Gain matched amplifier for light detection

Abstract

Aspects of the invention include methods for adjusting the sensitivity of a photodiode in a light detection system. Methods according to certain embodiments include detecting light with a light detection system having a photodiode and an amplifier; determining the responsivity of the photodiode at a plurality of wavelengths of light; and adjusting one or more parameters of the amplifier with respect to the responsivity of the photodiode at the plurality of wavelengths of light. Systems (e.g., particle analyzers) having a light source and a light detection system including a photodiode and an amplifier for implementing the subject methods are also described. Non-transitory computer readable storage media are also provided.

Description

[0001] Cross-referencing of related patent applications

[0002] This patent application relates to U.S. Provisional Patent Application No. 62 / 990,804, filed on March 17, 2020, the contents of which are incorporated herein by reference.

[0003] introduce

[0004] Optical detection is commonly used to characterize the components of samples (e.g., biological samples), for example, when the sample is used for disease or symptom diagnosis. When a sample is irradiated, it can scatter, transmit, and emit light (e.g., in the form of fluorescence). Changes in sample components (e.g., morphology, absorptivity, and the presence of fluorescent labels) can cause changes in the light scattered, transmitted, or emitted by the sample. These changes can be used to characterize and identify the presence of components in the sample. To quantify these changes, the light is collected and directed onto the detector surface.

[0005] The technique of characterizing components in a sample using optical detection is called flow cytometry. Using data generated based on the detected light, the distribution of components can be recorded, and desired materials can be classified. A flow cytometer typically includes a sample container for receiving a fluid sample (e.g., blood) and a sheath fluid container. The flow cytometer transports particles (including cells) from the fluid sample into a flow cell as a cell stream, while simultaneously guiding the sheath fluid into the flow cell. Within the flow cell, a liquid sheath forms around the cell stream to keep the cell stream flowing at a substantially uniform velocity. The flow cell hydrodynamically concentrates the cells in the stream so that they pass through the center of the light source within the flow cell. Light from the light source can be detected based on scattered or transmitted spectra, or it can be absorbed by one or more components in the sample and re-emitted as cold light. Summary of the Invention

[0006] The present invention includes a method for adjusting the sensitivity of a photodiode in a photodetector system. According to some embodiments, the method includes detecting light using a photodetector system having a photodiode and an amplifier; determining the responsivity of the photodiode at various light wavelengths; and adjusting one or more parameters of the amplifier for the responsivity of the photodiode at the various light wavelengths. The invention also describes a method for implementing the invention, a system having a light source and a photodetector system (e.g., a particle analyzer), the photodetector system including a photodiode and an amplifier. The invention also provides a non-transitory computer-readable storage medium.

[0007] In embodiments, light is detected by a light detection system (e.g., a light detection system in a particle analyzer) having photodiodes and an amplifier (e.g., a transimpedance amplifier) ​​electrically connected to the photodiodes. In some embodiments, the light detection system includes multiple photodiodes, such as 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, and even 1000 or more. In some embodiments, the light detection system includes a photodetector array. When implementing the standard method, each photodiode detects light at multiple wavelengths and determines the responsivity of the photodiode at the multiple wavelengths. In some embodiments, the responsivity of each photodiode is determined at a wavelength spectrum, for example, where the spectrum covers 200 or more wavelengths. In some cases, the responsivity of each photodiode is determined at a spectrum with a wavelength range of 200 nm to 1500 nm, for example, 400 nm to 1100 nm. In some cases, the method includes plotting the responsivity of the photodiode within the wavelength range of the light to generate a responsivity curve for each photodiode in the optical detection system.

[0008] In some embodiments, the method includes determining the average gain of the photodiode at a plurality of wavelengths. In some cases, the average gain of the photodiode is determined individually at each wavelength. In other cases, the average gain of the photodiode is determined over a range of wavelengths (e.g., within a wavelength spectrum). Based on the determined gain of the photodiode (e.g., the average gain of the amplifier at the plurality of light wavelengths) and the determined responsivity, the resistance of the amplifier is calculated. In some cases, the determined resistance is calculated as the resistance of a feedback resistor used in the amplifier. In some embodiments, the resistance of the amplifier is calculated according to the following formula:

[0009] R f XR(λ)=G t

[0010] Where R f R(λ) is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; G t It is the average gain of the photodiode at the various light wavelengths.

[0011] 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 generate a predetermined bandwidth for each photodiode. In some cases, the capacitance of each amplifier is adjusted according to the following formula to generate the predetermined bandwidth:

[0012]

[0013] Where BW is bandwidth; R f It is the resistance of the amplifier; C f It is the capacitance of the amplifier.

[0014] In some embodiments, the method includes determining one or more parameters of a photodetector (e.g., a photodetector in a particle analyzer) by irradiating particles in a fluid medium, wherein the particles comprise one or more fluorophores. In some cases, the particles are magnetic beads (e.g., polystyrene magnetic beads). In some cases, the method for determining the parameters of the photodetector includes irradiating a fluid medium having particles (including one or more fluorophores) at a first intensity for a first predetermined time period and at a second intensity for a second predetermined time period; detecting light from the fluid medium with the photodetector (with a light source); generating a data signal through the photodetector at the first irradiation intensity and at the second irradiation intensity; and determining one or more parameters of the photodetector based on the data signals generated at the first and second intensities. In some cases, the method includes determining the average fluorescence intensity of the particles at the first and second irradiation intensities. In some cases, the method includes determining the variance of the average fluorescence intensity at the first and second irradiation intensities. In some cases, the method includes determining the statistical photoelectrons (SPE) at the first and second irradiation intensities. In some cases, the method further includes calculating the detector efficiency (Q) of the photodetector for each fluorophore on the particle based on the statistical photoelectrons of the fluorophore and the determined average fluorescence intensity. det In some embodiments, the method includes determining the detector efficiency of each detector channel of the photodetector. In some embodiments, the method further includes determining the background signal of each photodetector. In some embodiments, the method further includes determining the electronic noise of each photodetector. In some embodiments, the method further includes determining the detection limit of the photodetector. In some embodiments, the method further includes determining the detector photosensitivity of one or more photodetectors. In some embodiments, determining the detector photosensitivity of the photodetector includes setting an initial detector gain for the photodetector.

[0015] The invention also includes systems (e.g., particle analyzers) having a light source and a light detection system, the light detection system comprising photodiodes and amplifiers. In some embodiments, the light detection system includes multiple photodiodes, such as 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, and up to 1000 or more. In some embodiments, the light detection system further includes multiple amplifiers (each electrically connected to at least one photodiode), such as 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, and up to 1000 or more. In some embodiments, the light detection system includes a photodetector array. In some cases, the light detection system includes a photodetector array with N photodiodes and an amplifier assembly with M amplifiers, where N is an integer between 4 and 10000, and M is an integer between 4 and 10000. In some cases, the number of photodiodes in the array is the same as the number of amplifiers (i.e., N equals 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 of multiple wavelengths. In some embodiments, the photodiodes in the light detection system are configured to detect light within a wavelength spectrum, for example, where the spectrum covers 200 or more wavelengths. In some cases, the photodiodes are configured to detect light within a wavelength range of 200 nm to 1500 nm, for example, 400 nm to 1100 nm.

[0016] In some embodiments, the system further includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following operations: determine the responsivity of the photodiode at multiple wavelengths of light from the light source; and adjust one or more parameters of the amplifier for the responsivity of the photodiode at the multiple wavelengths. In some embodiments, the memory includes instructions for determining the average gain of the photodiode at the multiple wavelengths. In some cases, the memory includes instructions for individually determining the gain of the photodiode at each wavelength. In other cases, the memory includes instructions for determining the average gain of the photodiode over a wavelength range (e.g., within a wavelength spectrum).

[0017] In some embodiments, the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the resistance of one or more amplifiers. In these embodiments, the memory includes instructions for calculating the resistance of each amplifier based on the determined gain of each photodiode (e.g., the average gain of the amplifier across the wavelength spectrum) and the determined responsivity. In some cases, the memory includes instructions for calculating the resistance of a feedback resistor to be used in an amplifier electrically connected to a photodiode. In some embodiments, the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the resistance of the amplifier according to the following formula:

[0018] R f XR(λ)=G t

[0019] Where R f R(λ) is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; G t It is the average gain of the photodiode at the various light wavelengths.

[0020] In some embodiments, the memory includes instructions for adjusting the capacitance of each amplifier based on the calculated resistance. In some cases, the memory includes instructions for adjusting the capacitance to generate a predetermined bandwidth for each photodiode. In some cases, the memory includes instructions for adjusting the capacitance of each amplifier according to the following formula to generate a predetermined bandwidth:

[0021]

[0022] Where BW is bandwidth; R f It is the resistance of the amplifier; C f It is the capacitance of the amplifier.

[0023] The invention also includes a non-transitory computer-readable storage medium for adjusting the sensitivity of a light detection system (e.g., a light detection system in a particle analyzer). According to some embodiments, the non-transitory computer-readable storage medium includes an algorithm for detecting light using a light detection system comprising a photodiode and an amplifier; an algorithm for determining the responsivity of the photodiode at a variety of light wavelengths; and an algorithm for adjusting one or more parameters of the amplifier for the responsivity of the photodiode at the variety of light wavelengths. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for determining the average gain of the photodiode at the variety of wavelengths. In some cases, the non-transitory computer-readable storage medium includes an algorithm for individually determining the gain of the photodiode at each wavelength. In other cases, the non-transitory computer-readable storage medium includes an algorithm for determining the average gain of the photodiode over a wavelength range (e.g., within a wavelength spectrum).

[0024] In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the resistance of one or more amplifiers. In these embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the resistance of each amplifier based on the determined gain of each photodiode (e.g., the average gain of the amplifier over the wavelength spectrum) and the determined responsivity. In some cases, the non-transitory computer-readable storage medium includes an algorithm for calculating the resistance of a feedback resistor to be used in an amplifier electrically connected to a photodiode. In some embodiments, the non-transitory computer-readable storage medium includes an algorithm for calculating the resistance of the amplifier according to the following formula:

[0025] R f XR(λ)=G t

[0026] Where R f R(λ) is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; G t It is the average gain of the photodiode at the various light wavelengths.

[0027] 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 some embodiments, the non-transitory computer-readable storage medium includes an algorithm for adjusting the capacitance of each amplifier according to the following formula to generate a predetermined bandwidth:

[0028]

[0029] Where BW is bandwidth; R f It is the resistance of the amplifier; C f It is the capacitance of the amplifier.

[0030] In some embodiments, aspects of the invention also include multispectral particles (e.g., magnetic beads) having one or more fluorophores for implementing one or more target methods. According to some embodiments, the multispectral particles include one or more fluorophores, such as two or more, three or more, five or more, and even ten or more fluorophores. In some cases, the target particles comprise single-peak multifluorophore magnetic beads that provide a bright photodetector signal at all light source wavelengths (e.g., all LEDs or lasers of the system) and the detection wavelength of the photodetector. Attached Figure Description

[0031] A full understanding of the invention can be obtained by reading the following detailed description in conjunction with the accompanying drawings. The drawings include the following illustrations:

[0032] Figure 1 Examples of wavelength-dependent response curves for photodiodes according to certain embodiments are depicted.

[0033] Figure 2 An amplifier for adjusting the responsivity of a photodiode according to certain embodiments is described.

[0034] Figure 3A A flowchart is depicted to adjust the sensitivity of a photodiode in a light detection system according to certain embodiments.

[0035] Figure 3B A diagram is depicted for setting the initial detector gain of a photodetector according to certain embodiments.

[0036] Figure 4A A functional block diagram of a particle analysis system according to certain embodiments for computation-based sample analysis and particle characterization is depicted.

[0037] Figure 4B A functional block diagram of an example sorting control system according to certain embodiments is depicted.

[0038] Figure 5 A functional block diagram of an example particle analyzer control system according to certain embodiments is depicted.

[0039] Figure 6A A schematic diagram of a particle sorting system according to certain embodiments is depicted.

[0040] Figure 6BA schematic diagram of a particle sorting system according to certain embodiments is depicted.

[0041] Figure 7 A block diagram of a computing system according to certain embodiments is depicted. Detailed Implementation

[0042] The present invention includes a method for adjusting the sensitivity of a photodiode in a photodetector system. According to some embodiments, the method includes detecting light using a photodetector system having a photodiode and an amplifier; determining the responsivity of the photodiode at various light wavelengths; and adjusting one or more parameters of the amplifier for the responsivity of the photodiode at the various light wavelengths. The invention also describes a method for implementing the invention, a system having a light source and a photodetector system (e.g., a particle analyzer), the photodetector system including a photodiode and an amplifier. The invention also provides a non-transitory computer-readable storage medium.

[0043] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as differences will certainly exist in actual implementation. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the inventive concept; the scope of the invention will be defined only by the appended claims.

[0044] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within that range, is included within the scope of this invention. Unless the context explicitly specifies otherwise, each intermediate value should be as low as one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also within this invention, subject to the requirements of any specifically excluded limits within the range. Where the range includes one or two limits, the range excluding any one or both of the included limits is also included within this invention.

[0045] Certain ranges presented in this paper are preceded by the term "approximately". The purpose of using the term "approximately" in this paper is to provide textual support for the precise figures that follow and for figures that are close to or approximate to the figures following the term. In determining whether a figure is close to or approximate to a specifically listed figure, an unlisted figure that is close to or approximates can be substantially equivalent to the specifically listed figure in its context.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials may be used in the implementation or testing of this invention, representative exemplary methods and materials are described below.

[0047] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and the purpose of their inclusion is to disclose and describe methods and / or materials relating to the cited publications. References to any publication refer to its content disclosed prior to the filing date and should not be construed as an admission that the present invention is not entitled to precede such publication by prior invention. Furthermore, the publication dates provided may differ from the actual publication dates and may require separate verification.

[0048] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a precondition for the use of specialized terms related to the stated elements of a claim, such as “alone,” “only,” or the use of the limiting word “negative.”

[0049] Upon reading this invention, the following will be apparent to those skilled in the art: each individual embodiment described and listed herein has hierarchical components and features that can be quickly decomposed or combined with features of any of the other embodiments without departing from the scope and spirit of the invention. Any of the stated methods may be implemented in the order of the stated events or in any other logically possible order.

[0050] Although the device and method have been or will be described and their functions explained for grammatical fluency, it should be clearly understood that, unless expressly provided for in Chapter 35 of the United States Code, the claims shall not in any case be construed as necessarily being limited to “method” or “step”, but shall conform to the judicial principles of equivalence and the meaning and full scope of the equivalent as defined in the claims. When the claims are explicitly prepared in accordance with the provisions of Section 112 of Chapter 35 of the United States Code, the claims shall conform in full to the legal equivalents in Section 112 of Chapter 35 of the United States Code.

[0051] As described above, the present invention provides a method for adjusting the sensitivity of a photodiode in a photodetector system. In further describing embodiments of the invention, the method for achieving the following is first described in more detail: detecting light using a photodetector system having a photodiode and an amplifier; determining the responsivity of the photodiode at various light wavelengths; and adjusting one or more parameters of the amplifier for the responsivity of the photodiode at the various light wavelengths. Next, the invention describes a system for implementing the method, including a light source and a photodetector system having a photodiode and an amplifier. The invention also describes a non-transitory computer-readable storage medium.

[0052] Methods for adjusting the sensitivity of photodiodes in optical detection systems

[0053] Aspects of the present invention include a method for adjusting the sensitivity of a photodiode in a light detection system. As used herein, the term "sensitivity" refers to the ratio between a detector output and a detector input, according to conventional usage. In some embodiments, the sensitivity of the photodiode refers to the ratio between the current output by the photodiode and the current generated by the light detected by the photodiode. In embodiments, the method results in an increase of 5% or more in the output (e.g., detector signal amplitude) of one or more photodiodes, such as 10% or more, 25% or more, 50% or more, 75% or more, 90% or more, and including 99% or more. In some cases, the method results in an increase of 2 times or more in the detector output, such as 3 times or more, 4 times or more, 5 times or more, and including 10 times or more. In some embodiments, the method results in an increase in the signal-to-noise ratio (SNR) of one or more photodiodes, wherein the SNR increase is 5% or more, such as 10% or more, 25% or more, 50% or more, 75% or more, 90% or more, and including 99% or more. In some cases, the method of the present invention increases the signal-to-noise ratio of the one or more photodiodes by 2 times or more, for example, 3 times or more, 4 times or more, 5 times or more, and including 10 times or more. In some embodiments, the method of the present invention is sufficient to expand the intensity detection and quantification range by 2 times or more, for example, 3 times or more, 5 times or more, 10 times or more, 25 times or more, 50 times or more, and including 100 times or more.

