Improving broadband signal acquisition efficiency in flow cytometers and cell sorters

Abstract

According to one embodiment, a method includes exciting different fluorescent dyes marking a plurality of mobile cells to emit fluorescence; collecting a broadband optical signal having a broadband wavelength range from the fluorescence of the different fluorescent dyes; dividing the wavelength range in the broadband optical signal into smaller wavelength ranges in optical signal paths; detecting optical signals in each of the smaller wavelength ranges among the smaller wavelength ranges in the optical signal paths separately with different photodetectors; generating digital signals from the detected optical signals in the smaller wavelength ranges in the optical signal paths with analog-to-digital converters; arranging the digital signals over the broadband wavelength range; and combining the arranged digital signals together into a full optical spectrum response based on the broadband optical signal having the broadband wavelength range.

Description

[0001] Cross-references to related applications

[0002] This U.S. patent application is a non-provisional patent application claiming priority to U.S. Provisional Patent Application No. 63 / 606,037, filed December 4, 2023, entitled "Improving BROADBAND SIGNAL COLLECTION EFFICIENCY IN FLOW CYTOMETERS ANDCELL SORTERS" by inventors Bing Shan et al., which is incorporated herein by reference for all intents and purposes. This U.S. patent application also relates to U.S. Patent Application No. 15 / 659,610, filed October 5, 2017, entitled "COMPACT DETECTION MODULE FOR FLOW CYTOMETERS" by inventors Ming Yan et al., which is incorporated herein by reference for all intents and purposes. This U.S. patent application also relates to U.S. Patent Application No. 15 / 942,430, filed March 30, 2018, entitled COMPACT MULTI-COLORFLOW CYTOMETER HAVING COMPACT DETECTION MODULE by inventors Ming Yan et al., which is also incorporated herein by reference for all intents and purposes. Technical Field

[0003] The embodiments of the present invention generally relate to flow cytometers and cell sorting systems. Background Technology

[0004] Flow cytometry is a technique that provides rapid analysis of the physical and chemical properties of biological cells moving in a stream of sample solution. Cell sorting further separates selected cells of interest into different containers (e.g., test tubes) for further use (e.g., testing) or counting. Laboratory instruments that perform these tasks are called flow cytometers and cell sorters, also known as sorting flow cytometers.

[0005] Biological cells or other particles are typically stained or labeled with one or more fluorescent dyes (fluorescent pigments), which can attach to cells or other particles at the labeled sites in the sample to make them more identifiable. Different fluorescent dyes can be excited by different lasers with different center wavelengths to better identify unknown cells and particles in the test sample. Flow cytometry uses one or more lasers as light sources to illuminate biological cells and fluorescent dyes to generate scattered light signals and fluorescence signals, respectively. The fluorescence signals and scattered light signals are received by photodetectors (such as photodiodes or photomultiplier tubes) to form electrical signals, which are then digitized to form digital signals that can be analyzed.

[0006] Cell populations in sample solutions can be analyzed and / or purified based on detected fluorescence or light scattering properties. Flow cytometry provides a method for identifying cells in solution and their characteristics, and is commonly used to evaluate biological cells in peripheral blood, bone marrow, and other body fluids. Summary of the Invention

[0007] The following claims best summarize these embodiments. However, simply put, in some aspects, the technology described herein relates to a method for flow cytometry, the method comprising the steps of: collecting fluorescence from one or more moving cells and side-scattered light exiting the one or more moving cells over a full bandwidth range between a minimum and a maximum wavelength to be detected using an objective lens; converging the fluorescence and the side-scattered light over the full bandwidth range toward a focal point along a first optical axis outside the objective lens to form a spot size; coupling first light from a first wavelength range from the minimum wavelength to the first wavelength within the full bandwidth range to a first light detection device using a first optical coupling device, and allowing the remaining light of the full bandwidth range to pass toward a final light detection device; converting the first light into a first plurality of digital signals representing discrete wavelength portions of the first light over the first wavelength range using the first light detection device; converting the remaining light into a final plurality of digital signals representing discrete wavelength portions of the remaining light over the full bandwidth range using the final light detection device; and receiving the first plurality of digital signals and the final plurality of digital signals using a processor, and processing the plurality of digital signals into a combined spectral signal over the full bandwidth range.

