes an algorithm for adjusting the capacitance of the amplifier to produce a wavelength responsivity of the photodiode that is substantially the same across multiple wavelengths (e.g., across a spectrum of wavelengths). In other cases, the computer program includes an algorithm for adjusting the capacitance of the amplifier to produce a desired responsivity of the photodiode at one or more predetermined detection wavelengths.

[0179] In some cases, the computer program includes an algorithm for adjusting the capacitance in a manner sufficient to produce a predetermined bandwidth for each photodiode. In particular cases, the computer program includes an algorithm for adjusting the capacitance of each amplifier to a predetermined bandwidth according to the following equation: where BW is the bandwidth and R f is the resistance of the amplifier, and C f is the capacitance of the amplifier.

[0180]

number

[0181] The non-transitory 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, etc. The processing module includes a processor that accesses a memory having stored instructions to perform the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, and an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are available or become available. The processor executes an operating system, which interfaces with firmware and hardware in a well-known manner to facilitate the processor's coordination and execution of functions of various computer programs, which may be written in various programming languages ​​such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as 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.

[0182] Multispectral fluorescent particles As summarized above, aspects of the present disclosure also include particles (e.g., beads) having one or more fluorophores for performing 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., all LEDs or lasers in the system) and across the detection wavelengths of the photodetector.

[0183] In embodiments, the particles of the present subject matter 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, such as two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more, or eleven or more, or twelve or more, or thirteen or more, or fourteen or more, or fifteen or more, sixteen or more, or seventeen or more, or eighteen or more, or nineteen or more, or twenty or more, or twenty-five or more, or thirty or more, or thirty-five or more, or forty or more, or forty-five or more, or fifty or more different types of fluorophores. For example, each particle may contain two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or nineteen, or twenty different types of fluorophores.

[0184] In embodiments, each fluorophore is stably associated with the particle. Stably associated means that the fluorophore does not easily dissociate from the particle due to contact with a liquid medium, such as an 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 fluorophores are covalently bound to the particle and one or more fluorophores are physically associated with the particle.

[0185] In some embodiments, each particle contains two or more different types of fluorophores. Any two fluorophores are considered different and distinct if they differ from each other by one or more of the following: molecular formula, excitation maximum, and emission maximum. Thus, different or distinct fluorophores may differ from each other in terms of chemical composition or one or more properties of the fluorophores. For example, different fluorophores may differ from each other by at least one of excitation maximum and emission maximum. In some cases, different fluorophores differ from each other by their excitation maximum. In some cases, different fluorophores differ from each other by their emission maximum. In some cases, different fluorophores differ from each other by both their excitation maximum and emission maximum. Thus, in embodiments containing first and second fluorophores, the first and second fluorophores may differ from each other by at least one of excitation maximum and emission maximum. For example, the first and second fluorophores may differ from each other by their excitation maximum, emission maximum, or both excitation and emission maximum. A given set of fluorophores may be considered distinct if they differ from one another with respect to excitation or emission maxima, the magnitude of such difference being, in some cases, 5 nm or more, including, for example, 10 nm or more, and 15 nm or more, and in some cases the magnitude of the difference being in the range of, for example, 10-200 nm, for example, 5-400 nm, including, for example, 15-100 nm, such as, for example, 25-50 nm.