[0054] In implementing the target method, light is detected by photodiodes electrically connected to an amplifier in a light detection system (e.g., the light detection system in a particle analyzer or flow cytometer, described in more detail below). In some embodiments, the light originates from an irradiated sample in a fluid medium. The light detected therefrom can be emitted light, transmitted light, scattered light, or a combination thereof. In embodiments, each photodiode detects multiple wavelengths of light. In some embodiments, the light detected by each photodiode includes 10 or more different wavelengths of light, such as 15 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 1500 or more, 2500 or more, and includes 5000 or more different wavelengths of light. In some embodiments, the light detected by each photodiode includes light within a spectrum, such as wavelengths spanning 50 nm or more, for example, 100 nm or more, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, 1000 nm or more, and including 1500 nm or more. For example, the wavelength range of light detected by each photodiode according to the standard method may be 200 nm to 1500 nm, for example, 400 nm to 1100 nm.

[0055] In some embodiments, light is detected using a light detection system that includes a photodiode and an amplifier electrically connected to the photodiode. In some embodiments, the photodiode is an avalanche photodiode. In some embodiments, the method includes detecting light using a light detection system having multiple photodiodes (e.g., a photodiode array). In these embodiments, the photodiode array may include four or more photodiodes, such as 10 or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, 750 or more, and may include 1000 or more photodiodes.

[0056] The photodiodes can be arranged in any geometric configuration as needed, including but not limited to square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The photodiodes in the photodiode array may be oriented at an angle to another photodiode (as described in the XZ plane), the angle ranging from 10° to 180°, for example 15° to 170°, 20° to 160°, 25° to 150°, 30° to 120°, and including 45° to 90°. The photodiode array can be any suitable shape, and can be a shape enclosed by straight lines (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a shape enclosed by curves (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom connecting to a planar top). In some embodiments, the photodiode array has a rectangular active surface.

[0057] The active surface width of each photodiode in the array ranges from 5m to 250m, for example, 10m to 225m, 15m to 200m, 20m to 175m, 25m to 150m, 30m to 125m, and includes 50m to 100m; its length ranges from 5m to 250m, for example, 10m to 225m, 15m to 200m, 20m to 175m, 25m to 150m, 30m to 125m, and includes 50m to 100m; wherein the surface area of ​​each photodiode in the array ranges from 25m². 2 Up to 10000m 2 For example, 50m 2 up to 9000m 2 75m 2 up to 8000m 2 100m 2 up to 7000m 2 150m 2 up to 6000m 2 And including 200m 2 up to 5000m 2 .

[0058] The size of the photodiode array can vary depending on the amount and intensity of light, the number of photodiodes, and the required sensitivity, and its length ranges from 0.01 mm to 100 mm, for example, 0.05 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, 2 mm to 50 mm, 3 mm to 40 mm, 4 mm to 30 mm, and includes 5 mm to 25 mm. The width of the photodiode array may also vary, ranging from 0.01 mm to 100 mm, for example, 0.05 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, 2 mm to 50 mm, 3 mm to 40 mm, 4 mm to 30 mm, and includes 5 mm to 25 mm. Therefore, the surface area of ​​the active surface of the photodiode array ranges from 0.1 mm². 2 Up to 10000mm 2 For example, 0.5mm 2 Up to 5000mm 2 1mm 2 Up to 1000mm 2 5mm 2 Up to 500mm 2 And including 10mm 2 Up to 100mm 2 .

[0059] The method may include measuring light continuously or at discrete time intervals. In some cases, the target photodiode is configured to continuously measure the collected light. In other cases, the light detection system is configured to perform measurements at discrete time intervals, such as 0.001 ms, 0.01 ms, 0.1 ms, 1 ms, 10 ms, 100 ms, and including 1000 ms or some other time interval.

[0060] In implementing the target method, according to certain embodiments, each photodiode detects light of multiple wavelengths and determines the responsivity of the photodiode at said multiple wavelengths. As used herein, the term "responsivity" refers to the ratio between the photocurrent generated by the photodiode and the optical power of the incident light, according to conventional understanding. In some embodiments, the responsivity of each photodiode is determined at 10 or more different wavelengths, such as 15 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 1500 or more, 2500 or more, and including 5000 or more different wavelengths. In some embodiments, the responsivity of each photodiode in a spectrum is determined, for example, wherein the spectrum includes wavelengths spanning 50 nm or more, such as 100 nm or more, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, 1000 nm or more, and includes 1500 nm or more. For example, the responsivity of each photodiode in a spectrum according to the target method is determined, wherein the wavelength range of the spectrum is 200 nm to 1500 nm, for example, 400 nm to 1100 nm.

[0061] In some cases, the method includes plotting the responsivity of the photodiode within the wavelength range of the light to generate a responsivity curve for each photodiode in the optical detection system. Figure 1 Examples of wavelength-dependent response curves for photodiodes according to certain embodiments are depicted. Figure 1 In the study, the responsivity of photodiodes increases between 450 nm and approximately 875 nm, and decreases between 875 nm and 1100 nm. As shown in the responsivity curves, the photodiode detector exhibits a responsivity at 875 nm that is nearly 10 times higher than that at 450 nm.

[0062] In some embodiments, the method includes using the responsivity of the photodiode at a variety of wavelengths to determine the average gain of the photodiode at the variety of wavelengths. In some cases, the average gain of the photodiode at each wavelength is determined individually. For example, in determining the responsivity of the photodiode in a wavelength spectrum (e.g., wavelength ranges of 200 nm to 1500 nm, 400 nm to 1100 nm), the method includes determining the average gain of the photodiode at every 1 nm in the wavelength spectrum, such as every 2 nm, every 5 nm, every 10 nm, and including every 25 nm or some other interval. In other cases, the average gain of the photodiode is determined over a range of wavelengths, such as 1 nm or longer, 2 nm or longer, 5 nm or longer, 10 nm or longer, 25 nm or longer, 50 nm or longer, 100 nm or longer, 250 nm or longer, 500 nm or longer, and including 1000 nm or longer.

[0063] Based on the determined gain of the photodiode (e.g., the average gain of the amplifier across the various light wavelengths) and the determined responsivity, the resistance of the amplifier is calculated. In some cases, the determined resistance is calculated as the resistance of the feedback resistor used in the amplifier. In some embodiments, the resistance of the amplifier is calculated according to the following formula:

[0064] R f XR(λ)=G t

[0065] Where R f R(λ) is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; G t It is the average gain of the photodiode at the various light wavelengths.

[0066] In some embodiments, the capacitance of each amplifier is adjusted based on the calculated resistance. In some embodiments, the capacitance adjustment is 5% or more, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, and including 95% or more. In some cases, the capacitance of the amplifier is adjusted to compensate for the wavelength-dependent responsivity of the photodiode.

[0067] 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 some cases, the capacitance of the amplifier is adjusted so that the photodiode produces substantially the same wavelength responsivity at a variety of wavelengths (e.g., in the wavelength spectrum). In other cases, the capacitance of the amplifier is adjusted so that the photodiode produces the desired responsivity at one or more predetermined detection wavelengths.

[0068] Figure 2 An amplifier for adjusting the responsivity of a photodiode according to certain embodiments is depicted. Amplifier 201 is electrically connected to photodiode 202 via 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 at the various wavelengths.

[0069] In some cases, the capacitor is adjusted in a manner sufficient to generate a predetermined bandwidth for each photodiode. In other cases, the capacitor of each amplifier is adjusted according to the following formula to generate the predetermined bandwidth:

[0070]

[0071] Where BW is bandwidth; R f It is the resistance of the amplifier; C f It is the capacitance of the amplifier.

[0072] Depending on the average gain of the amplifier, the predetermined bandwidth can be 10 kHz or greater, for example 25 kHz or greater, 50 kHz or greater, 100 kHz or greater, 150 kHz or greater, 200 kHz or greater, 250 kHz or greater, 500 kHz or greater, and includes a predetermined bandwidth of 1000 kHz or greater. For example, in the case where the average gain of the amplifier is 10... 6 In one example of volts / amperes, the predetermined bandwidth can be 250 kHz or greater, such as 275 kHz or greater, 300 kHz or greater, 325 kHz or greater, 350 kHz or greater, 375 kHz or greater, 400 kHz or greater, 425 kHz or greater, 450 kHz or greater, 475 kHz or greater, and includes a predetermined bandwidth of 500 kHz or greater.

[0073] Figure 3AA flowchart illustrating the adjustment of the sensitivity of a photodiode in a light detection system according to certain embodiments is provided. In step 301, light from a light source is detected by a photodiode electrically connected to an amplifier. In step 302, the responsivity of the photodiode at multiple wavelengths (e.g., within a wavelength spectrum) is determined. In some embodiments, determining the responsivity of the photodiode includes generating wavelength-correlated responsivity curves that compare the responsivity of the photodiode at different light wavelengths. In step 303, the average gain of the amplifier at multiple wavelengths (e.g., within a wavelength spectrum) is determined, and the resistance of the amplifier is calculated based on the determined responsivity of the photodiode and the average gain of the amplifier. Based on the calculated resistance, the capacitance of each amplifier is adjusted in step 304. In some cases, the capacitance of the amplifier is adjusted so that the photodiode produces substantially the same wavelength responsivity at the multiple wavelengths (e.g., within a wavelength spectrum). In other cases, the capacitance of the amplifier is adjusted so that the photodiode produces a desired responsivity at one or more predetermined detection wavelengths.

[0074] In an embodiment, the light detected by the photodiode according to the target method originates from a light source, such as a broadband light source, which emits light with a wide range of wavelengths, for example, spanning 50 nm or more, 100 nm or more, 150 nm or more, 200 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, and including spans of 500 nm or more. For example, a suitable broadband light source emits light with a wavelength range of 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with a wavelength range of 400 nm to 1000 nm. When the light source is a broadband light source, the desired broadband light source scheme may include, but is not limited to: halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with a continuous spectrum, superluminescent diodes, semiconductor light-emitting diodes, broadband LED white light sources, multi-LED integrated light sources, and other broadband light sources or any combination thereof.

[0075] In other embodiments, the light source is a narrowband light source that emits light of a specific wavelength or a narrow range of wavelengths. For example, a light source emitting light of a narrow range of wavelengths, such as 50 nm or less, 40 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, 2 nm or less, and includes light sources that emit light of a specific wavelength (i.e., monochromatic light). When the method includes irradiation with a narrowband light source, the desired narrowband light source may include, but is not limited to: narrow-wavelength LEDs, laser diodes, or broadband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0076] In some embodiments, the light source comprises one or more lasers, such as gas lasers, for example, helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon fluoride (ArF) excimer lasers, krypton fluoride (KrF) excimer lasers, xenon chloride (XeCl) excimer lasers, or xenon fluoride (XeF) excimer lasers, or combinations thereof. In other cases, the method comprises irradiating a fluid medium 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, or combinations thereof. In other cases, the light source includes solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulim YAG lasers, YAG ytterbium lasers, ytterbium trioxide lasers, or cerium-doped lasers and combinations thereof.

[0077] The light source may include any combination of light source types. The light source can be configured to irradiate with light in the wavelength range of 200 nm to 1500 nm, for example, 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, including 400 nm to 800 nm. For example, the light source can be a broadband light source irradiating with light in the wavelength range of 200 nm to 900 nm. In other cases, the light source includes multiple narrowband light sources that can irradiate with light in a specific wavelength range of 200 nm to 900 nm. For example, the light source can be multiple narrowband LEDs (1 nm–25 nm), each emitting light with a wavelength between 200 nm and 900 nm. In other embodiments, the narrowband light source includes one or more lasers (e.g., a laser array) that irradiate with lasers in a specific wavelength range of 200 nm to 700 nm, for example, using a laser array having the aforementioned gas lasers, excimer lasers, dye lasers, metal vapor lasers, and solid-state lasers.

[0078] When using more than one light source, the light sources can be configured to irradiate simultaneously, sequentially, or in combination. In one example, the light sources are configured to irradiate simultaneously. In other embodiments, the light sources are configured to irradiate sequentially. When using more than one light source for sequential irradiation, the irradiation time of each light source can be 0.001 microseconds or longer, for example, 0.01 microseconds or longer, 0.1 microseconds or longer, 1 microsecond or longer, 5 microseconds or longer, 10 microseconds or longer, 30 microseconds or longer, and including 60 microseconds or longer. For example, the light source (e.g., a laser) can be configured to irradiate for a period of time, the duration ranging from 0.001 microseconds to 100 microseconds, for example, 0.01 microseconds to 75 microseconds, 0.1 microseconds to 50 microseconds, 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. The irradiation duration of each light source can be the same or different.

[0079] The time interval between irradiations from each light source may also vary as needed, and may be 0.001 microseconds or longer, for example, 0.01 microseconds or longer, 0.1 microseconds or longer, 1 microsecond or longer, 5 microseconds or longer, 10 microseconds or longer, 15 microseconds or longer, 30 microseconds or longer, and including 60 microseconds or longer. For example, the time interval between irradiations from each light source may range from 0.001 microseconds to 60 microseconds, for example, 0.01 microseconds to 50 microseconds, 0.1 microseconds to 35 microseconds, 1 microsecond to 25 microseconds, including 5 microseconds to 10 microseconds. In some embodiments, the time interval between irradiations from each light source is 10 microseconds. The delay time between irradiations from each light source may be the same or different.

[0080] Irradiation can be continuous or at discrete time intervals. In some cases, the light source irradiates continuously. In other cases, the light source irradiates at discrete time intervals, for example, irradiation time intervals of 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, including 1000 milliseconds or some other time interval.

[0081] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams. In some cases, the beam generator includes a laser and a radio frequency (RF) generator, the RF generator being configured to apply an RF drive signal to an acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the laser in the target beam generator can 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 CO laser, an argon fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon fluoride (XeF) excimer laser or a combination thereof; or a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser. Helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof; or solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulim YAG lasers, YAG ytterbium lasers, ytterbium trioxide lasers, or cerium-doped lasers, and combinations thereof.

[0082] The acousto-optic device can be any suitable acousto-optic scheme configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the target system is configured to generate an angle-deflected laser beam using light from the laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital frequency 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 angle-deflected laser beams in the output laser beam, for example, to apply 3 or more radio frequency drive signals, 4 or more radio frequency drive signals, 5 or more radio frequency drive signals, 6 or more radio frequency drive signals, 7 or more radio frequency drive signals, 8 or more radio frequency drive signals, 9 or more radio frequency drive signals, 10 or more radio frequency drive signals, 15 or more radio frequency drive signals, 25 or more radio frequency drive signals, 50 or more radio frequency drive signals, and includes being configured to apply 100 or more radio frequency drive signals.

[0084] In some cases, in order to obtain the intensity distribution of the angle-deflected laser beam in the output laser beam, the controller is configured to apply radio frequency drive signals with different amplitudes, for example, the amplitude range being about 0.001V to about 500V, about 0.005V to about 400V, about 0.01V to about 300V, about 0.05V to about 200V, about 0.1V to about 100V, about 0.5V to about 75V, about 1V to 50V, about 2V to 40V, 3V to about 30V, and including about 5V to about 25V. In some embodiments, each applied radio frequency drive signal has a frequency ranging from about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, about 0.01 MHz to about 300 MHz, about 0.05 MHz to about 200 MHz, about 0.1 MHz to about 100 MHz, about 0.5 MHz to about 90 MHz, about 1 MHz to about 75 MHz, about 2 MHz to about 70 MHz, about 3 MHz to about 65 MHz, about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0085] 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 irradiating particles in a fluid medium, wherein the particles comprise one or more fluorophores. In some cases, the particles are magnetic beads (e.g., polystyrene magnetic beads), which are described in more detail below. In some cases, the standard method described below can be used to determine parameters of the photodetector, including a specified relative fluorescence unit (e.g., ABD unit) for each photodetector, robust coefficient of variation (rCV) for one or more photodetectors, maximum and minimum linearity for each photodetector, relative change of rCV from a baseline, relative change of detector gain from a baseline, and imaging specifications of the photodetector (e.g., RF power or axial optical loss).