[0008] In some aspects, the technology described herein relates to a method that further includes: before conversion using the final light detection device: coupling a second light from a second wavelength range, from one or more nanometers above the maximum wavelength of the first light to a second wavelength within the full bandwidth, to the second light detection device using a second coupling device, and allowing the remaining light within the full bandwidth to pass toward the final light detection device; converting the second light into a second plurality of digital signals representing discrete wavelength portions of the second light within the second wavelength range using the second light detection device; and receiving the second plurality of digital signals using the signal processor, and processing the first plurality of digital signals, the second plurality of digital signals, and the final plurality of digital signals into a combined spectral signal over the full bandwidth.

[0009] In some aspects, the technology described herein relates to a method further comprising: after conversion using the second optical detection device and before conversion using the final optical detection device: coupling a third light from one or more nanometers above the maximum wavelength of the second light to a third wavelength range within the full bandwidth to the third optical detection device using a third coupling device, and allowing the remaining light within the full bandwidth range to pass toward the final optical detection device; converting the third light into a third plurality of digital signals representing discrete wavelength portions of the third light within the third wavelength range using the third optical detection device; and receiving the third plurality of digital signals using the signal processor, and processing the first plurality of digital signals, the second plurality of digital signals, the third plurality of digital signals, and the final plurality of digital signals into a combined spectral signal over the full bandwidth range.

[0010] In some respects, the techniques described herein relate to a method in which the first optical coupling device is selected from one or more of the group consisting of a lens, an optical fiber, a beam splitter, a Bragg grating, and / or a mirror.

[0011] In some respects, the techniques described herein relate to a method in which: each optical coupling device is selected from one or more of a group consisting of lenses, optical fibers, beam splitters, Bragg gratings and / or mirrors.

[0012] In some respects, the technology described herein relates to a method in which the first optical coupling device is a first beamsplitter, and the coupling comprises: using the first beamsplitter to separate the first light in the first wavelength range from the minimum wavelength to the first wavelength in the full bandwidth range, and allowing the remaining light in the full bandwidth range to pass through; and using the first beamsplitter to redirect the first light in the first wavelength range along a second optical axis toward the first optical detection device at a 45-degree angle.

[0013] In some aspects, the technology described herein relates to a method in which: the first optical coupling device is a first beamsplitter, and the first coupling includes: using the first beamsplitter to separate the first light in a first wavelength range from the minimum wavelength to a first wavelength in the full bandwidth range, and allowing the remaining light in the full bandwidth range to pass through; and using the first beamsplitter to redirect the first light in the first wavelength range toward a first photodetector along a second optical axis at an angle; and the second optical coupling device is a second beamsplitter, and the second coupling includes: using the second beamsplitter to separate the second light in a second wavelength range from one or more nanometers above the maximum wavelength of the first light to a second wavelength in the full bandwidth range, and allowing the remaining light in the full bandwidth range to pass toward a third photodetector; and using the second beamsplitter to redirect the second light in the second wavelength range toward the second photodetector along a third optical axis at a 45-degree angle.

[0014] In some aspects, the technology described herein relates to a method in which the first optical coupling device is a first dichroic mirror and a first optical fiber, and the coupling includes: using the first dichroic mirror to separate the first light in a first wavelength range from the minimum wavelength to a first wavelength in the full bandwidth range, and allowing the remaining light in the full bandwidth range to pass through; using the first dichroic mirror to redirect the first light in the first wavelength range along a second optical axis toward a first end of the first optical fiber at an angle of 45 degrees; and using the first end of the first optical fiber to receive the first light and to transmit the first light from a second end of the first optical fiber to a first optical detection device.