[0186] For example, fluorophores of interest according to certain embodiments may have excitation maxima ranging from 100 nm to 800 nm, including, for example, 150 nm to 750 nm, e.g., 200 nm to 700 nm, e.g., 250 nm to 650 nm, e.g., 300 nm to 600 nm, and 400 nm to 500 nm. Fluorophores of interest according to certain embodiments may have emission maxima ranging from 400 nm to 1000 nm, including, for example, 450 nm to 950 nm, e.g., 500 nm to 900 nm, e.g., 550 nm to 850 nm, and 600 nm to 800 nm. In certain cases, the fluorophore is a luminescent dye, such as a fluorescent dye having a peak emission wavelength of 200 nm or greater, including, for example, 250 nm or greater, for example, 300 nm or greater, for example, 350 nm or greater, for example, 400 nm or greater, for example, 450 nm or greater, for example, 500 nm or greater, for example, 550 nm or greater, for example, 600 nm or greater, for example, 650 nm or greater, for example, 700 nm or greater, for example, 750 nm or greater, for example, 800 nm or greater, for example, 850 nm or greater, for example, 900 nm or greater, for example, 1000 nm or greater, and 1050 nm or greater. For example, the fluorophore can be a fluorescent dye having a peak emission wavelength ranging from 200 nm to 1200 nm, including, for example, fluorescent dyes having peak emission wavelengths of 300 nm to 1100 nm, for example, 400 nm to 1000 nm, for example, 500 nm to 900 nm, and 600 nm to 800 nm. In certain embodiments, the subject multispectral particles provide stable excitation with lasers emitting at wavelengths near about 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 instances, the subject multispectral particles provide stable excitation with light sources across the entire spectral detection band, such as from 350 nm to 850 nm.

[0187] 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, quinoneimine 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, bodipys, squaraineoxythenes, naphthalenes, coumarins, oxadiazoles, anthracenes, pyrenes, acridines, arylmethines, or tetrapyrroles, and combinations thereof. In certain embodiments, the conjugate may include two or more dyes, such as 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, quinoneimine dyes, azine dyes, eurodine 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, bodipys, squaraineoxythenes, naphthalenes, coumarins, oxadiazoles, anthracenes, pyrenes, acridines, arylmethines, or tetrapyrroles, and combinations thereof.

[0188] In certain embodiments, fluorophores of interest 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-Cy7) 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 cases, 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]formamide), 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 mFluor780NS).

[0189] The particles may be of any convenient shape for illumination by a light source, as described above. In some cases, the particles are solid supports formed or configured as disks, spheres, ovals, cubes, blocks, cones, etc., and irregular shapes. The mass of the particles may vary in some cases, ranging from 0.01 mg to 20 mg, including, for example, 0.05 mg to 19.5 mg, for example, 0.1 mg to 19 mg, for example, 0.5 mg to 18.5 mg, for example, 1 mg to 18 mg, for example, 1.5 mg to 17.5 mg, for example, 2 mg to 15 mg, and 3 mg to 10 mg. The particles may be, for example, 0.05 mm, as determined, for example, using a vertex system or equivalent. 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 mm2 More than, for example, 2.5 mm 2 More than, for example, 3 mm 2 More than, for example, 3.5 mm 2 More than, for example, 4 mm 2 More than, for example, 4.5 mm 2 or more, and 5 mm 2 Including above 0.01mm 2 The surface area may be greater than or equal to 1000 nm.

[0190] The size of the particles can vary as desired, and in some cases the particles have a longest dimension in the range of 0.01 mm to 10 mm, including, for example, 0.05 mm to 9.5 mm, e.g., 0.1 mm to 9 mm, e.g., 0.5 mm to 8.5 mm, e.g., 1 mm to 8 mm, e.g., 1.5 mm to 7.5 mm, e.g., 2 mm to 7 mm, e.g., 2.5 mm to 6.5 mm, and 3 mm to 6 mm. In certain cases, the particles have a shortest dimension in the range of 0.01 mm to 5 mm, including, for example, 0.05 mm to 4.5 mm, e.g., 0.1 mm to 4 mm, e.g., 0.5 mm to 3.5 mm, and 1 mm to 3 mm.

[0191] In certain cases, the particles of interest are porous, e.g., when the particles have a porosity in the range of 5 μm to 100 μm, including, e.g., 10 μm to 90 μm, e.g., 15 μm to 85 μm, e.g., 20 μm to 80 μm, e.g., 25 μm to 75 μm, and 30 μm to 70 μm, e.g., 50 μm, as determined using, e.g., a capillary flow porometer or equivalent.