[0086] In some cases, methods for determining parameters of a photodetector include irradiating a fluid medium having particles (including one or more fluorophores) at a first intensity for a first predetermined time period and at a second intensity for a second predetermined time period; detecting light from the fluid medium using the photodetector (with a light source); generating a data signal through the photodetector at the first irradiation intensity and at the second irradiation intensity; and determining one or more parameters of the photodetector based on the data signals generated at the first and second intensities.

[0087] In some embodiments, the method includes determining the average fluorescence intensity (M) of the particles under the first irradiation intensity and the second irradiation intensity. In some cases, the method includes determining the variance (V(M)) of the average fluorescence intensity under the first irradiation intensity and the second irradiation intensity. In some cases, the method includes determining the %rCV (robust coefficient of variation) of the photodetector. In some embodiments, a linear fit of the variance is calculated according to the following formula:

[0088]

[0089] Q led The variance, derived from 1 / c1, refers to the statistical photoelectrons per unit of average fluorescence intensity (M) (i.e., SPE / MFI). In some embodiments, the variance is plotted to determine a linear fit to the variance according to the following formula:

[0090] y = c1x + c0

[0091] In an embodiment, the average fluorescence intensity and variance can be determined for a variety of different irradiation intensities, such as two or more irradiation intensities, for example three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, and including 15 or more different irradiation intensities.

[0092] In some embodiments, the method includes determining the statistical photoelectrons (SPEs) at one or more irradiation intensities, for example, at least at a first irradiation intensity and a second irradiation intensity. In some cases, the method further includes calculating the detector efficiency (Q) of the photodetector for the particle based on the statistical photoelectrons per particle and the determined average fluorescence intensity. detIn some embodiments, the method includes determining the detector efficiency of one or more detector channels in the photodetector based on the statistical photoelectrons of the particles and the determined average fluorescence intensity, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 20 or more, 24 or more, 36 or more, 48 or more, 72 or more, and includes determining the detector efficiency of 96 or more detector channels in the photodetector. In some cases, the method further includes determining the detector efficiency of all detector channels in the photodetector for each particle based on the statistical photoelectrons of each particle and the determined average fluorescence intensity. In some embodiments, the detector efficiency of the photodetector is determined according to the following formula:

[0093]

[0094] Where SPE stands for Statistical Photoelectrons, MFI is the Average Fluorescence Intensity, and ABD is the unit allocated to each channel for each particle batch.

[0095] In some embodiments, the method further includes determining background signals for one or more photodetectors. In some cases, the background signals are determined under one or more irradiation intensities, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and including 10 or more different irradiation intensities. In some cases, the background signals are determined under all applied irradiation intensities. Similarly, the background signals can be determined in one or more detector channels of the photodetector, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 20 or more, 24 or more, 36 or more, 48 or more, 72 or more, and include determining the background signals in 96 or more detector channels of the photodetector, wherein in some cases, the background signals are determined in all detector channels of the photodetector. In some cases, the background signal is determined based on the statistical photoelectrons of the photodetector and the detector efficiency. In other cases, the background signal is determined according to the following formula:

[0096] B SD =B SD,MFI ×Q led

[0097]

[0098] In some embodiments, the method further includes determining the electronic noise of one or more photodetectors. In some cases, the electronic noise of the photodetector is determined under one or more irradiation intensities, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and includes 10 or more different irradiation intensities. In some cases, the electronic noise of the photodetector is determined under all applied irradiation intensities. Similarly, the electronic noise can be determined in one or more detector channels of the photodetector, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 20 or more, 24 or more, 36 or more, 48 or more, 72 or more, and includes determining the electronic noise in 96 or more detector channels of the photodetector, wherein in some cases, the electronic noise is determined in all detector channels of the photodetector. In some cases, the electronic noise is determined based on the statistical photoelectrons of the photodetector and the detector efficiency. In other cases, the electronic noise is determined according to the following formula:

[0099] ENS SD =ENS SD,MFI ×Q led

[0100]

[0101] In some embodiments, the method further includes determining the detection limit of one or more photodetectors. In some cases, the detection limit of the photodetector is determined in one or more detector channels of the photodetector, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 20 or more, 24 or more, 36 or more, 48 or more, 72 or more, and includes determining the detection limit of the photodetector in 96 or more detector channels of the photodetector, wherein in some cases, the detection limit is determined in all detector channels of the photodetector. In some cases, the detection limit of each photodetector is determined according to the following formula:

[0102] 2 + 2SD = 4(1 + B) SD )

[0103] In some embodiments, the method further includes determining the detector photosensitivity of one or more photodetectors. In some embodiments, determining the detector photosensitivity of the photodetector includes setting an initial detector gain for the photodetector. In some cases, the method includes irradiating the photodetector with the light source (see above) at multiple different light intensities; generating data signals through the photodetector for the multiple light intensities at one or more detector gains of the photodetector; and determining the minimum light irradiance intensity at which a data signal resolvable from the background data signal can be generated at each detector gain. In some cases, the method includes determining the minimum light irradiance intensity at which a data signal reduced by two standard deviations compared to the background data signal can be generated at each detector gain. In some cases, the method includes setting the detector gain to achieve a plateau at the minimum light irradiance intensity that, when plotted as a function of light intensity, is capable of generating a data signal resolvable from the background data signal. Figure 3B A diagram depicting the initial detector gain for setting a photodetector according to certain embodiments is shown. Figure 3B As shown, the detector gain of the photodetector is plotted as a function of the irradiance of light (e.g., LED) from two different fluorophores (e.g., fluorophores stably bound to particles, which are described in more detail below). When setting the initial detector gain of the photodetector, the detector gain is determined such that the lowest light irradiance at which it can produce a data signal that can be resolved from the background data signal reaches a plateau. Figure 3B The volts are approximately 575.

[0104] System for adjusting the sensitivity of photodiodes

[0105] As described above, aspects of the invention also include a system having a light source and a light detection system (e.g., a particle analyzer), the light detection system including a photodiode and an amplifier. As described above, the term "sensitivity" as used herein refers to the ratio between a detector output and a detector input. In some embodiments, the sensitivity of the photodiode refers to the ratio between the current output by the photodiode and the current generated by the light detected by the photodiode. A system according to certain embodiments includes a light source; a light detection system having a photodiode and an amplifier (e.g., a light detection system located within the housing of the particle analyzer); and a processor including a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to perform the following operations: determine the responsivity of the photodiode at various light wavelengths from the light source; and adjust one or more parameters of the amplifier for the responsivity of the photodiode at the various light wavelengths.

[0106] In embodiments, the light source can be any suitable broadband or narrowband light source. Depending on the components in the sample (e.g., cells, magnetic beads, non-cellular particles, etc.), the light source can be configured to emit light of different wavelengths ranging from 200 nm to 1500 nm, such as 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, and including 400 nm to 800 nm. For example, the light source can include a broadband light source emitting light with a wavelength range of 200 nm to 900 nm. In other cases, the light source includes a narrowband light source emitting light with a wavelength range of 200 nm to 900 nm. For example, the light source can be a narrowband LED (1 nm–25 nm) emitting light with a wavelength range of 200 nm to 900 nm. In some embodiments, the light source is a laser. In some cases, the target system includes gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon fluoride (ArF) excimer lasers, krypton fluoride (KrF) excimer lasers, xenon chloride (XeCl) excimer lasers, or xenon fluoride (XeF) excimer lasers, or combinations thereof. In other cases, the target system includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In still other cases, the target laser includes metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, or combinations thereof. In other cases, the target system includes solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulim YAG lasers, YAG ytterbium lasers, ytterbium trioxide lasers, or cerium-doped lasers and combinations thereof.

[0107] In other embodiments, the light source is a non-laser light source, such as lamps (including but not limited to halogen lamps, deuterium arc lamps, xenon arc lamps), or light-emitting diodes (e.g., broadband LEDs with continuous spectrum, superluminescent LEDs, semiconductor light-emitting diodes, broadband LED white light sources, and multi-LED integrated light sources). In some cases, the non-laser light source is a stable fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0108] The light source can be positioned at any suitable distance from the sample (e.g., the fluid medium in a flow cytometer), such as 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, and including a distance of 100 mm or more. Furthermore, the light source irradiates the sample at any suitable angle (e.g., relative to the vertical axis of the fluid medium), for example, angles ranging from 10° to 90°, 15° to 85°, 20° to 80°, 25° to 75°, including 30° to 60°, such as a 90° angle.

[0109] The light source can be configured to irradiate the sample continuously or at discrete time intervals. In some cases, the system includes a light source configured to continuously irradiate the sample, for example, using a continuous-wave laser that can continuously irradiate the fluid medium in the flow cytometer at the interrogation point. In other cases, the target system includes a light source configured to irradiate the sample at discrete time intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, including every 1000 milliseconds or some other time interval. When the light source is configured to irradiate the sample at discrete time intervals, the system may include one or more other components to intermittently irradiate the sample using the light source. For example, in these embodiments, the target system may include one or more laser beam choppers, manually or computer-controlled beam stops for shielding the sample and exposing the sample to the light source.

[0110] In some embodiments, the light source is a laser. The target laser may include a pulsed laser or a continuous-wave laser. 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 CO laser, an argon fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon fluoride (XeF) excimer laser, or a combination thereof; or a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury laser, or a helium-mercury laser. HeHg lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; or solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulim YAG lasers, YAG ytterbium lasers, ytterbium trioxide lasers, or cerium-doped lasers and combinations thereof; or semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or second or third harmonic embodiments of any of the above lasers.

[0111] In some embodiments, the light source is a beam generator configured to generate two or more frequency-shifted beams. In some cases, the beam generator includes a laser and a radio frequency (RF) generator, the RF generator being configured to apply an RF drive signal to an acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, the laser can be a pulsed laser or a continuous-wave laser. For example, the laser in the target beam generator can 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 CO laser, an argon fluoride (ArF) excimer laser, a krypton fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon fluoride (XeF) excimer laser or a combination thereof; or a dye laser, such as a stilbene, coumarin, or rhodamine laser; or a metal vapor laser, such as a helium-cadmium (HeCd) laser. Helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, and combinations thereof; or solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, Ti:sapphire lasers, thulim YAG lasers, YAG ytterbium lasers, ytterbium trioxide lasers, or cerium-doped lasers, and combinations thereof.

[0112] The acousto-optic device can be any suitable acousto-optic scheme configured to frequency-shift a laser using applied acoustic waves. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the target system is configured to generate an angle-deflected laser beam using light from the laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital frequency synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0113] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angle-deflected laser beams in the output laser beam, for example, to apply 3 or more radio frequency drive signals, 4 or more radio frequency drive signals, 5 or more radio frequency drive signals, 6 or more radio frequency drive signals, 7 or more radio frequency drive signals, 8 or more radio frequency drive signals, 9 or more radio frequency drive signals, 10 or more radio frequency drive signals, 15 or more radio frequency drive signals, 25 or more radio frequency drive signals, 50 or more radio frequency drive signals, and includes being configured to apply 100 or more radio frequency drive signals.

[0114] In some cases, in order to obtain the intensity distribution of the angle-deflected laser beam in the output laser beam, the controller is configured to apply radio frequency drive signals with different amplitudes, for example, the amplitude range being about 0.001V to about 500V, about 0.005V to about 400V, about 0.01V to about 300V, about 0.05V to about 200V, about 0.1V to about 100V, about 0.5V to about 75V, about 1V to 50V, about 2V to 40V, 3V to about 30V, and including about 5V to about 25V. In some embodiments, each applied radio frequency drive signal has a frequency ranging from about 0.001 MHz to about 500 MHz, such as about 0.005 MHz to about 400 MHz, about 0.01 MHz to about 300 MHz, about 0.05 MHz to about 200 MHz, about 0.1 MHz to about 100 MHz, about 0.5 MHz to about 90 MHz, about 1 MHz to about 75 MHz, about 2 MHz to about 70 MHz, about 3 MHz to about 65 MHz, about 4 MHz to about 60 MHz, and including about 5 MHz to about 50 MHz.

[0115] In some embodiments, the controller has a processor having a memory operatively coupled thereto, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with angle-deflected laser beams (having a desired intensity distribution). For example, the memory may include instructions for generating two or more angle-deflected laser beams with the same intensity, such as 3 or more, 4 or more, 5 or more, 10 or more, 25 or more, 50 or more, and may include memory for generating 100 or more angle-deflected laser beams with the same intensity. In other embodiments, the memory may include instructions for generating two or more angle-deflected laser beams with different intensities, such as 3 or more, 4 or more, 5 or more, 10 or more, 25 or more, 50 or more, and may include memory for generating 100 or more angle-deflected laser beams with different intensities.

[0116] In some embodiments, the controller has a processor having a memory operatively coupled thereto, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity along the horizontal axis gradually increases from its edge to its center. In these cases, the intensity of the angle-deflected laser beam measured at the center of the output beam can be from 0.1% to about 99% of the intensity of the angle-deflected laser beam measured along the horizontal axis at the edge of the output laser beam, for example, from 0.5% to about 95%, 1% to about 90%, about 2% to about 85%, about 3% to about 80%, about 4% to about 75%, about 5% to about 70%, about 6% to about 65%, about 7% to about 60%, about 8% to about 55%, and includes about 10% to about 50% of the intensity of the angle-deflected laser beam measured along the horizontal axis at the edge of the output laser beam. In other embodiments, the controller has a processor having a memory operatively coupled thereto, such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam whose intensity gradually increases along a horizontal axis from its edge to its center. In these cases, the intensity of the angle-deflected laser beam measured at the edge of the output beam can be from 0.1% to about 99% of the intensity of the angle-deflected laser beam measured along the horizontal axis at the center of the output laser beam, for example, from 0.5% to about 95%, 1% to about 90%, about 2% to about 85%, about 3% to about 80%, about 4% to about 75%, about 5% to about 70%, about 6% to about 65%, about 7% to about 60%, about 8% to about 55%, and includes about 10% to about 50% of the intensity of the angle-deflected laser beam measured along the horizontal axis at the center of the output laser beam. In other embodiments, the controller has a processor with a memory operatively coupled thereto, such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with an intensity distribution along the horizontal axis conforming to a Gaussian distribution. In some embodiments, the controller has a processor with a memory operatively coupled thereto, such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with a top-hat shaped intensity distribution along the horizontal axis.

[0117] In embodiments, the target beam generator can be configured to generate spatially separated angle-deflected laser beams within the output laser beam. Depending on the applied radio frequency drive signal and the desired irradiation profile of the output laser beam, the spacing between the angle-deflected laser beams can be 0.001 m or more, for example, 0.005 m or more, 0.01 m or more, 0.05 m or more, 0.1 m or more, 0.5 m or more, 1 m or more, 5 m or more, 10 m or more, 100 m or more, 500 m or more, 1000 m or more, and includes 5000 m or more. In some embodiments, the system is configured to generate overlapping angle-deflected laser beam overlaps within the output laser beam, for example, overlaps with adjacent angle-deflected laser beams distributed along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be 0.001m or more, such as 0.005m or more, 0.01m or more, 0.05m or more, 0.1m or more, 0.5m or more, 1m or more, 5m or more, 10m or more, and includes 100m or more.

[0118] In some cases, beam generators configured to produce two or more frequency-shifted beams include laser excitation modules as described in the following publications: U.S. Patents Nos. 9,423,353, 9,784,661, and 10,006,852; and U.S. Patent Publications Nos. 2017 / 0133857 and 2017 / 0350803, the contents of which are incorporated herein by reference.

[0119] In some embodiments, the system includes a light detection system having one or more photodiodes. In some embodiments, the photodiode is an avalanche photodiode. In some embodiments, the light detection system includes multiple photodiodes, such as a photodiode array. In these embodiments, the photodiode array may include four or more photodiodes, such as 10 or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, 750 or more, and may include 1000 or more photodiodes.

[0120] The photodiodes can be arranged in any geometric configuration as needed, including but not limited to square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The photodiodes in the photodiode array may be oriented at an angle to another photodiode (as described in the XZ plane), the angle ranging from 10° to 180°, for example 15° to 170°, 20° to 160°, 25° to 150°, 30° to 120°, and including 45° to 90°. The photodiode array can be any suitable shape, and can be a shape enclosed by straight lines (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a shape enclosed by curves (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom connecting to a planar top). In some embodiments, the photodiode array has a rectangular active surface.