[0015] In some aspects, the technology described herein relates to a method in which: the first optical coupling device is a first dichroic mirror and a first optical fiber, and the first coupling includes: using the first dichroic mirror to separate the first light in a first wavelength range from the minimum wavelength to a first wavelength in the full bandwidth range, and allowing the remaining light in the full bandwidth range to pass through; using the first dichroic mirror to redirect the first light in the first wavelength range along a second optical axis toward a first end of the first optical fiber at a first angle; and using the first end of the first optical fiber to receive the first light, and emitting the first light from a second end of the first optical fiber to a first optical detection device; and the second optical coupling device is a second dichroic mirror and a second optical fiber, and the second coupling includes: using the second dichroic mirror to separate the second light in a second wavelength range from the minimum wavelength to a first wavelength in the full bandwidth range, and allowing the remaining light in the full bandwidth range to pass through; using the second dichroic mirror to redirect the second light in the second wavelength range along a second optical axis toward a first end of the second optical fiber at a second angle; and using the first end of the second optical fiber to receive the second light, and emitting the second light from a second end of the second optical fiber to a second optical detection device.

[0016] In some respects, the technology described herein relates to a method in which: each optical detection device is a photodiode array detector having a plurality of mirrors in a first row on one side, and a plurality of wavelength filters and a plurality of photodetectors arranged in rows on the opposite side to detect discrete wavelength portions over an optical bandwidth.

[0017] In some respects, the technology described herein relates to a method in which: each of the plurality of photodetectors is an avalanche photodiode.

[0018] In some aspects, the technology described herein relates to a method in which: various optical detection devices include: a first grating that receives light coupled into the optical detection device along a first axis and reflects and first expands the light along a second axis; a second grating that receives the first expansion of the light along the second axis and reflects and second expands the light along a third axis; and a photodiode array detector having a plurality of mirrors in a first row on one side and a plurality of wavelength filters and a plurality of photodetectors arranged in rows on the opposite side to detect discrete wavelength portions over an optical bandwidth.

[0019] In some respects, the technology described herein relates to a method in which: each of the plurality of photodetectors in the photodiode array detector is an avalanche photodiode.

[0020] In some respects, the technique described herein relates to a method in which: each light detection device is a photomultiplier tube (PMT).

[0021] In some aspects, the technology described herein relates to a flow cytometer comprising: a flow cell receiving mobile biological cells in a sample fluid and a sheath fluid encapsulating the biological cells, the flow cell having a questioning region receiving one or more laser beams from one or more lasers to strike the mobile biological cells and generate fluorescence, side-scattered light, and forward-scattered light; an objective lens adjacent to the flow cell, the objective lens collecting the fluorescence and the side-scattered light over the full bandwidth range between a minimum and a maximum wavelength to be detected, and converging the fluorescence and the side-scattered light over the full bandwidth range outside the objective lens along a first optical axis toward a focal point to form a spot size; a first optical coupling device preceding the focal point along the first optical axis, the first optical coupling device along a second optical axis converging a first wavelength within the first wavelength range from the minimum wavelength to the full bandwidth. A first light in a wavelength range is redirected, and a first optical coupling device allows the remaining light in the full bandwidth range to pass along the first optical axis; a first optical detection device receives the first light along a second optical axis and converts the first light into a first plurality of digital signals representing discrete wavelength portions of the first light in the first wavelength range; a last optical detection device receives the remaining light along the first optical axis, and a third optical detection device converts the remaining light into a last plurality of digital signals representing discrete wavelength portions of the remaining light in the full bandwidth range; and a processor communicatively coupled to the first optical detection device and the last optical detection device, the signal processor receiving the first plurality of digital signals and the last plurality of digital signals, and processing the digital signals into a combined spectral signal over the full bandwidth range.

[0022] In some aspects, the technology described herein relates to a flow cytometer, the flow cytometer further comprising: a second optical coupling device located along a first optical axis between the first optical coupling device and the focal point, the first optical coupling device reorienting second light along a third optical axis in a second wavelength range from one or more nanometers above the maximum wavelength of the first light to a second wavelength in the full bandwidth range, the second optical coupling device allowing the remaining light in the full bandwidth range to pass along the first optical axis; a second light detection device receiving the second light along the third optical axis, the second light detection device converting the second light into a second plurality of digital signals representing discrete wavelength portions of the second light in the second wavelength range, wherein a signal processor is also communicatively coupled to the second light detection device to receive the second plurality of digital signals, and wherein the signal processor processes the first plurality of digital signals, the second plurality of digital signals, and the last plurality of digital signals into the combined spectral signal in the full bandwidth range.