[0192] The particles can be formed from any convenient material. In some embodiments, the particles of interest are particles with low or no autofluorescence, such as beads. Suitable materials include, but are not limited to, glass materials (e.g., silicates), ceramic materials (e.g., calcium phosphates), metal materials, and polymeric materials, such as polyethylene, polypropylene, polytetrafluoroethylene, and polyvinylidine fluoride. In some cases, the particles are formed from a solid support, such as a porous matrix described in U.S. Published Application 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, e.g., 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 frit may have a chemical composition that varies depending on the type of sintered particles used to prepare the frit, and frits that may be employed include, but are not limited to, frits made from aluminosilicate, boron trioxide, borophosphosilicate glass, borosilicate glass, ceramic glaze, cobalt glass, cranberry glass, fluorine phosphate glass, fluorine silicate glass, fused quartz, germanium dioxide, metal and sulfide embedded borosilicate, lead glass, phosphate glass, phosphorus pentoxide glass, phosphosilicate glass, potassium silicate, sodium lime glass, sodium hexametaphosphate glass, sodium silicate, tellurite glass, uranium glass, bright carbon, and combinations thereof. In some embodiments, the porous matrix is ​​a glass frit, such as a borosilicate, aluminosilicate, fluorine silicate, potassium silicate, or borophosphosilicate glass frit.

[0193] In some embodiments, the particles are formed from porous organic polymers. The porous organic polymers of interest 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), ethylene vinyl acetate (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, e.g., 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, e.g., vinyl chloride, vinylidene chloride, and chloromethylstyrene; acrylic compounds, e.g., acrylic and methacrylate styrene; and vinyl acetate, vinyl propionate resin, n-octadecyl acrylamide, ethylene, propylene, and butane, and combinations thereof.

[0194] 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 metal-organic matrix is ​​an organosiloxane polymer, including, but not limited to, polymers of methyltrimethoxysilane, dimethyldimethoxysilane, tetraethoxysilane, methacryloxypropyltrimethoxysilane, bis(triethoxysilyl)ethane, bis(triethoxysilyl)butane, bis(triethoxysilyl)pentane, bis(triethoxysilyl)hexane, bis(triethoxysilyl)heptane, bis(triethoxysilyl)heptane, bis(triethoxysilyl)octane, and combinations thereof.

[0195] kit Aspects of the present disclosure further include kits, which include one or more of the components of the optical detection systems described herein. In some embodiments, the kits include the photodiode array, amplifier components, and programming for the subject system, such as in the form of a computer-readable medium (e.g., a flash drive, USB storage, a compact disc, a DVD, a Blu-ray disc, etc.) or instructions for downloading the programming from an Internet web protocol or cloud server. The kits may also include light conditioning components, such as lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimating probes, and combinations thereof.

[0196] The kit may further include instructions for practicing the subject method. These instructions may be present in the subject kit in various 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 one or more sheets of paper on which the information is printed, in the kit packaging, in a package insert, etc. Another form in which these instructions may be present is as a computer-readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Another form in which these instructions may be present is as a website address that can be used via the Internet to access the information at a remote site.

[0197] Utilities The subject methods, systems, and computer systems are used in a variety of applications where it is desirable to calibrate or optimize photodetectors, such as in particle analyzers. The subject methods and systems also find use 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 cytometers where it is desirable to provide a flow cytometer with improved cell sorting accuracy, enhanced particle collection, reduced energy consumption, particle charging efficiency, more accurate particle charging, and enhanced 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 so that adjustments to the flow cytometer during use require little or no human input.

[0198] Aspects (including embodiments) of the present subject matter described herein may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the present disclosure, numbered 1 through 95, 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 can be used or combined with any of the preceding or subsequent individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0199] 1. A method for adjusting the sensitivity of an optical detection system of a particle analyzer, comprising: detecting light with a light detection system of the particle analyzer, the light detection system comprising a photodiode and an amplifier; determining a responsivity of the photodiode across multiple wavelengths of light; adjusting one or more parameters of the amplifier in response to the response of the photodiode across multiple wavelengths of light; A method comprising: 2. The method of claim 1, wherein the particle analyzer is incorporated into a flow cytometer. 3. The method of claim 1 or 2, wherein a photodiode is positioned within the particle analyzer to detect light from particles within the flow stream. 4. The method of claim 1, comprising determining the responsivity of the photodiode across a spectrum of wavelengths of light. 5. The method of claim 4, wherein the spectrum includes 200 or more wavelengths of light.