[0121] The active surface width of each photodiode in the array ranges from 5m to 250m, for example, 10m to 225m, 15m to 200m, 20m to 175m, 25m to 150m, 30m to 125m, and includes 50m to 100m; its length ranges from 5m to 250m, for example, 10m to 225m, 15m to 200m, 20m to 175m, 25m to 150m, 30m to 125m, and includes 50m to 100m; wherein the surface area of ​​each photodiode in the array ranges from 25m². 2 Up to 10000m 2 For example, 50m 2 up to 9000m 2 75m 2 up to 8000m 2 100m 2 up to 7000m 2 150m 2 up to 6000m 2 And including 200m 2 up to 5000m 2 .

[0122] The size of the photodiode array can vary depending on the amount and intensity of light, the number of photodiodes, and the required sensitivity, and its length ranges from 0.01 mm to 100 mm, for example, 0.05 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, 2 mm to 50 mm, 3 mm to 40 mm, 4 mm to 30 mm, and includes 5 mm to 25 mm. The width of the photodiode array may also vary, ranging from 0.01 mm to 100 mm, for example, 0.05 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, 2 mm to 50 mm, 3 mm to 40 mm, 4 mm to 30 mm, and includes 5 mm to 25 mm. Therefore, the surface area of ​​the active surface of the photodiode array ranges from 0.1 mm². 2 Up to 10000mm 2 For example, 0.5mm 2 Up to 5000mm 2 1mm 2 Up to 1000mm 2 5mm 2 Up to 500mm 2 And including 10mm 2 Up to 100mm 2 .

[0123] The target photodiode is configured to measure light collected at one or more wavelengths, such as two or more wavelengths, five or more different wavelengths, ten or more different wavelengths, such as 15 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 1500 or more, 2500 or more, and including 5000 or more different wavelengths. In some embodiments, the photodiode is configured to measure a spectrum, such as the spectrum including wavelengths spanning 50 nm or more, such as 100 nm or more, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, 1000 nm or more, and including 1500 nm or more. For example, a photodiode is configured to measure light in the wavelength range of 200 nm to 1500 nm (e.g., 400 nm to 1100 nm).

[0124] The light detection system is configured to measure light continuously or at discrete time intervals. In some cases, the target photodiode is configured to continuously measure the collected light. In other cases, the light detection system is configured to perform measurements at discrete time intervals, such as 0.001 ms, 0.01 ms, 0.1 ms, 1 ms, 10 ms, 100 ms, and including 1000 ms or some other time interval.

[0125] As described above, the light detection system also includes an amplifier assembly. In one embodiment, the amplifier assembly is configured to amplify the output signal from the photodiode in response to detected light. In some embodiments, the amplifier assembly includes a current-to-voltage converter, such as a transimpedance amplifier. In other embodiments, the amplifier assembly includes an operational amplifier circuit, such as a summing amplifier. In one embodiment, the output current from the photodiode is converted into a voltage, and in some cases, combined with the summing amplifier and transmitted to a processor for outputting a data signal.

[0126] Based on the number of photodiodes used in the optical detection system, the amplifier assembly may include two or more amplifiers, such as three or more amplifiers, four or more amplifiers, five or more amplifiers, six or more amplifiers, seven or more amplifiers, eight or more amplifiers, nine or more amplifiers, ten or more amplifiers, fifteen or more amplifiers, twenty-five or more amplifiers, fifty or more amplifiers, one hundred or more amplifiers, twenty-five or more amplifiers, twenty-five or more amplifiers, fifty or more amplifiers, seven hundred or more amplifiers, seven hundred or more amplifiers, and may include one hundred or more amplifiers. In some embodiments, the amplifier assembly includes two or more transimpedance amplifiers, such as three or more transimpedance amplifiers, four or more transimpedance amplifiers, five or more transimpedance amplifiers, six or more transimpedance amplifiers, seven or more transimpedance amplifiers, eight or more transimpedance amplifiers, nine or more transimpedance amplifiers, ten or more transimpedance amplifiers, fifteen or more transimpedance amplifiers, twenty-five or more transimpedance amplifiers, fifty or more transimpedance amplifiers, one hundred or more transimpedance amplifiers, twenty-five or more transimpedance amplifiers, fifty or more transimpedance amplifiers, seven hundred or more transimpedance amplifiers, or even one hundred or more transimpedance amplifiers. In other embodiments, the amplifier assembly includes two or more summing amplifiers, such as three or more summing amplifiers, four or more summing amplifiers, five or more summing amplifiers, six or more summing amplifiers, seven or more summing amplifiers, eight or more summing amplifiers, nine or more summing amplifiers, ten or more summing amplifiers, fifteen or more summing amplifiers, twenty-five or more summing amplifiers, fifty or more summing amplifiers, one hundred or more summing amplifiers, twenty-five or more summing amplifiers, fifty or more summing amplifiers, seven hundred or more summing amplifiers, and even one hundred or more summing amplifiers.

[0127] In some embodiments, the light detection system includes a number of amplifiers corresponding to the number of photodiodes. For example, the light detection system may include N photodiodes and N amplifiers, where N is an integer between 2 and 1024, N is an integer between 4 and 512, N is an integer between 8 and 256, and includes where N is an integer between 16 and 128. In some cases, N is 4 (i.e., the light detection system includes 4 photodiodes and 4 amplifiers). In other cases, N is 8. In other cases, N is 16. In other cases, N is 32. In other embodiments, the light detection system includes N photodiodes and 2N amplifiers, where N is an integer between 2 and 1024, N is an integer between 4 and 512, N is an integer between 8 and 256, and includes where N is an integer between 16 and 128. For example, the light detection system may include a transimpedance amplifier and a summing amplifier for each photodiode.

[0128] The amplifier assembly (e.g., a transimpedance amplifier) ​​is electrically connected to the plurality of photodiodes. In some embodiments, the amplifier assembly is directly electrically connected to the plurality of photodiodes (i.e., immediately downstream). In other embodiments, the amplifier assembly is electrically connected to the plurality of photodiodes via an electronic switch assembly. In some embodiments, the target light detection system includes a first amplifier assembly electrically positioned between the photodiodes and the electronic switch assembly and a second amplifier assembly electrically positioned downstream of the electronic switch. In some cases, the first amplifier assembly includes a plurality of transimpedance amplifiers configured to receive output signals from the photodiodes, while the second amplifier assembly includes a plurality of summing amplifiers configured to receive output signals from the electronic switch.

[0129] The amplifier assembly is electrically connected to each amplifier within the assembly, and each amplifier can be configured to receive electronic signals from one or more photodiodes. For example, when the photodiodes comprise N photodiodes, each amplifier in the amplifier assembly 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 other portions. In one example, the light detection system comprises 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 light detection system comprises 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 light detection system comprises 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 assembly can multiplex or inverse multiplex the electronic signal from the photodiode, and can transmit the multiplexed or inverse multiplexed electronic signal from the photodiode.

[0130] In some embodiments, the system is configured to adjust the sensitivity of photodiodes in the light detection system. In some embodiments, the system includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine the responsivity of the photodiodes at various wavelengths of light from the light source. As used herein, the term "responsivity" refers to the ratio between the photocurrent generated by the photodiode and the optical power of the incident light, according to conventional usage. In some embodiments, the memory includes instructions for determining the responsivity of each photodiode at 10 or more different wavelengths of light, such as 15 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 1500 or more, 2500 or more, and including 5000 or more different wavelengths of light. In some embodiments, the memory includes methods for determining responsivity in a spectrum, for example, where the spectrum includes wavelengths spanning 50 nm or more, such as 100 nm or more, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, 1000 nm or more, and including 1500 nm or more. For example, the memory includes methods for determining responsivity in a spectrum with wavelengths ranging from 200 nm to 1500 nm, for example, 400 nm to 1100 nm. In some cases, the system includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to graphically represent the responsivity of the photodiode within the light wavelength range to generate a wavelength responsivity curve for each photodiode in the photodetector system. In some embodiments, the memory includes instructions for determining the responsivity of the photodiode at a predetermined wavelength using the wavelength responsivity curve.

[0131] In some embodiments, the system includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine the average gain of the photodiode at the multiple wavelengths using the responsivity of the photodiode at the multiple wavelengths. In some cases, the memory includes instructions for individually determining the average gain of the photodiode at each wavelength. For example, in determining the responsivity of the photodiode in a wavelength spectrum (e.g., wavelength ranges of 200 nm to 1500 nm, 400 nm to 1100 nm), the memory includes instructions for determining the average gain of the photodiode at every 1 nm in the wavelength spectrum, such as every 2 nm, every 5 nm, every 10 nm, and including every 25 nm or some other interval. In other cases, the memory includes instructions for determining the average gain of the photodiode over a wavelength range, for example, the range being 1 nm or longer, 2 nm or longer, 5 nm or longer, 10 nm or longer, 25 nm or longer, 50 nm or longer, 100 nm or longer, 250 nm or longer, 500 nm or longer, and including 1000 nm or longer.

[0132] In some embodiments, the system includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to calculate the resistance of the amplifier based on the determined gain of the photodiode (e.g., the average gain of the amplifier across the multiple light wavelengths) and the determined responsivity. In some cases, the memory includes instructions for calculating the resistance of a feedback resistor used in the amplifier. In some embodiments, the memory includes instructions for calculating the resistance of the amplifier according to the following formula:

[0133] R f XR(λ)=G t

[0134] Where R f R(λ) is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; G t It is the average gain of the photodiode at the various light wavelengths.

[0135] In some embodiments, the system includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to adjust the capacitance of each amplifier based on the calculated resistance. In some embodiments, the memory includes instructions for adjusting the capacitance by 5% or more, such as 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, and including 95% or more.

[0136] In some cases, the memory includes instructions for adjusting 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 some cases, the memory includes instructions for adjusting the capacitance of the amplifier so that the photodiode produces substantially the same wavelength responsivity at the various wavelengths (e.g., in the wavelength spectrum). In other cases, the memory includes instructions for adjusting the capacitance of the amplifier so that the photodiode produces a desired responsivity at one or more predetermined detection wavelengths.

[0137] In some cases, the system includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to adjust the capacitance in a manner sufficient to generate a predetermined bandwidth for each photodiode. In other cases, the memory includes instructions for adjusting the capacitance of each amplifier according to the following formula to generate a predetermined bandwidth:

[0138]

[0139] Where BW is bandwidth; R f It is the resistance of the amplifier; C f It is the capacitance of the amplifier.

[0140] In some embodiments, the system includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to adjust the capacitance of the amplifier to obtain a predetermined bandwidth of 10 kHz or greater, such as 25 kHz or greater, 50 kHz or greater, 100 kHz or greater, 150 kHz or greater, 200 kHz or greater, 250 kHz or greater, 500 kHz or greater, and including a predetermined bandwidth of 1000 kHz or greater. For example, in the case where the average gain of the amplifier is 10... 6 In one example of volt / ampere, the memory includes a capacitor for adjusting the amplifier to obtain a predetermined bandwidth of 250 kHz or greater, such as 275 kHz or greater, 300 kHz or greater, 325 kHz or greater, 350 kHz or greater, 375 kHz or greater, 400 kHz or greater, 425 kHz or greater, 450 kHz or greater, 475 kHz or greater, and includes a predetermined bandwidth of 500 kHz or greater.

[0141] In some embodiments, the system includes a processor having a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to determine one or more parameters of a photodetector, wherein the memory includes instructions for irradiating particles in a fluid medium, wherein the particles (e.g., multispectral magnetic beads described below) comprise one or more fluorophores. In some cases, the memory includes instructions that, when executed by the processor, cause the processor to determine parameters of the photodetector, including relative fluorescence units (e.g., ABD units) specified for each photodetector, robust coefficient of variation (rCV) of one or more photodetectors, maximum and minimum linearity of each photodetector, relative change of rCV from a baseline, relative change of detector gain from a baseline, and imaging specifications of the photodetector (e.g., RF power or axial optical loss).

[0142] In some cases, the memory includes instructions for determining parameters of a photodetector, the parameter determination method comprising irradiating a fluid medium having particles (including one or more fluorophores) at a first intensity for a first predetermined time period and at a second intensity for a second predetermined time period; detecting light from the fluid medium using the photodetector (with a light source); generating a data signal through the photodetector at the first irradiation intensity and at the second irradiation intensity; and determining one or more parameters of the photodetector based on the data signals generated at the first and second intensities.

[0143] In some embodiments, the memory includes instructions for determining the average fluorescence intensity (M) of the particles under the first irradiation intensity and the second irradiation intensity. In some cases, the memory includes instructions for determining the variance (V(M)) of the average fluorescence intensity under the first irradiation intensity and the second irradiation intensity. In some cases, the memory includes instructions for determining the %rCV (robust coefficient of variation) of the photodetector. In some embodiments, the memory includes instructions that, when executed by the processor, cause the processor to calculate a linear fit of the variance according to the following formula:

[0144]

[0145] Q led The value derived from 1 / c1 refers to the statistical photoelectrons per unit average fluorescence intensity (M) (i.e., SPE / MFI). In some embodiments, the memory includes instructions for graphically displaying the variance to determine a linear fit to the variance according to the following formula:

[0146] y = c1x + c0

[0147] In an embodiment, the average fluorescence intensity and variance can be determined for a variety of different irradiation intensities, such as two or more irradiation intensities, for example three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, and including 15 or more different irradiation intensities.

[0148] In some embodiments, the memory includes instructions for determining the statistical photoelectrons (SPEs) at one or more irradiation intensities, for example, at least at a first irradiation intensity and a second irradiation intensity. In some cases, the memory includes instructions for calculating the detector efficiency (Q) of the photodetector for the particle based on the statistical photoelectrons per particle and the determined average fluorescence intensity. detIn some embodiments, the memory includes instructions for determining the detector efficiency of one or more detector channels in the photodetector based on the statistical photoelectrons of the particle and the determined average fluorescence intensity, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 20 or more, 24 or more, 36 or more, 48 or more, 72 or more, and includes instructions for determining the detector efficiency of 96 or more detector channels in the photodetector. In some cases, the memory includes instructions for determining the detector efficiency of all detector channels in the photodetector for each particle based on the statistical photoelectrons of each particle and the determined average fluorescence intensity. In some embodiments, the memory includes instructions that, when executed by the processor, cause the processor to determine the detector efficiency according to the following formula:

[0149]

[0150] Where SPE stands for Statistical Photoelectrons, MFI is the Average Fluorescence Intensity, and ABD is the unit allocated to each channel for each particle batch.

[0151] In some embodiments, the memory includes instructions for determining background signals for one or more photodetectors. In some cases, the background signals are determined under one or more irradiation intensities, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and include 10 or more different irradiation intensities. In some cases, the memory includes instructions for determining background signals under all applied irradiation intensities. In some embodiments, the memory includes instructions for determining the background signals in one or more detector channels of the photodetector, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 20 or more, 24 or more, 36 or more, 48 or more, 72 or more, and the memory includes instructions for determining the background signals in 96 or more detector channels of the photodetector. In some cases, the memory includes instructions for determining the background signal in all detector channels of the photodetector. In some cases, the memory includes instructions, when executed by the processor, that cause the processor to determine the background signal based on the statistical photoelectrons of the photodetector and the detector efficiency. In some cases, the memory includes instructions for determining the background signal according to the following formula:

[0152] B SD =B SD,MFI ×Q led

[0153]

[0154] In some embodiments, the memory includes instructions for determining the electronic noise of one or more photodetectors. In some cases, the electronic noise of the photodetector is determined under one or more irradiation intensities, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, and includes ten or more different irradiation intensities. In some cases, the electronic noise of the photodetector is determined under all applied irradiation intensities. Similarly, the electronic noise can be determined in one or more detector channels of the photodetector, such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, sixteen or more, twenty or more, twenty-four or more, thirty-six or more, four eight or more, seventy-two or more, and includes determining the electronic noise in 96 or more detector channels of the photodetector. In some cases, the memory includes instructions for determining the electronic noise in all detector channels of the photodetector. In some cases, the memory includes instructions for determining the electronic noise based on the statistical photoelectrons of the photodetector and the detector efficiency. In other cases, the memory includes instructions for determining the electronic noise according to the following formula:

[0155] EN SD =EN SD,MFI ×Q led

[0156]

[0157] In some embodiments, the memory includes instructions for determining the detection limits of one or more photodetectors. In some cases, the memory includes instructions for determining the detection limits of the photodetectors in one or more detector channels of the photodetectors, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 16 or more, 20 or more, 24 or more, 36 or more, 48 or more, 72 or more, and the memory includes instructions for determining the detection limits of the photodetectors in 96 or more detector channels of the photodetectors. In some cases, the memory includes instructions for determining the detection limits in all detector channels of the photodetectors. In some cases, the memory includes instructions for determining the detection limit of each photodetector according to the following formula:

[0158] 2 + 2SD = 4(1 + B)SD )

[0159] In some embodiments, the memory includes instructions for determining the detector photosensitivity of one or more photodetectors. In some embodiments, the memory includes instructions for setting an initial detector gain for the photodetector. In some cases, the memory includes instructions for performing the following operations: irradiating the photodetector with the light source (see above) at multiple different light intensities; generating data signals through the photodetector for the multiple light intensities at one or more detector gains; and determining the minimum light irradiance intensity at which a data signal resolvable from the background data signal can be generated at each detector gain. In some cases, the memory includes instructions for determining the minimum light irradiance intensity at which a data signal reduced by two standard deviations compared to the background data signal can be generated at each detector gain. In some cases, the memory includes instructions for setting the detector gain to achieve a plateau in the minimum light irradiance intensity that, when plotted as a function of light intensity, is capable of generating a data signal resolvable from the background data signal.

[0160] In some embodiments, the photodetector system having one or more photodiodes and amplifier assemblies as described above is part of or located within a particle analyzer (e.g., a particle sorter). In some embodiments, the target system is a flow cytometry system for detecting light emitted from a sample in a fluid medium, which includes the aforementioned photodiodes and amplifier assemblies (as part of the photodetector system). Suitable flow cytometry systems may include, but are not limited to, those described in the following publications: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods of Molecular Biology, Vol. 91, Humana Press (1997); Practical Flow Cytometry, 3rd Edition, Wiley-Liss (1995); Virgo et al. (2012), Annals of Clinical Biochemistry, Jan. 49 (pt 1): 17-28; Linden et al., Symposium on Thrombosis and Hemostasis, Oct. 2004; 30 (5): 502-11; Alison et al., Journal of Pathology, Dec. 2010; 222 (4): 335-344; and Herbig et al. (2007), Reviews of Therapeutic Drug Carrier Systems, 24 (3): 203-255; the contents of these publications are incorporated herein by reference. In some cases, the target flow cytometry system includes BD Biosciences FACSCanto TMII flow cytometer, BD Accuri TM Flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortess TM X-20 flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorting instrument, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorting instrument, BDBiosciences Aria TM Cell sorting instrument and BD Biosciences FACSMelody TM Cell sorting instruments, etc.

[0161] In some embodiments, the target particle analyzer system is a flow cytometry system, such as the system described in the following U.S. patents: 10,006,852; 9,952,076; 9,933,341; 9,784,661; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753,5 The contents of these documents are incorporated herein by reference in their entirety: 73; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039;

[0162] In some embodiments, the target system is a flow cytometry system with an excitation module that utilizes radio frequency multiplexed excitation to generate multiple frequency-shifted beams. In other cases, the target system is a flow cytometry system, the contents of which are described in the following publications: U.S. Patents Nos. 9,423,353 and 9,784,661; and U.S. Patent Publications Nos. 2017 / 0133857 and 2017 / 0350803, the contents of which are incorporated herein by reference.

[0163] In some embodiments, the target system is a particle sorting system configured to sort particles using a closed particle sorting module, for example, as described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the contents of which are incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) in a sample, for example, as described in U.S. Provisional Patent Application No. 62 / 803,264, filed February 8, 2019, the contents of which are incorporated herein by reference. In some embodiments, a method for sorting sample components includes sorting particles (e.g., cells in a biological sample) using a particle sorting module having deflection plates, for example, as described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the contents of which are incorporated herein by reference.

[0164] In some embodiments, the target system includes a particle analysis system that can be used for particle analysis and characterization, regardless of whether the particles are physically sorted into a collection container. Figure 4A A functional block diagram illustrating an example particle analysis system is shown. In some embodiments, particle analysis system 401 is a flow cytometry system. For example, Figure 4A The particle analysis system 401 shown can be configured to perform all or part of the methods described herein. The particle analysis system 401 includes a fluid system 402. The fluid system 402 may include, or be coupled to, a sample tube 405 and a moving liquid column within the sample tube, in which particles 403 (e.g., cells) in a sample move along a common sample path 409.

[0165] The particle analysis system 401 includes a detection system 404 configured to collect signals from each particle as it passes through one or more detection stations along the common sample path. Detection station 408 typically refers to a monitored area 407 of the common sample path. In some embodiments, detection may include detecting light or one or more other properties of the particle as it passes through the monitored area 407. Figure 4AThe image shows a monitoring station 408 with a monitored area 407. Some implementations of the particle analysis system 401 may include multiple monitoring stations. Furthermore, some monitoring stations may monitor more than one area.

[0166] Each signal is assigned a signal value to constitute a data point for each particle. As mentioned above, this data can be referred to as event data. The data points can be multi-dimensional data points, which include values ​​for each measured characteristic of the particle. The detection system 404 is configured to collect a series of said data points within a first time interval.

[0167] The particle analysis system 401 also includes a control system 306. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The control system is operationally associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on a Poisson distribution and the number of data points collected by the detection system 404 within the first time interval. The control system 406 may be further configured to generate an experimental signal frequency based on the number of data points within said portion of the first time interval. Furthermore, the control system 406 may compare the experimental signal frequency with a calculated signal frequency or a predetermined signal frequency.

[0168] Figure 4B A system 400 for flow cytometry according to an illustrative embodiment of the invention is shown. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collecting lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long-pass (“LP”) filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0169] The 115a-c laser is excited to emit light in the form of a laser beam. Figure 4B In the example system shown, the laser beams emitted from excitation lasers 415a-415c have wavelengths of 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first guided through one or more beam splitters 445a and 445b. Beam splitter 445a transmits light with a wavelength of 488 nm and reflects light with a wavelength of 633 nm. Beam splitter 445b transmits UV light (light with a wavelength range of 10 to 400 nm) and reflects light with wavelengths of 488 nm and 633 nm.

[0170] The laser beam is then directed to a focusing lens 420, which focuses the beam onto a portion of the fluid medium containing the sample particles within a flow chamber 425. The flow chamber is part of a fluid system that guides particles (typically one at a time in the flow) to the focused laser beam for interrogation. The flow chamber may comprise a flow cell in a benchtop cytometer or a nozzle head in an air-flow cytometer.

[0171] The light from the laser beam interacts with the particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various wavelengths depending on the characteristics of the particles (e.g., their size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particles). The fluorescence emission, as well as the diffracted, refracted, reflected, and scattered light, can be guided to one or more of the following: a beam splitter 445a-445g, a bandpass filter 450a-450e, a long-pass filter 455a-455b, and a fluorescence collecting lens 440. The light is directed to one or more of the following: a forward scattering detector 430, a side scattering detector 435, and one or more fluorescence detectors 460a-460f.

[0172] A fluorescence collecting lens 440 collects light emitted through particle-laser beam interaction and directs the light to one or more beam splitters and filters. Bandpass filters (e.g., bandpass filters 450a-450e) allow a narrow range of wavelengths to pass through. 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, on each side of the center of the spectral band, the 510 / 20 filter increases the wavelength by 10 nm, or increases the wavelength from 500 nm to 520 nm. Short-pass filters transmit light with wavelengths equal to or less than a specified wavelength. Long-pass filters (e.g., long-pass filters 455a-455b) transmit light with wavelengths equal to or longer than a specified wavelength. For example, long-pass filter 455a (a 670 nm long-pass filter) transmits light with wavelengths equal to or longer than 670 nm. Filters are typically selected to optimize the detector's specificity for a particular fluorescent dye. The filter can be configured such that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0173] 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 shorter and reflects light with wavelengths longer than 620 nm in different directions. In one embodiment, beam splitters 445a-445g may include optical mirrors, such as dichroic mirrors.

[0174] A forward scattering detector 430 is positioned slightly away from the direct beam passing through the flow cell and is configured to detect diffracted light, primarily forward-directed excitation light traveling through or around the particle. The intensity of the light detected by the forward scattering detector depends on the overall size of the particle. The forward scattering detector may include a photodiode. A side scattering detector 435 is configured to detect refracted and reflected light from the particle surface and internal structure, and its number tends to increase with increasing particle structural complexity. One or more fluorescence detectors 460a-460f can detect fluorescence emission from fluorescent molecules associated with the particle. The side scattering detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected at the forward scattering detector 430, the side scattering detector 435, and the fluorescence detector can be converted into electronic signals (voltages) by the detectors. This data can provide information about the sample.

[0175] Those skilled in the art will recognize that the flow cytometer according to embodiments of the present invention is not limited to Figure 4B The flow cytometer shown 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, which have various wavelengths and various different configurations.

[0176] During operation, the cytometer is controlled by a controller / processor 490, and measurement data from the detector can be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive the output signal therefrom, and may also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control the laser, fluid flow parameters, etc. Input / output (I / O) functionality 497 may also be provided in the system. Memory 495, controller / processor 490, and I / O 497 may be provided as integral components of the flow cytometer 410. In such embodiments, a display may also be part of the I / O functionality 497 for presenting experimental data to the user of the cytometer 400. Alternatively, some or all of memory 495 and controller / processor 490 and I / O functionality may be part of one or more external devices (e.g., a general-purpose computer). In some embodiments, some or all of memory 495 and controller / processor 490 may communicate wirelessly or wiredly with the cytometer 410. The controller / processor 490, together with the memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0177] Figure 4BThe system shown includes six different detectors capable of detecting fluorescence in six different wavelength bands (which may be referred to herein as “filter windows” for a given detector), as defined by the configuration of filters and / or separators in the beam path between flow cell 425 and each detector. Different fluorescent molecules used in flow cytometry experiments emit light within their own characteristic wavelength bands. The specific fluorescent label used for the experiment and its associated fluorescence emission band can be selected to generally correspond to the filter windows of the detectors. However, with more detectors and more labels, a perfect correspondence between filter windows and fluorescence emission spectra cannot be achieved. In fact, while the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of a particular detector, some emission spectra of the label may also overlap with the filter windows of one or more other detectors. This can be referred to as spillover. I / O 497 can be configured to receive data for flow cytometry experiments having a set of fluorescent labels and multiple cell populations (with multiple markers), each cell population having a subset of multiple markers. I / O 497 can also be configured to receive biological data (involving the assignment of one or more markers to one or more cell populations), marker density data, emission spectral data, data relating to the assignment of markers to one or more markers, and cytometer configuration data. Flow cytometry experimental data (e.g., marker spectral characteristics and flow cytometry configuration data) can also be stored in memory 495. Controller / processor 490 can be configured to evaluate the assignment of one or more markers to the markers.

[0178] Figure 5 A functional block diagram illustrating an example of a particle analyzer control system (e.g., analyzer controller 500) for analyzing and displaying biological events is shown. Analyzer controller 500 can be configured to implement various processes for controlling the graphical display of biological events.

[0179] The particle analyzer or sorting system 502 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 502 can be configured to provide the biological event data to an 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 said data communication channel.

[0180] Analysis controller 500 may be configured to receive bioevent data from particle analyzer or sorting system 502. The bioevent data received from particle analyzer or sorting system 502 may include flow cytometry event data. Analysis controller 500 may be configured to provide a graphical display including a first graph of the bioevent data to display device 506. For example, analysis controller 500 may be further configured to render target regions as gates around multiple bioevent data points shown by display device 506, overlaying the first graph. In some embodiments, the gates may be a logical combination of one or more target graphical regions plotted on a single-parameter histogram or bivariate graph. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0181] Alternatively, the analysis controller 500 may be further configured to display the biological event data on the display device 506 inside the door in a manner different from other events in the biological event data outside the door. For example, the analysis controller 500 may be configured to make the color of the biological event data contained inside the door different from the color of the biological event data outside the door. The display device 506 may be implemented in the form of a monitor, tablet computer, smartphone, or other electronic device configured to display a graphical interface.

[0182] The analysis controller 500 may be configured to receive a door selection signal from a first input device to identify the door. For example, the first input device may be implemented in the form of a mouse 510. The mouse 510 may issue a door selection signal to the analysis controller 500 to determine the door to be displayed on or manipulated via the display device 506 (e.g., clicking on or inside the desired door when the cursor is present). In some embodiments, the first device may be implemented in the form of a keyboard 508 or other means for providing input signals to the analysis controller 500 (e.g., a touchscreen, stylus, optical detector, or voice recognition system). Some input devices may include multiple input functions. In such embodiments, each of the input functions may be considered as an input device. For example, such as... Figure 5 As shown, the mouse 510 may include a right mouse button and a left mouse button, both of which can generate trigger events.

[0183] The triggering event may cause the analysis controller 500 to change the way the data is displayed (actually displaying a portion of the data on the display device 506), and / or provide input for further processing, such as selecting a target population for particle sorting.

[0184] In some embodiments, the analysis controller 500 may be configured to detect the time when the mouse 510 initiates a gate selection. The analysis controller 500 may be further configured to automatically modify the graph visualization to facilitate the gate selection process. The modification may be based on a specific distribution of the biological event data received by the analysis controller 500.

[0185] The analysis controller 500 may be connected to a storage device 504. The storage device 504 may be configured to receive and store biological event data from the analysis controller 500. The storage device 504 may also be configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 may also be configured to allow the analysis controller 500 to retrieve biological event data, such as flow cytometry event data.

[0186] Display device 506 may be configured to receive display data from analysis controller 500. The display data may include a biological event data graph and gates summarizing portions of the graph. Display device 506 may be further configured to change the displayed information based on input received from analysis controller 500 and input received from particle analyzer 502, storage device 504, keyboard 508, and / or mouse 510.

[0187] In some implementations, the analysis controller 500 may generate a user interface to receive example events for sorting. For example, the user interface may include controls for receiving example events or example images. Example events, images, or example gates may be provided before acquiring event data for the sample or based on an initial set of events for a portion of the sample.

[0188] In some embodiments, the target system includes a particle sorting system. Figure 6A This is a schematic diagram of a particle sorting system 600 (e.g., a particle analyzer or sorting system 502) according to one embodiment shown herein. In some embodiments, the particle sorting system 600 is a cell sorting system. Figure 6A As shown, a droplet formation sensor 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601 (which may be coupled to, or may include, a nozzle 603). Within the fluid conduit 601, a sheath fluid 604 hydrodynamically concentrates a sample fluid 606 (containing particles 609) into a moving liquid column 608 (e.g., a flow). Within the moving liquid column 608, the particles 609 (e.g., cells) are aligned to pass through a monitored area 611 (e.g., a laser flow intersection) irradiated by an irradiation source 612 (e.g., a laser). Vibration of the droplet formation sensor 602 causes the moving liquid column 608 to break into multiple droplets 610, some of which contain particles 609.

[0189] During operation, detection station 614 (e.g., an event detector) determines when a target particle (or target cell) crosses the monitored area 611. Detection station 614 feeds into timing circuit 628, which in turn feeds into instantaneous charging circuit 630. At the droplet breakup point, after a timed droplet delay (Δt) notification, an instantaneous charge can be applied to the moving liquid column 608 to charge the target droplet. The target droplet may include one or more particles or cells to be sorted. The charged droplet can then be sorted by enabling a deflection plate (not shown), deflecting the droplet into a container such as a collection tube or a porous or microporous sample plate, where the pores or micropores can be associated with a specific target droplet. Figure 6A As shown, the droplets can be collected in the discharge container 638.

[0190] A detection system 616 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a target particle passes through the monitored region 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 616 allows the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may be fed an amplitude signal 620 and / or a phase signal 618, which are then sequentially (via amplifier 622) fed into an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624, in turn, control the droplet formation sensor 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be included in a control system.