[0200] 6. The method of any one of claims 1 to 5, comprising determining the responsivity of the photodiode over wavelengths of light from 400 nm to 1100 nm. 7. The method of any one of claims 1 to 6, comprising determining an average gain of the photodiode across multiple wavelengths. 8. The method of claim 7, further comprising calculating a resistance of the amplifier based on the determined responsivity and average gain of the photodiode across multiple wavelengths of light. 9. The resistance of the amplifier is R f ×R(λ)=G t is calculated according to In the formula, R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, G t 9. The method of claim 8, wherein: ∑ i = 1 i ⁢ ... 10. The method of claim 8 or 9, further comprising adjusting the capacitance of the amplifier based on the calculated resistance.

[0201] 11. The method of claim 10, wherein the capacitance of the amplifier is adjusted in a manner sufficient to produce a predetermined bandwidth of the photodiode. 12. The capacitance of the amplifier is adjusted to a given bandwidth according to the following formula:

[0202]

number

[0203] where BW is the bandwidth, C f 12. The method of claim 11, wherein: is the capacitance of the amplifier. 13. The method of any one of claims 1 to 12, wherein the amplifier is one or more transimpedance amplifiers. 14. The optical detection system a photodiode array including a plurality of photodiodes; a plurality of amplifiers, each photodiode being in electrical communication with an amplifier; 14. The method according to any one of claims 1 to 13, comprising: 15. The method of claim 14, comprising determining the responsivity of each photodiode in the photodiode array across multiple wavelengths of light.

[0204] 16. The method of claim 11, comprising independently determining the responsivity of two or more of the photodiodes in the photodiode array across multiple wavelengths of light. 17. The method of any one of claims 14-16, further comprising determining an average gain of each photodiode in the photodiode array across multiple wavelengths. 18. The method of claim 17, further comprising independently calculating the resistance of each amplifier based on the determined responsivity and average gain of each photodiode across multiple wavelengths of light. 19. The method of claim 18, further comprising adjusting the capacitance of each amplifier based on the calculated resistance.

[0205] 20. Detecting light with a light detection system including a photodiode and an amplifier; determining a responsivity of the photodiode across multiple wavelengths of light; adjusting one or more parameters of the amplifier in response to the response of the photodiode across multiple wavelengths of light; A method comprising: 21. The method of claim 20, comprising determining the responsivity of the photodiode across a spectrum of wavelengths of light. 22. The method of claim 21, wherein the spectrum includes 200 or more wavelengths of light. 23. The method of any one of claims 20-22, comprising determining the responsivity of the photodiode over wavelengths of light from 400 nm to 1100 nm. 24. The method of any one of claims 20-23, comprising determining an average gain of the photodiode across multiple wavelengths.

[0206] 25. The method of claim 24, further comprising calculating a resistance of the amplifier based on the determined responsivity and average gain of the photodiode across multiple wavelengths of light. 26. The method of claim 25, further comprising calculating a resistance of the amplifier based on the determined responsivity and average gain of the photodiode across multiple wavelengths of light. 27. The resistance of an amplifier is R f ×R(λ)=G t is calculated according to In the formula, R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, G t 27. The method of claim 26, wherein ∑ i is the average gain of the photodiode over multiple wavelengths of light. 28. The method of claim 26 or 27, further comprising adjusting the capacitance of the amplifier based on the calculated resistance. 29. The method of claim 28, wherein the capacitance of the amplifier is adjusted in a manner sufficient to produce a predetermined bandwidth of the photodiode.