[0191] In some embodiments, sorting electronics (e.g., detection system 616, detection station 614, processor 640) may be coupled to a memory configured to store detected events and sorting decisions based thereon. The sorting decisions may be included in the event data of the particles. In some embodiments, detection system 616 and detection station 614 may be implemented as a single detection unit or communicatively coupled such that event measurements can be collected by one of detection system 616 or detection station 614 and provided to the non-collecting element.

[0192] Figure 6B This is a schematic diagram of a particle sorting system according to one embodiment shown herein. Figure 6B The particle sorting system 600 shown includes deflection plates 652 and 654. Charge is applied via a current-charged wire in a barb. This produces a droplet stream 610 containing particles 610 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to produce light scattering and generate fluorescence information. This particle information is then processed by sorting electronics or other detection systems. Figure 6B(Not shown in the image) for analysis. Deflection plates 652 and 654 can be independently controlled to attract or repel the charged droplets, guiding them to a destination collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, deflector plates 652 and 654 can be controlled to guide particles toward container 674 along a first path 662 or toward container 678 along a second path 668. If the particles are not target particles (e.g., within a specified sorting range, no scattering or irradiation information is displayed), the deflector plates can allow the particles to continue flowing along flow path 664. Such uncharged droplets can be introduced into the waste container via a device such as a suction device 670.

[0193] The sorting electronics may include the sorting electronics to begin collecting measurements, receive fluorescence signals from the particles, and determine how to adjust the deflection plate to sort the particles. Figure 6B The example implementation of the embodiments shown includes the BD FACSAria™ series flow cytometer, commercially available from Becton, Dickinson and Company (Franklin Lake, New Jersey).

[0194] Computer control system

[0195] The invention further includes a computer control system, wherein the system further includes one or more computers for achieving full or partial automation. In some embodiments, the system includes a computer having a computer-readable storage medium storing a computer program, wherein the computer program includes instructions, when loaded onto the computer, to perform the following operations: detect light using a photodiode electrically connected to an amplifier; determine the responsivity of the photodiode at various light wavelengths; and adjust one or more parameters of the amplifier for the responsivity of the photodiode at the various light wavelengths. In some embodiments, the computer program includes instructions for determining the responsivity of the photodiode at the wavelength spectrum of the light. In some embodiments, the computer program includes instructions for determining the average gain of the photodiode at the various wavelengths. In some cases, the computer program includes instructions for calculating the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at the various light wavelengths. In some cases, the computer program includes instructions for calculating the resistance of the amplifier according to the following formula.

[0196] R f XR(λ)=G t

[0197] Where R fR(λ) is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; G t This is the average gain of the photodiode at the various light wavelengths. In some embodiments, the computer program includes instructions for adjusting the capacitance of each amplifier based on the calculated resistance. In some cases, the computer program includes instructions for adjusting 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 some cases, the computer program includes instructions for adjusting the capacitance of the amplifier so that the photodiode produces substantially the same wavelength responsivity at the various wavelengths (e.g., in the wavelength spectrum). In other cases, the computer program includes instructions for adjusting the capacitance of the amplifier so that the photodiode produces a desired responsivity at one or more predetermined detection wavelengths.

[0198] In some cases, the computer program includes instructions for adjusting the capacitance in a manner sufficient to generate a predetermined bandwidth for each photodiode. In other cases, the computer program includes instructions for adjusting the capacitance of each amplifier according to the following formula to generate a predetermined bandwidth:

[0199]

[0200] Where BW is bandwidth; R f It is the resistance of the amplifier; C f It is the capacitance of the amplifier.

[0201] In an embodiment, the system includes an input module, a processing module, and an output module. The target system may include hardware and software components, wherein the hardware components may be one or more platforms, such as servers, that enable the system's functional elements (i.e., elements in the system that perform specific tasks (e.g., managing the input and output of information, processing information, etc.)) to function by executing software applications on one or more computer platforms on which the system is equipped.

[0202] The system may include a display and an operator input device. The operator input device may be a keyboard, mouse, etc. The processing module includes a processor that can access memory having instructions stored thereon for performing the target method steps. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or one of other processors that are already available or will soon be available. As is known in the art, the processor executes an operating system, which is connected to firmware and hardware in a well-known manner and helps the processor coordinate and execute the functions of various computer programs written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof. The operating system typically works with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, communication control, and related services according to known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that allow a user to manually align the light source with the fluid medium based on the first and second optical signals. In some embodiments, the processor includes analog electronics that provide feedback control (e.g., negative feedback control).

[0203] The system memory can be any of a variety of known or future memory storage devices. Examples include any generally available random access memory (RAM), magnetic media (e.g., resident hard disks or magnetic tapes), optical media (e.g., optical discs), flash memory devices, or other memory storage devices. The memory storage device can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, removable hard disk drives, or floppy disk drives. Memory storage devices of this type typically read content from and / or write content to program storage media (not shown), including optical discs, magnetic tapes, removable hard disks, or floppy disks. Any of these program storage media, or other media currently in use or that may be developed in the future, can be considered a computer program product. It is understood that these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0204] In some embodiments, a computer program product is described that includes a computer-usable medium storing control logic (computer software program, including program code). When executed by a computer processor, the control logic enables the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using a hardware state machine. Enabling a hardware state machine to perform the functions described herein will be apparent to those skilled in the art.

[0205] The memory can be any suitable device in which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including disks, optical discs, magnetic tapes, or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor that has been appropriately programmed based on a computer-readable medium carrying the necessary program code. The programmed information can be provided to the processor remotely via a communication channel or pre-stored in a computer program product using any device connected to the memory, such as memory or certain other portable or fixed computer-readable storage media. For example, a disk or optical disc can carry the programmed information and can be read using a disk writer / reader. The system of the present invention also includes a programmed information for implementing the methods described above, such as a computer program product or algorithm. The programmed information according to the present invention can be recorded in a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROMs; portable flash drives; and hybrids of these categories, such as magnetic / optical storage media.

[0206] The processor can also access communication channels to communicate with users located in remote locations. A remote location refers to a location where the user has no direct contact with the system but instead forwards input information from external devices (e.g., connected to a wide area network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including mobile phones (i.e., smartphones)) to the input manager.

[0207] In some embodiments, the system according to the invention can be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface can be configured for wired or wireless communication, including but not limited to: radio frequency (RF) communication (e.g., radio frequency identification (RFID), Zigbee communication protocol, WiFi, infrared communication, wireless universal serial bus (USB), ultra-wideband (UWB)). Communication protocols and cellular communications, such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM).

[0208] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces (e.g., USB ports, RS-232 ports) or any other suitable electrical connection ports, to enable data communication between the target system and any external device (e.g., a computer terminal configured to enable similar complementary data communication (e.g., in a physician's office or hospital environment)).

[0209] In one embodiment, the communication interface is configured for infrared communication. Communication or any other suitable wireless communication protocol that enables the target system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user can use in conjunction with them.

[0210] In one embodiment, the communication interface is configured to provide data transmission connectivity via mobile phone networks or SMS service using Internet Protocol (IP); to provide wireless connectivity to personal computers (PCs) within a local area network (LAN) connected to the Internet; or to provide WiFi connectivity to a WiFi hotspot for connecting to the Internet.

[0211] In one embodiment, the target system is configured to wirelessly communicate with a server device via a communication interface, for example, using a common standard such as 802.11 or... The server device may be an RF protocol or an IrDA infrared protocol. It can also be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop; or a larger device, such as a desktop computer, instrument, etc. In some embodiments, the server device includes a display (e.g., a liquid crystal display (LCD)) and input devices (e.g., buttons, keyboard, mouse, or touchscreen).

[0212] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the target system (e.g., stored in an optional data storage unit) with a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0213] The output controller may include a controller for any of a variety of known display devices to provide information to local or remote users (whether human or machine). If one of the display devices provides visual information, the information may typically be logically and / or physically organized into an array of image elements. The graphical user interface (GUI) controller may include any of a variety of known or future-developed software programs to provide a graphical input and output interface between the system and the user, and to process user input. Functional elements of the computer may communicate with each other via a system bus. Some of this communication may be implemented using a network or other types of remote communication in alternative embodiments. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite networks, according to known technologies. The output manager may implement data display according to a variety of known technologies. In some examples, the data may include SQL, HTML, or XML documents, emails, or other files or other forms of data. The data may include Internet URLs so that the user can retrieve other SQL, HTML, XML, or other documents or data from a remote source. One or more platforms in the target system may be any type of known or future-developed computer platform, although they typically belong to a certain class of computers (often referred to as servers). However, the platform can also be a mainframe computer, workstation, or other computer type. They can be connected via any known or future-to-be-developed cable or other communication system (including wireless systems connected by networking or other means). They can be located in the same location or physically separated. Various operating systems can be used on any computer platform, depending on the type and / or brand of the chosen platform. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, etc.

[0214] Figure 7 The overall architecture of an example computing device 800 according to certain embodiments is described. Figure 7 The overall architecture of the computing device 700 shown includes the arrangement of computer hardware and software components. The computing device 700 may include, compared to... Figure 7The components shown may include more (or fewer) components. However, it is not necessary to show all such conventional components when providing implementation disclosure. As shown, computing device 700 includes processing unit 710, network interface 720, computer-readable media drive 730, input / output device interface 740, display 750, and input device 760, all of which can communicate with another device via a communication bus. Network interface 720 can be connected to one or more networks or computing systems. Processing unit 710 can therefore receive information and instructions from other computing systems or services via the network. Processing unit 710 can also communicate with memory 770 and can further provide output information to optional display 750 via input / output device interface 740. Input / output device interface 740 can also accept input from optional input device 760, such as keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope, or other input devices.

[0215] Memory 770 may contain computer program instructions (grouped into modules or components in some embodiments) executed by processing unit 710 to implement one or more embodiments. Memory 770 typically includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store operating system 772, which provides computer program instructions for use by processing unit 710 in the routine management and operation of computing device 700. Memory 770 may further include computer program instructions and other information for implementing aspects of the invention. A non-transitory computer-readable storage medium for adjusting the sensitivity of photodiodes in a light detection system.

[0216] The invention further includes a non-transitory computer-readable storage medium having instructions for implementing the method herein. The computer-readable storage medium can be employed on one or more computers, enabling a system for implementing the methods described herein to be fully or partially automated. In some embodiments, instructions according to the methods described herein can be encoded in a “programmed” form into a computer-readable medium, wherein the term “computer-readable medium” as used herein refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network-attached storage devices (NAS), whether such devices are internal or external to a computer. Files containing information can be “stored” on a computer-readable medium, where “stored” means recording information so that a computer can later access and retrieve it. The computer-implemented methods described herein can be implemented using programming capable of being written in one or more of any number of computer programming languages. For example, such languages ​​include Java (Sun Microsystems, Inc., Santa Clara, California), Visual Basic (Microsoft Corp., Redmond, Washington), and C++ (AT&T Corp., Bedminster, New Jersey), as well as many other languages.

[0217] In some embodiments, the intended computer-readable storage medium includes a computer program stored thereon, wherein the computer program includes instructions, when loaded onto the computer, to have an algorithm for performing the following operations: detecting light with a photodiode electrically connected to an amplifier; determining the responsivity of the photodiode at a variety of light wavelengths; and adjusting one or more parameters of the amplifier for the responsivity of the photodiode at the variety of light wavelengths.

[0218] In some embodiments, the intended computer-readable storage medium includes a computer program stored thereon, wherein the computer program includes instructions, when loaded onto the computer, for an algorithm to determine the responsivity of the photodiode at various wavelengths of light. In some embodiments, the computer program includes an algorithm for determining the average gain of the photodiode at various wavelengths. In some cases, the computer program includes an algorithm for calculating the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at various wavelengths of light. In some cases, the computer program includes an algorithm for calculating the resistance of the amplifier according to the following formula.

[0219] R fXR(λ)=G t

[0220] Where R f R(λ) is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; G t This is the average gain of the photodiode across the various light wavelengths. 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 some cases, the computer program includes an algorithm for adjusting the capacitance of the amplifier so that the photodiode produces substantially the same wavelength responsivity across the various wavelengths (e.g., in the wavelength spectrum). In other cases, the computer program includes an algorithm for adjusting the capacitance of the amplifier so that the photodiode produces a desired responsivity at one or more predetermined detection wavelengths.

[0221] In some cases, the computer program includes an algorithm for adjusting the capacitance in a manner sufficient to generate a predetermined bandwidth for each photodiode. In other cases, the computer program includes an algorithm for adjusting the capacitance of each amplifier according to the following formula to generate a predetermined bandwidth:

[0222]

[0223] Where BW is bandwidth; R f It is the resistance of the amplifier; C f It is the capacitance of the amplifier.

[0224] The non-transitory computer-readable storage medium can be used in one or more computer systems having displays and operator input devices. Operator input devices may be keyboards, mice, etc. The processing module includes a processor that can access memory having instructions stored thereon for performing the target method steps. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or one of other processors that are already available or will soon be available. As is known in the art, the processor executes an operating system, which is connected to firmware and hardware in a well-known manner and helps the processor coordinate and execute the functions of various computer programs written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof. The operating system typically cooperates with the processor to coordinate and execute the functions of other computer components. The operating system also provides scheduling, input / output control, file and data management, memory management, communication control, and related services according to known techniques.

[0225] Multispectral fluorescent particles

[0226] As described above, aspects of the invention also include particles (e.g., magnetic beads) having one or more fluorophores for carrying out certain methods described herein. According to some embodiments, the target particles may include single-peak multifluorophore magnetic beads that provide a bright photodetector signal at all light source wavelengths (e.g., all LEDs or lasers of the system) and the detection wavelength of the photodetector.

[0227] In an embodiment, target particles are prepared (e.g., in a fluid composition) and allowed to flow in a fluid medium irradiated by a light source as described above. Each particle may have one or more different types of fluorophores, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more different types of fluorophores. For example, each particle may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different types of fluorophores.

[0228] In some embodiments, each fluorophore is stably bound to the particle. Stable binding means that the fluorophore does not easily detach from the particle before contact with a liquid medium (e.g., an aqueous medium). In some embodiments, one or more fluorophores are covalently bound to the particle. In other embodiments, one or more fluorophores are physically bound to the particle (i.e., non-covalently coupled). In still other embodiments, one or more fluorophores are covalently bound to the particle, and one or more fluorophores are physically bound to the particle.

[0229] In some embodiments, each particle comprises two or more different types of fluorophores. Any two fluorophores are considered different and distinguishable if they differ from each other in one or more of the following: molecular formula, excitation maxima, and emission maxima. Therefore, different or distinguishable fluorophores may differ from each other in chemical composition or in one or more of the properties of the fluorophores. For example, different fluorophores may differ from each other in at least one of the excitation maxima and emission maxima. In some cases, different fluorophores differ from each other in their excitation maxima. In some cases, different fluorophores differ from each other in their emission maxima. In some cases, different fluorophores differ from each other in both their excitation maxima and emission maxima. Therefore, in embodiments including first and second fluorophores, the first and second fluorophores may differ from each other in at least one of the excitation maxima and emission maxima. For example, the first and second fluorophores may differ from each other in excitation maxima, emission maxima, or both excitation and emission maxima. If a given set of fluorophores differs from each other in terms of excitation or emission maxima, they can be considered distinct, wherein the magnitude of such difference is 5 nm or greater in some cases, such as 10 nm or greater, including 15 nm or greater, and wherein in some cases, the magnitude of the difference ranges from 5 to 400 nm, such as 10 to 200 nm, including 15 to 100 nm, such as 25 to 50 nm.

[0230] The excitation maxima of the target fluorophore according to some embodiments range from 100 nm to 800 nm, for example, 150 nm to 750 nm, 200 nm to 700 nm, 250 nm to 650 nm, 300 nm to 600 nm, and includes 400 nm to 500 nm. The emission maxima of the target fluorophore according to some embodiments range from 400 nm to 1000 nm, for example, 450 nm to 950 nm, 500 nm to 900 nm, 550 nm to 850 nm, and includes 600 nm to 800 nm. In some cases, the fluorophore is a luminescent dye, such as a fluorescent dye with an emission peak wavelength of 200 nm or longer, including 250 nm or longer, 300 nm or longer, 350 nm or longer, 400 nm or longer, 450 nm or longer, 500 nm or longer, 550 nm or longer, 600 nm or longer, 650 nm or longer, 700 nm or longer, 750 nm or longer, 800 nm or longer, 850 nm or longer, 900 nm or longer, 950 nm or longer, 1000 nm or longer, and including 1050 nm or longer. For example, the fluorophore can be a fluorescent dye with an emission peak wavelength range of 200 nm to 1200 nm, such as 300 nm to 1100 nm, 400 nm to 1000 nm, 500 nm to 900 nm, including fluorescent dyes with an emission peak wavelength range of 600 nm to 800 nm. In some embodiments, the target multispectral particles are stably excited by a laser irradiated at wavelengths of approximately 349 nm (UV laser), 488 nm (blue laser), 532 nm (Nd:YAG solid-state laser), 640 nm (red laser), and 405 nm (violet laser). In other cases, the target multispectral particles are stably excited by a light source across the full spectrum of detection (e.g., 350 nm to 850 nm).

[0231] The target fluorophore may include, but is not limited to, bodipy dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazo dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, anthocyanin dyes, asymmetric anthocyanin dyes, quinone imine dyes, azazine dyes, diaminoacrazine dyes, safranin dyes, indole, indophenol dyes, fluorine dyes, oxazine dyes, oxazolone dyes, thiazine dyes, thiazolium dyes, xanthan dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, squaric acid cyanine, bodipys, squaric acid cyanine rotaxane, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethylene or tetrapyrrole and combinations thereof. In some embodiments, the conjugate may include two or more dyes, such as two or more dyes selected from the following: bodipy dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazo dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone imine dyes, azazine dyes, diaminoacrazine dyes, safranin dyes, indole, indophenol dyes, fluorine dyes, oxazine dyes, oxazolone dyes, thiazine dyes, thiazolium dyes, xanthonium dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, squaric acid cyanine, bodipys, squaric acid cyanine rotaxane, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethylene or tetrapyrrole, and combinations thereof.

[0232] In some embodiments, the target fluorophore may include, but is not limited to, fluorescein isothiocyanate (FITC), phycoerythrin (PE) dye, polydinophyte chlorophyll-cyanin dye (e.g., PerCP-Cy5.5), phycoerythrin-cyanin (PE-Cy) dye (PE-Cy7), allophycocyanin (APC) dye (e.g., APC-R700), allophycocyanin-cyanin dye (e.g., APC-Cy7), and coumarin dye (e.g., V450 or V500). In some cases, the fluorophore may include one or more of the following: 1,4-bis-(o-methylstyryl)-benzene (bis-MSB 1,4-bis[2-(2-methylphenyl)vinyl]-benzene), C510 dye, C6 dye, Nile Red dye, T614 dye (e.g., N-[7-(methanesulfonyl)-4-oxo-6-phenoxybenzopyran-3-yl]carboxamide), LDS821 dye ((2-(6-(p-dimethylaminophenyl)-2,4-neopentenyl-1,3,5-hextrienyl)-3-ethylbenzothiazolyl perchlorate), and mFluor dye (e.g., mFluor Red dye, such as mFluor 780NS).

[0233] The particles can be of any suitable shape and are irradiated by the light source as described above. In some cases, the particles are solid supports, and their shape or structure is disc-shaped, spherical, oval, cubic, blocky, conical, etc., as well as irregular shapes. The mass of the particles can vary, and in some cases, the mass ranges from 0.01 mg to 20 mg, for example, 0.05 mg to 19.5 mg, 0.1 mg to 19 mg, 0.5 mg to 18.5 mg, 1 mg to 18 mg, 1.5 mg to 17.5 mg, 2 mg to 15 mg, and includes 3 mg to 10 mg. The surface area of ​​the particles can be 0.01 mm. 2 Or larger, for example, 0.05mm 2 Or larger, 0.1mm 2 Or larger, 0.5mm 2 or larger, 1mm 2 or larger, 1.5mm 2 Or larger, 2mm 2 Or larger, 2.5mm 2 Or larger, 3mm 2 Or larger, 3.5mm 2 Or larger, 4mm 2 Or larger, 4.5mm 2 Or larger, and including 5mm 2 Or larger, for example, the surface area determined using a Vertex system or an equivalent system.

[0234] The size of the particles can also vary as needed. In some cases, the longest particle size ranges from 0.01 mm to 10 mm, for example, 0.05 mm to 9.5 mm, 0.1 mm to 9 mm, 0.5 mm to 8.5 mm, 1 mm to 8 mm, 1.5 mm to 7.5 mm, 2 mm to 7 mm, 2.5 mm to 6.5 mm, and includes 3 mm to 6 mm. In other cases, the shortest particle size ranges from 0.01 mm to 5 mm, for example, 0.05 mm to 4.5 mm, 0.1 mm to 4 mm, 0.5 mm to 3.5 mm, and includes 1 mm to 3 mm.

[0235] In some cases, the target particles are porous particles, for example, the porosity of said particles ranges from 5 μ to 100 μ, such as 10 μ to 90 μ, 15 ​​μ to 85 μ, 20 μ to 80 μ, 25 μ to 75 μ, and includes 30 μ to 70 μ, such as 50 μ, for example, the porosity determined using a capillary flow pore size analyzer or equivalent.

[0236] The particles can be composed of any suitable material. In some embodiments, particles with low or no autofluorescence, such as magnetic beads, are of interest. Suitable materials include, but are not limited to, glass materials (e.g., silicates), ceramic materials (e.g., calcium phosphate), metallic materials, and polymeric materials such as polyethylene, polypropylene, polytetrafluoroethylene, and polyvinylidene fluoride. In some cases, the particles are composed of a solid support, such as the porous matrix described in U.S. Patent Application Publication No. 9,797,899, the contents of which are incorporated herein by reference. Thus, the surface area of ​​the particles can be any suitable macroporous or microporous substrate, wherein suitable macroporous and microporous substrates include, but are not limited to, ceramic matrices, glass frits (e.g., sintered glass), polymeric matrices, and organometallic polymeric matrices. In some embodiments, the porous matrix is ​​a glass frit. As used herein, the term "glass frit" in its conventional sense refers to a porous composition composed of sintered granular solids such as glass. The glass frit may have a chemical composition that varies depending on the type of sintered particles used to prepare the glass frit. Possible glass frits include, but are not limited to, glass frits composed of: aluminosilicate, boron trioxide, borophosphosilicate glass, borosilicate glass, ceramic glaze, cobalt glass, amber glass, fluorophosphate glass, fluorosilicate glass, fused silica, germanium dioxide, metal and sulfide-embedded borosilicate, lead glass, phosphate glass, phosphorus pentoxide glass, phosphosilicate glass, potassium silicate, soda-lime glass, sodium hexametaphosphate glass, sodium silicate, tellurite glass, uranium glass, micromirror glass, and combinations thereof. In some embodiments, the porous matrix is ​​a glass frit, such as borosilicate, aluminosilicate, fluorosilicate, potassium silicate, or borophosphosilicate glass frit.

[0237] In some embodiments, the particles are composed of a porous organic polymer. The target porous organic polymer varies depending on the sample volume, the components in the sample, and the assay reagents present, and may include, but is not limited to, porous polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), ethyl vinyl acetate (EVA), polycarbonate, polycarbonate alloys, polyurethane, polyethersulfone, copolymers, and combinations thereof. For example, the desired porous polymers include homopolymers, heteropolymers, and copolymers composed of: monomer units such as styrene; monoalkyleneallyl monomers such as ethylstyrene, α-methylstyrene, vinyltoluene, and vinylethylbenzene; (meth)acrylates such as methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, isodecyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, cyclohexyl (meth)acrylate, and benzyl (meth)acrylate; chlorinated monomers such as vinyl chloride, vinylidene chloride, and chloromethylstyrene; acrylonitrile compounds such as acrylonitrile and methacrylonitrile; and vinyl acetate, vinyl propionate, n-octadecylacrylamide, ethylene, propylene, and butane, and combinations thereof.

[0238] In some embodiments, the particles are composed of a metal-organic polymer matrix, such as an organic polymer matrix having a framework structure containing metals 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 composed of: methyltrimethoxysilane, dimethyldimethoxysilane, tetraethoxysilane, methacryloxypropyltrimethoxysilane, bis(triethoxysilyl)ethane, bis(triethoxysilyl)butane, bis(triethoxysilyl)pentane, bis(triethoxysilyl)hexane, bis(triethoxysilyl)heptane, bis(triethoxysilyl)octane, and combinations thereof.

[0239] kit

[0240] The invention further includes a kit, wherein the kit comprises one or more components of the optical detection system described herein. In some embodiments, the kit includes a photodiode array, an amplifier assembly, and instructions for programming the target system, for example, in the form of a computer-readable medium (e.g., a flash drive, USB memory, optical disc, DVD, Blu-ray disc, etc.) or for downloading the programming from the Internet Web Protocol or a cloud server. The kit may include optical adjustment components, such as lenses, mirrors, filters, optical fibers, wavelength splitters, pinholes, slits, collimation schemes, and combinations thereof.

[0241] The kit may further include instructions for implementing the target method. These instructions may exist in various forms within the target kit, including one or more. One form of these instructions may be printed information on a suitable medium or substrate (e.g., a sheet or several sheets of paper with information printed on it), kit packaging, packaging instructions, etc. Another form of these instructions may be computer-readable media on which information is already recorded, such as floppy disks, optical discs (CDs), portable flash drives, etc. Yet another form of these instructions may be a URL, allowing access to information on a remote website via the Internet.

[0242] utility

[0243] The proposed methods, systems, and computer systems can be used in a variety of applications requiring calibration or optimization of photodetectors (e.g., photodetectors in particle analyzers). The proposed methods and systems can also be used in photodetectors used to analyze and sort particulate components in samples (e.g., biological samples) within a fluid medium. The invention can also be used in flow cytometry, where a flow cytometer is required to meet the following requirements: improved cell sorting accuracy; enhanced particle collection capacity; reduced energy consumption; improved particle charging efficiency; more accurate particle charging; and enhanced particle deflection during cell sorting. In embodiments, the invention reduces the need for user input or manual adjustments during sample analysis using a flow cytometer. In some embodiments, the proposed methods and systems provide a fully automated process, requiring virtually no adjustments to the flow cytometer during use, or manual input if necessary.

[0244] The aspects of the aforementioned subject matter (including embodiments) may be used alone or in combination with one or more aspects or embodiments. Without limiting the scope of this document, certain non-limiting aspects of claims 1-95 are provided below. Upon reading this invention, the following will be apparent to those skilled in the art: each individually numbered item may be used alone or in combination with any preceding or following individually numbered item. This is intended to support all such combinations of the items and is not limited to combinations of the items explicitly provided below:

[0245] 1. A method for adjusting the sensitivity of an optical detection system in a particle analyzer, the method comprising:

[0246] Light is detected using a photodetector system in a particle analyzer, the photodetector system comprising a photodiode and an amplifier; the responsivity of the photodiode at various light wavelengths is determined; and...

[0247] Adjust one or more parameters of the amplifier to adjust the responsivity of the photodiode at the various light wavelengths.

[0248] 2. The method according to 1, wherein the particle analyzer is incorporated into a flow cytometer.

[0249] 3. The method according to any one of 1-2, wherein the photodiode is located in the particle analyzer for detecting light from particles in the fluid medium.

[0250] 4. The method according to 1, wherein the method comprises determining the responsivity of the photodiode in the wavelength spectrum of the light.

[0251] 5. The method according to 4, wherein the spectrum comprises light of 200 or more wavelengths.

[0252] 6. The method according to any one of 1-5, wherein the method comprises determining the responsivity of the photodiode in the range of 400 nm to 1100 nm.

[0253] 7. The method according to any one of 1-6, wherein the method comprises determining the average gain of the photodiode at the plurality of wavelengths.

[0254] 8. The method according to 7, wherein the method further comprises calculating the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at the multiple light wavelengths.

[0255] 9. The method according to 8, wherein the resistance of the amplifier is calculated according to the following formula:

[0256] R f XR(λ)=G t

[0257] Where R f It is the resistance of the amplifier;

[0258] R(λ) is the responsivity of the photodiode at each wavelength; and

[0259] G t It is the average gain of the photodiode at the various light wavelengths.

[0260] 10. The method according to any one of 8-9, wherein the method further comprises adjusting the capacitance of the amplifier based on the calculated resistance.

[0261] 11. The method according to 10, wherein the capacitance of the amplifier is adjusted in a manner sufficient to generate a predetermined bandwidth for the photodiode.

[0262] 12. The method according to 11, wherein the capacitance of the amplifier is adjusted according to the following formula to generate a predetermined bandwidth:

[0263]

[0264] Where BW is bandwidth; and

[0265] C f It is the capacitance of the amplifier.

[0266] 13. The method according to any one of 1-12, wherein the amplifier is a transimpedance amplifier.

[0267] 14. The method according to any one of 1-13, wherein the light detection system comprises:

[0268] A photodiode array containing multiple photodiodes; and

[0269] Multiple amplifiers

[0270] Each photodiode is electrically connected to the amplifier.

[0271] 15. The method of 14, wherein the method comprises determining the responsivity of each photodiode in the photodiode array at the plurality of light wavelengths.

[0272] 16. The method of 11, wherein the method comprises individually determining the responsivity of two or more photodiodes in the photodiode array at the various light wavelengths.

[0273] 17. The method according to any one of 14-16, wherein the method further comprises determining the average gain of each photodiode in the photodiode array at the plurality of wavelengths.

[0274] 18. The method of 17, wherein the method further comprises calculating the resistance of each amplifier individually based on the determined responsivity and average gain of each photodiode at the multiple light wavelengths.

[0275] 19. The method according to 18, wherein the method further comprises adjusting the capacitance of each amplifier based on the calculated resistance.

[0276] 20. A method comprising:

[0277] Light is detected using a light detection system, which includes a photodiode and an amplifier;

[0278] Determine the responsivity of the photodiode at various light wavelengths;

[0279] Adjust one or more parameters of the amplifier to adjust the responsivity of the photodiode at the various light wavelengths.

[0280] 21. The method of 20, wherein the method comprises determining the responsivity of the photodiode at the wavelength spectrum of the light.

[0281] 22. The method according to 21, wherein the spectrum comprises light of 200 or more wavelengths.

[0282] 23. The method according to any one of 20-22, wherein the method comprises determining the responsivity of the photodiode in the range of 400 nm to 1100 nm.

[0283] 24. The method according to any one of 20-23, wherein the method comprises determining the average gain of the photodiode at the plurality of wavelengths.

[0284] 25. The method according to 24, wherein the method further comprises calculating the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at the various light wavelengths.

[0285] 26. The method according to 25, wherein the method further comprises calculating the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at the multiple light wavelengths.

[0286] 27. The method according to 26, wherein the resistance of the amplifier is calculated according to the following formula:

[0287] R f XR(λ)=G t

[0288] Where R f It is the resistance of the amplifier;

[0289] R(λ) is the responsivity of the photodiode at each wavelength; and

[0290] G t It is the average gain of the photodiode at the various light wavelengths.

[0291] 28. The method according to any one of 26-27, wherein the method further comprises adjusting the capacitance of the amplifier based on the calculated resistance.

[0292] 29. The method according to 28, wherein the capacitance of the amplifier is adjusted in a manner sufficient to generate a predetermined bandwidth for the photodiode.

[0293] 30. The method according to 29, wherein the capacitance of the amplifier is adjusted according to the following formula to generate a predetermined bandwidth:

[0294]

[0295] Where BW is bandwidth; and

[0296] C f It is the capacitance of the amplifier.

[0297] 31. The method according to any one of 20-30, wherein the amplifier is a transimpedance amplifier.

[0298] 32. The method according to any one of 20-31, wherein the light detection system comprises:

[0299] A photodiode array containing multiple photodiodes; and

[0300] Multiple amplifiers

[0301] Each photodiode is electrically connected to the amplifier.

[0302] 33. The method of 32, wherein the method comprises determining the responsivity of each photodiode in the photodiode array at the plurality of light wavelengths.

[0303] 34. The method of 33, wherein the method comprises individually determining the responsivity of two or more photodiodes in the photodiode array at the multiple light wavelengths.

[0304] 35. The method according to any one of 32-34, wherein the method further comprises determining the average gain of each photodiode in the photodiode array at the plurality of wavelengths.