[0207] 30. The capacitance of an amplifier is adjusted to a given bandwidth according to the following formula:

[0208]

number

[0209] where BW is the bandwidth, C f 20. The method of claim 29, wherein: is the capacitance of the amplifier. 31. The method of any one of appendixes 20 to 30, wherein the amplifiers are 20 or more transimpedance amplifiers. 32. An optical detection system a photodiode array including a plurality of photodiodes; a plurality of amplifiers, each photodiode being in electrical communication with an amplifier; 32. The method according to any one of appendices 20 to 31, comprising: 33. The method of claim 32, comprising determining the responsivity of each photodiode in the photodiode array across multiple wavelengths of light. 34. The method of claim 33, comprising independently determining the responsivity of two or more of the photodiodes in the photodiode array across multiple wavelengths of light.

[0210] 35. The method of any one of claims 32-34, further comprising determining an average gain of each photodiode in the photodiode array across multiple wavelengths. 36. The method of claim 35, further comprising independently calculating the resistance of each amplifier based on the determined responsivity and average gain of each photodiode across multiple wavelengths of light. 37. The method of claim 36, further comprising adjusting the capacitance of each amplifier based on the calculated resistance. 38. The method of any one of claims 20 to 37, wherein the optical detection system is positioned within a flow cytometer. 39. The method of claim 38, wherein the flow cytometer includes a flow cell for propagating particles within the flow stream.

[0211] 40. A particle analyzer comprising: A light source and a light detection system positioned within the particle analyzer housing, the light detection system including a photodiode and an amplifier; 1. A processor comprising: a memory operatively coupled to the processor, the memory including instructions stored therein, the instructions, when executed by the processor, causing the processor to: determining a responsivity of the photodiode across multiple wavelengths of light from the light source; adjusting one or more parameters of the amplifier in response to the response of the photodiode across multiple wavelengths of light; a processor and A particle analyzer comprising: 41. The particle analyzer of claim 40, incorporated into a flow cytometer. 42. The particle analyzer of claim 40 or 41, wherein a photodiode is positioned within the particle analyzer to detect light from particles within the flow stream. 43. A particle analyzer according to any one of claims 40 to 42, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine the responsivity of the photodiode across a spectrum of wavelengths of light. 44. The particle analyzer of claim 43, wherein the spectrum includes 200 or more wavelengths of light.

[0212] 45. A particle analyzer according to any one of claims 40 to 44, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine the responsivity of the photodiode across wavelengths of light between 400 nm and 1100 nm. 46. ​​A particle analyzer according to any one of claims 40 to 45, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine an average gain of the photodiode across multiple wavelengths. 47. The particle analyzer of claim 46, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to calculate a resistance of the amplifier based on the determined responsivity and average gain of the photodiode across multiple wavelengths of light. 48. The memory includes instructions stored in the memory, which, when executed by the processor, cause the processor to f ×R(λ)=G t Calculate the amplifier resistance according to In the formula, R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, G t is the average gain of the photodiode over multiple wavelengths of light 48. The particle analyzer of claim 47. 49. The particle analyzer of claim 47 or 48, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacitance of the amplifier based on the calculated resistance.

[0213] 50. The particle analyzer of claim 49, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to adjust a capacitance of the amplifier sufficient to produce a predetermined bandwidth of the photodiode. 51. The memory includes instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacitance of the amplifier according to the following formula:

[0214]

number

[0215] where BW is the bandwidth, C f 51. The method of claim 50, wherein: is the capacitance of the amplifier. 52. The particle analyzer of any one of appendices 40-51, wherein the amplifier is a transimpedance amplifier. 53. An optical detection system a photodiode array including a plurality of photodiodes; a plurality of amplifiers, each photodiode being in electrical communication with an amplifier; 53. The particle analyzer of any one of appendices 40 to 52, comprising: 54. The particle analyzer of claim 53, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine the responsivity of each photodiode in the photodiode array across multiple wavelengths of light.