[0305] 36. The method of 35, wherein the method further comprises calculating the resistance of each amplifier individually based on the determined responsivity and average gain of each photodiode at the multiple light wavelengths.

[0306] 37. The method according to 36, wherein the method further comprises adjusting the capacitance of each amplifier based on the calculated resistance.

[0307] 38. The method according to any one of 20-37, wherein the light detection system is located in a flow cytometer.

[0308] 39. The method of 38, wherein the flow cytometer comprises a flow cell for diffusing particles in a fluid medium.

[0309] 40. A particle analyzer, comprising:

[0310] light source;

[0311] A photodetector system located within the housing of the particle analyzer, the photodetector system comprising a photodiode and an amplifier; and

[0312] A processor includes memory operatively coupled to the processor, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to perform the following operations:

[0313] Determine the responsivity of the photodiode at various wavelengths of light from the light source; and

[0314] Adjust one or more parameters of the amplifier to adjust the responsivity of the photodiode at the various light wavelengths.

[0315] 41. The particle analyzer according to 40, wherein the particle analyzer is incorporated into a flow cytometer.

[0316] 42. The particle analyzer according to any one of 40-41, wherein the photodiode is located in the particle analyzer for detecting light from particles in a fluid medium.

[0317] 43. The particle analyzer according to any one of 40-42, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to determine the responsivity of the photodiode in the wavelength spectrum of the light.

[0318] 44. The particle analyzer according to 43, wherein the spectrum contains light of 200 or more wavelengths.

[0319] 45. The particle analyzer according to any one of 40-44, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to determine the responsivity of the photodiode in the range of 400 nm to 1100 nm.

[0320] 46. ​​The particle analyzer according to any one of 40-45, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the average gain of the photodiode at the multiple wavelengths.

[0321] 47. The particle analyzer of claim 46, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to calculate the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at the various light wavelengths.

[0322] 48. The particle analyzer according to claim 47, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to calculate the resistance according to the following formula:

[0323] R f XR(λ)=G t

[0324] Where R f It is the resistance of the amplifier;

[0325] R(λ) is the responsivity of the photodiode at each wavelength; and

[0326] G t It is the average gain of the photodiode at the various light wavelengths.

[0327] 49. The particle analyzer according to any one of 47-48, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to adjust the capacitance of the amplifier based on the calculated resistance.

[0328] 50. The particle analyzer according to claim 49, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to adjust the capacitance of the amplifier in a manner sufficient to generate a predetermined bandwidth for the photodiode.

[0329] 51. The particle analyzer of claim 50, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to adjust the capacitance of the amplifier according to the following formula:

[0330]

[0331] Where BW is bandwidth; and

[0332] C f It is the capacitance of the amplifier.

[0333] 52. The particle analyzer according to any one of 40-51, wherein the amplifier is a transimpedance amplifier.

[0334] 53. The particle analyzer according to any one of 40-52, wherein the optical detection system comprises:

[0335] A photodiode array containing multiple photodiodes; and

[0336] Multiple amplifiers

[0337] Each photodiode is electrically connected to the amplifier.

[0338] 54. The particle analyzer of claim 53, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the responsivity of each photodiode in the photodiode array at the various light wavelengths.

[0339] 55. The particle analyzer of claim 54, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to individually determine the responsivity of two or more photodiodes in the photodiode array at the various light wavelengths.

[0340] 56. The particle analyzer according to any one of 53-55, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the average gain of each photodiode in the photodiode array at the multiple wavelengths.

[0341] 57. The particle analyzer of claim 56, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to individually calculate the resistance of each amplifier based on the determined responsivity and average gain of each photodiode at the various light wavelengths.

[0342] 58. The particle analyzer of claim 57, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to adjust the capacitance of each amplifier based on the calculated resistance.

[0343] 59. A system comprising:

[0344] light source;

[0345] A photodetector system comprising a photodiode and an amplifier; and

[0346] A processor includes memory operatively coupled to the processor, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to perform the following operations:

[0347] Determine the responsivity of the photodiode at various wavelengths of light from the light source; and

[0348] Adjust one or more parameters of the amplifier to adjust the responsivity of the photodiode at the various light wavelengths.

[0349] 60. The system of claim 59, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the responsivity of the photodiode in the wavelength spectrum of the light.

[0350] 61. The system according to 60, wherein the spectrum comprises light of 200 or more wavelengths.

[0351] 62. The system according to any one of 59-61, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the responsivity of the photodiode in the range of 400 nm to 1100 nm.

[0352] 63. The system according to any one of 59-62, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the average gain of the photodiode at the multiple wavelengths.

[0353] 64. The method according to 63, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to calculate the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at the various light wavelengths.

[0354] 65. The system of claim 64, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to calculate the resistance according to the following formula:

[0355] R f XR(λ)=G t

[0356] Where R f It is the resistance of the amplifier;

[0357] R(λ) is the responsivity of the photodiode at each wavelength; and

[0358] G t It is the average gain of the photodiode at the various light wavelengths.

[0359] 66. The particle analyzer according to any one of 64-65, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to adjust the capacitance of the amplifier based on the calculated resistance.

[0360] 67. The system of claim 66, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to adjust the capacitance of the amplifier in a manner sufficient to generate a predetermined bandwidth for the photodiode.

[0361] 68. The system of claim 67, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to adjust the capacitance of the amplifier according to the following formula:

[0362]

[0363] Where BW is bandwidth; and

[0364] C f It is the capacitance of the amplifier.

[0365] 69. The system according to any one of 59-68, wherein the amplifier is a transimpedance amplifier.

[0366] 70. The system according to any one of 59-69, wherein the light detection system comprises:

[0367] A photodiode array containing multiple photodiodes; and

[0368] Multiple amplifiers

[0369] Each photodiode is electrically connected to the amplifier.

[0370] 71. The system of claim 70, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the responsivity of each photodiode in the photodiode array at the various light wavelengths.

[0371] 72. The system of claim 71, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to individually determine the responsivity of two or more photodiodes in the photodiode array at the various light wavelengths.

[0372] 73. The system according to any one of 70-72, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the average gain of each photodiode in the photodiode array at the plurality of wavelengths.

[0373] 74. The system of claim 73, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to individually calculate the resistance of each amplifier based on the determined responsivity and average gain of each photodiode at the various light wavelengths.

[0374] 75. The system of claim 74, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to adjust the capacitance of each amplifier based on the calculated resistance.

[0375] 76. The system according to any one of 59-75, wherein the system is a flow cytometer.

[0376] 77. The system of claim 76, wherein the flow cytometer comprises a flow cell for diffusing particles in a fluid medium.

[0377] 78. The system according to 77, wherein the photodiode is positioned to detect light from particles in a fluid medium.

[0378] 79. A non-transitory computer-readable storage medium comprising instructions stored thereon for adjusting the sensitivity of an optical detection system in a particle analyzer, the instructions comprising:

[0379] An algorithm for detecting light using an optical detection system, which includes a photodiode and an amplifier;

[0380] An algorithm for determining the responsivity of the photodiode at various light wavelengths; and

[0381] An algorithm for adjusting one or more parameters of the amplifier in response to the responsivity of the photodiode at the various light wavelengths.

[0382] 80. The non-transitory computer-readable storage medium of claim 79, wherein the non-transitory computer-readable storage medium includes an algorithm for determining the responsivity of the photodiode at the wavelength spectrum of the light.

[0383] 81. The non-transitory computer-readable storage medium according to claim 80, wherein the spectrum comprises light of 200 or more wavelengths.

[0384] 82. The non-transitory computer-readable storage medium according to any one of claims 79-81, wherein the non-transitory computer-readable storage medium includes an algorithm for determining the responsivity of the photodiode in the range of 400 nm to 1100 nm.

[0385] 83. The non-transitory computer-readable storage medium according to any one of claims 79-82, wherein the non-transitory computer-readable storage medium includes an algorithm for determining the average gain of the photodiode at the multiple wavelengths.

[0386] 84. The non-transitory computer-readable storage medium of claim 83, wherein the non-transitory computer-readable storage medium includes an algorithm for calculating the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at the various light wavelengths.

[0387] 85. The non-transitory computer-readable storage medium according to claim 84, wherein the non-transitory computer-readable storage medium includes an algorithm for calculating the resistance according to the following formula:

[0388] R f XR(λ)=G t

[0389] Where R f It is the resistance of the amplifier;

[0390] R(λ) is the responsivity of the photodiode at each wavelength; and

[0391] G t It is the average gain of the photodiode at the various light wavelengths.

[0392] 86. The non-transitory computer-readable storage medium according to any one of 84-85, wherein the non-transitory computer-readable storage medium includes an algorithm for adjusting the capacitance of the amplifier based on the calculated resistance.

[0393] 87. The non-transitory computer-readable storage medium of claim 86, wherein the non-transitory computer-readable storage medium includes an algorithm for adjusting the capacitance of the amplifier in a manner sufficient to generate a predetermined bandwidth for the photodiode.

[0394] 88. The non-transitory computer-readable storage medium according to claim 87, wherein the non-transitory computer-readable storage medium includes an algorithm for adjusting the capacitance of the amplifier according to the following formula:

[0395]

[0396] Where BW is bandwidth; and

[0397] C f It is the capacitance of the amplifier.

[0398] 89. The non-transitory computer-readable storage medium according to any one of 79-88, wherein the non-transitory computer-readable storage medium comprises an algorithm, wherein the amplifier is a transimpedance amplifier.

[0399] 90. The non-transitory computer-readable storage medium according to any one of claims 79-89, wherein the non-transitory computer-readable storage medium comprises an algorithm, and wherein the optical detection system comprises:

[0400] A photodiode array containing multiple photodiodes; and

[0401] Multiple amplifiers

[0402] Each photodiode is electrically connected to the amplifier.

[0403] 91. The non-transitory computer-readable storage medium of claim 90, wherein the non-transitory computer-readable storage medium includes an algorithm for determining the responsivity of each photodiode in the photodiode array at the multiple light wavelengths.

[0404] 92. The non-transitory computer-readable storage medium of claim 91, wherein the non-transitory computer-readable storage medium includes an algorithm for individually determining the responsivity of two or more photodiodes in the photodiode array at the various light wavelengths.

[0405] 93. The non-transitory computer-readable storage medium according to any one of 90-92, wherein the non-transitory computer-readable storage medium includes an algorithm for determining the average gain of each photodiode in the photodiode array at the multiple wavelengths.

[0406] 94. The non-transitory computer-readable storage medium of claim 93, wherein the non-transitory computer-readable storage medium includes an algorithm for individually calculating the resistance of each amplifier based on the determined responsivity and average gain of each photodiode at the various light wavelengths.

[0407] 95. The non-transitory computer-readable storage medium of claim 94, wherein the non-transitory computer-readable storage medium includes an algorithm for adjusting the capacitance of each amplifier based on the calculated resistance.

[0408] Although the invention has been described in detail with illustrations and examples for the purpose of clarity, it will be apparent to those skilled in the art, given the edifying significance of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0409] Therefore, the foregoing only illustrates the principles of the invention. It should be understood that those skilled in the art can design various structures, although not explicitly stated or shown herein, but these designs reflect the principles of the invention and do not depart from its spirit and scope. Furthermore, all examples and conditional language listed herein are primarily intended to help the reader understand the principles of the invention and the inventors' ideas for further expanding the field, and should be interpreted as not being limited by these specifically listed examples and conditions. Moreover, all statements herein referencing the principles, aspects, and embodiments of the invention and their specific examples are intended to cover their structural and functional equivalents. Furthermore, the equivalents are intended to include currently known equivalents and those to be developed in the future, i.e., any functionally identical elements developed regardless of their structure. Moreover, no part of the invention will be disclosed to the public, whether or not it is explicitly stated in the claims.

[0410] Therefore, the scope of the invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is explicitly invoked only when the limiting phrase “means for…” or “steps for…” is explicitly used at the beginning of the limiting phrase of the claims; if such phrase is not used in the limiting phrase of the claims, then 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

Claims

1. A method for adjusting the sensitivity of an optical detection system in a particle analyzer, the method comprising: The light is detected by a light detection system in a particle analyzer, the light detection system comprising a photodiode and an amplifier; Determine the responsivity of the photodiode at various light wavelengths; Determine the average gain of the photodiode at the various wavelengths; and In response to the responsivity and average gain of the photodiode at the various light wavelengths, one or more parameters of the amplifier are adjusted. in, Adjusting one or more parameters of the amplifier includes: calculating the amplifier's resistance based on the determined responsivity and average gain of the photodiode at the various light wavelengths, according to the following formula: R f XR(λ) = G t Where R f It is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; and G t It is the average gain of the photodiode at the various light wavelengths.

2. The method of claim 1, wherein the method comprises determining the responsivity of the photodiode at the wavelength spectrum of the light.

3. The method of claim 1, wherein the method further comprises adjusting the capacitance of the amplifier according to the calculated resistance, based on the following formula, to generate a predetermined bandwidth: ; Where BW is bandwidth; and C f It is the capacitance of the amplifier.

4. The method according to any one of claims 1-2, wherein the light detection system comprises: A photodiode array containing multiple photodiodes; and Multiple amplifiers Each photodiode is electrically connected to the amplifier.

5. The method of claim 4, wherein the method further comprises: Determine the average gain of each photodiode in the photodiode array at the various wavelengths; Based on the determined responsivity and average gain of each photodiode at the various light wavelengths, the resistance of each amplifier is calculated individually; as well as Based on the calculated resistance, adjust the capacitance of each amplifier.

6. A system comprising: light source; A photodetector system comprising a photodiode and an amplifier; and A processor includes memory operatively coupled to the processor, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to perform the following operations: Determine the responsivity of the photodiode at various wavelengths of light from the light source; Determine the average gain of the photodiode at the various wavelengths; and In response to the responsivity and average gain of the photodiode at the various light wavelengths, one or more parameters of the amplifier are adjusted. in, Adjusting one or more parameters of the amplifier includes: calculating the amplifier's resistance based on the determined responsivity and average gain of the photodiode at the various light wavelengths, according to the following formula: R f XR(λ) = G t Where R f It is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; and G t It is the average gain of the photodiode at the various light wavelengths.

7. The system of claim 6, wherein the memory contains instructions stored thereon that, when executed by the processor, cause the processor to determine the responsivity of the photodiode in the wavelength spectrum of the light.

8. The system according to any one of claims 6-7, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to adjust the capacitance of the amplifier according to the following formula: ; Where BW is bandwidth; and C f It is the capacitance of the amplifier.

9. The system according to any one of claims 6-7, wherein the light detection system comprises: A photodiode array containing multiple photodiodes; and Multiple amplifiers Each photodiode is electrically connected to an amplifier; and A processor includes memory operatively coupled to the processor, wherein the memory contains instructions stored thereon, which, when executed by the processor, cause the processor to perform the following operations: Determine the responsivity of each photodiode in the photodiode array at the various light wavelengths; The responsivity of two or more photodiodes in the photodiode array at the various light wavelengths is determined individually; Determine the average gain of each photodiode in the photodiode array at the various wavelengths; The resistance of each amplifier is calculated based on the determined responsivity and average gain of each photodiode at the various light wavelengths. as well as Based on the calculated resistance, adjust the capacitance of each amplifier.

10. A non-transitory computer-readable storage medium comprising instructions stored thereon for adjusting the sensitivity of a photodetector system in a particle analyzer, the instructions comprising: An algorithm for detecting light using an optical detection system, which includes a photodiode and an amplifier; An algorithm for determining the responsivity of the photodiode at various light wavelengths; An algorithm for determining the average gain of the photodiode at the various wavelengths; as well as An algorithm for adjusting one or more parameters of the amplifier in response to the responsivity and average gain of the photodiode at various light wavelengths. The algorithm for adjusting one or more parameters of the amplifier includes: an algorithm for calculating the resistance of the amplifier based on the determined responsivity and average gain of the photodiode at the various light wavelengths, according to the following formula: R f XR(λ) = G t Where R f It is the resistance of the amplifier; R(λ) is the responsivity of the photodiode at each wavelength; and G t It is the average gain of the photodiode at the various light wavelengths.

11. The non-transitory computer-readable storage medium of claim 10, wherein the non-transitory computer-readable storage medium includes an algorithm for determining the responsivity of the photodiode at the wavelength spectrum of the light.

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