[0216] 55. The particle analyzer of claim 54, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to independently determine the responsivity of two or more of the photodiodes in the photodiode array across multiple wavelengths of light. 56. A particle analyzer according to any one of claims 53 to 55, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine an average gain of each photodiode in the photodiodes across a plurality of wavelengths. 57. The particle analyzer of claim 56, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to independently calculate the resistance of each amplifier based on the determined responsivity and average gain of each photodiode across multiple wavelengths of light. 58. The particle analyzer of claim 57, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacitance of each amplifier based on the calculated resistance.

[0217] 59. A system comprising: A light source and a light detection system comprising a photodiode and an amplifier; 1. A processor comprising: a memory operatively coupled to the processor, the memory including instructions stored therein, the instructions, when executed by the processor, causing the processor to: determining a responsivity of the photodiode across multiple wavelengths of light from the light source; adjusting one or more parameters of the amplifier in response to the response of the photodiode across multiple wavelengths of light; a processor and A system comprising: 60. The system of claim 59, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine the responsivity of the photodiode across a spectrum of wavelengths of light. 61. The system of claim 60, wherein the spectrum includes 200 or more wavelengths of light. 62. A system described in any one of appendices 59 to 61, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine the responsivity of the photodiode across wavelengths of light between 400 nm and 1100 nm. 63. The system of any one of notes 59-62, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine an average gain of the photodiode across multiple wavelengths.

[0218] 64. The system of claim 63, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to calculate a resistance of the amplifier based on the determined responsivity and average gain of the photodiode across multiple wavelengths of light. 65. The memory includes instructions stored in the memory, which, when executed by the processor, cause the processor to f ×R(λ)=G t Calculate the amplifier resistance according to In the formula, R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, G t 65. The system of claim 64, wherein ℓ is the average gain of the photodiode across multiple wavelengths of light. 66. The system of claim 64 or 65, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacitance of the amplifier based on the calculated resistance. 67. The system of claim 66, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to adjust a capacitance of the amplifier sufficient to produce a predetermined bandwidth of the photodiode. 68. The memory includes instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacitance of the amplifier according to the following formula:

[0219]

number

[0220] where BW is the bandwidth, C f 68. The system of claim 67, wherein: is the capacitance of the amplifier.

[0221] 69. The system of any one of notes 59 to 68, wherein the amplifier is a transimpedance amplifier. 70. An optical detection system a photodiode array including a plurality of photodiodes; a plurality of amplifiers, each photodiode being in electrical communication with an amplifier; 70. The system of any one of appendices 59 to 69, comprising: 71. The system of claim 70, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine the responsivity of each photodiode in the photodiode array across multiple wavelengths of light. 72. The system of claim 71, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to independently determine the responsivity of two or more of the photodiodes in the photodiode array across multiple wavelengths of light. 73. The system of any one of claims 70-72, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to determine an average gain of each photodiode in the photodiode array across multiple wavelengths.

[0222] 74. The system of claim 73, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to independently calculate the resistance of each amplifier based on the determined responsivity and average gain of each photodiode across multiple wavelengths of light. 75. The system of claim 74, wherein the memory includes instructions stored in the memory that, when executed by the processor, cause the processor to adjust the capacitance of each amplifier based on the calculated resistance. 76. A system described in any one of appendices 59 to 75, which is a flow cytometer. 77. The system of claim 76, wherein the flow cytometer includes a flow cell for propagating particles within the flow stream. 78. The system of claim 77, wherein the photodiode is positioned to detect light from particles in the flow stream.

[0223] 79. A non-transitory computer-readable storage medium containing stored instructions for adjusting the sensitivity of an optical detection system in a particle analyzer, comprising: The command, an algorithm for detecting light in a light detection system comprising a photodiode and an amplifier; an algorithm for determining the responsivity of the photodiode across multiple wavelengths of light; an algorithm for adjusting one or more parameters of the amplifier in response to the response of the photodiode across multiple wavelengths of light; 1. A non-transitory computer-readable storage medium comprising: 80. The non-transitory computer-readable storage medium of claim 79, comprising an algorithm for determining the responsivity of a photodiode across a spectrum of wavelengths of light. 81. The non-transitory computer-readable storage medium of claim 80, wherein the spectrum includes 200 or more wavelengths of light. 82. The non-transitory computer-readable storage medium of any one of Clauses 79-81, comprising an algorithm for determining the responsivity of a photodiode over wavelengths of light from 400 nm to 1100 nm. 83. The non-transitory computer-readable storage medium of any one of clauses 79-82, comprising an algorithm for determining the average gain of a photodiode across multiple wavelengths.

[0224] 84. The non-transitory computer-readable storage medium of claim 83, comprising an algorithm for calculating a resistance of an amplifier based on a determined responsivity and average gain of a photodiode across multiple wavelengths of light. 85.R f ×R(λ)=G t and an algorithm for calculating the resistance of the amplifier according to In the formula, R f is the resistance of the amplifier, R(λ) is the responsivity of the photodiode at each wavelength, G t 85. The non-transitory computer-readable storage medium of claim 84, wherein λ is the average gain of the photodiode across multiple wavelengths of light. 86. The non-transitory computer-readable storage medium of claim 84 or 85, wherein the non-transitory computer-readable storage medium includes an algorithm for adjusting the capacitance of the amplifier based on the calculated resistance. 87. The non-transitory computer-readable storage medium of claim 86, comprising an algorithm for adjusting the capacitance of an amplifier sufficient to produce a predetermined bandwidth of a photodiode. 88. Include an algorithm for adjusting the capacitance of an amplifier according to the following formula:

[0225]

number

[0226] where BW is the bandwidth, C f 88. The non-transitory computer-readable storage medium of claim 87, wherein:

[0227] 89. The non-transitory computer-readable storage medium of any one of clauses 79 to 88, including an algorithm for when the amplifier is a transimpedance amplifier. 90. An optical detection system a photodiode array including a plurality of photodiodes; a plurality of amplifiers, each photodiode being in electrical communication with an amplifier; 90. A non-transitory computer-readable storage medium according to any one of claims 79 to 89, comprising an algorithm for the case in which: 91. The non-transitory computer-readable storage medium of claim 90, comprising an algorithm for determining the responsivity of each photodiode in a photodiode array across multiple wavelengths of light. 92. The non-transitory computer-readable storage medium of claim 91, comprising an algorithm for independently determining the responsivity of two or more of the photodiodes in the photodiode array across multiple wavelengths of light. 93. The non-transitory computer-readable storage medium of any one of Clauses 90-92, comprising an algorithm for determining the average gain of each photodiode in a photodiode array across multiple wavelengths.

[0228] 94. The non-transitory computer-readable storage medium of claim 93, comprising an algorithm for independently calculating the resistance of each amplifier based on the determined responsivity and average gain of each photodiode across multiple wavelengths of light. 95. The non-transitory computer-readable storage medium of Claim 94, comprising an algorithm for adjusting the capacitance of each amplifier based on the calculated resistance.

[0229] 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.

[0230] Accordingly, the foregoing merely illustrates the principles of the present invention. It will be understood that those skilled in the art will be able to devise various arrangements, not explicitly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language set forth herein are intended primarily to aid the reader in understanding the principles of the present invention and concepts contributed by the inventors to further the art, and should not be construed as being limited to such specifically described examples and conditions. Furthermore, all statements herein describing principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, 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 recited in the claims.

[0231] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention 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; if such precise phrases are not used in the limitation in the claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0232] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Patent Application No. 62 / 990,804, filed March 17, 2020, the disclosure of which is incorporated herein by reference.

Claims

[Claim 1] 1. A method for adjusting the sensitivity of an optical detection system of a particle analyzer, comprising: detecting light with a light detection system of the particle analyzer, the light detection system comprising a photodiode and an amplifier; determining a responsivity of the photodiode across multiple wavelengths of light; adjusting one or more parameters of the amplifier in response to a response of the photodiode across a plurality of wavelengths of the light; A method comprising:

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