[0023] In some aspects, the technology described herein relates to a flow cytometer further comprising: a third optical coupling device located along a first optical axis between a second optical coupling device and the focal point, the third optical coupling device reorienting third light from a third wavelength range of a third wavelength within the full bandwidth range, from one or more nanometers above the maximum wavelength of the second light, along a fourth optical axis, the third optical coupling device allowing the remaining light within the full bandwidth range to pass along the first optical axis toward the final optical coupling device; a third light detection device receiving the third light along the fourth optical axis, the third light detection device converting the third light into a third plurality of digital signals representing discrete wavelength portions of the third light within the third wavelength range, and wherein a signal processor is also communicatively coupled to the third light detection device to receive the third plurality of digital signals, and wherein the signal processor processes the first plurality of digital signals, the second plurality of digital signals, the third plurality of digital signals, and the final plurality of digital signals into the combined spectral signal over the full bandwidth range.

[0024] In some respects, the technology described herein relates to a flow cytometer, wherein: the first optical coupling device is selected from one or more of the group consisting of lenses, Bragg gratings and / or dichroic mirrors and optical fibers.

[0025] In some respects, the technology described herein relates to a flow cytometer in which: each optical coupling device in the optical coupling apparatus is selected from one or more of the group consisting of lenses, Bragg gratings and / or dichroic mirrors and optical fibers.

[0026] In some respects, the technology described herein relates to a flow cytometer in which: each optical coupling device in the optical coupling apparatus is selected from one or more of the group consisting of lenses, Bragg gratings and / or dichroic mirrors and optical fibers.

[0027] In some aspects, the technology described herein relates to a method for improving a flow cytometer or cell sorter, the method comprising the steps of: exciting multiple different fluorescent dyes labeled with multiple moving cells to emit fluorescence using one or more laser beams; collecting a broadband optical signal having a broadband wavelength range from the fluorescence of the multiple different fluorescent dyes; dividing the wavelength range of the collected broadband optical signal into multiple smaller wavelength ranges in multiple optical signal paths; detecting the optical signal in each of the multiple smaller wavelength ranges in the multiple optical signal paths using multiple different photodetectors; generating multiple digital signals using multiple analog-to-digital converters based on the detected optical signals in each of the multiple smaller wavelength ranges in the multiple optical signal paths; arranging the multiple digital signals over the broadband wavelength range; and combining the arranged multiple digital signals together to form a full-spectrum response based on the broadband optical signal having the broadband wavelength range.

[0028] In some respects, the techniques described herein relate to a method in which the collected broadband optical signals have a broadband wavelength range of 320 nanometers to 1,000 nanometers.

[0029] In some respects, the technique described herein relates to a method in which the broadband wavelength range of the collected broadband optical signal is separated into two wavelength ranges in two optical signal paths.

[0030] In some respects, the technique described herein relates to a method in which: a first wavelength range in a first optical signal path is from 320 nanometers to 480 nanometers; and a second wavelength range in a second optical signal path is from 480 nanometers to 1,000 nanometers.

[0031] In some respects, the techniques described herein relate to a method in which: separating a wavelength range in a collected broadband optical signal into a plurality of smaller wavelength ranges in a plurality of optical signal paths further includes: collecting at least one of the plurality of different smaller wavelength ranges using a first end of an optical fiber, and redirecting the optical signal toward a first photodetector; and transmitting the optical signal from a second end of the optical fiber to the first photodetector.

[0032] In some respects, the techniques described herein relate to a method in which: the first photodetector is a linear array detector having a plurality of photodetectors arranged in a row.

[0033] According to one embodiment, a method includes the following steps: exciting different fluorescent dyes labeled with a plurality of mobile cells to emit fluorescence; collecting a broadband optical signal having a broadband wavelength range from the fluorescence of the different fluorescent dyes; dividing the wavelength range of the broadband optical signal into smaller wavelength ranges in the optical signal path; detecting the optical signal in each of the smaller wavelength ranges in the optical signal path using different photodetectors; generating a digital signal using an analog-to-digital converter based on the detected optical signal in the smaller wavelength range of the optical signal path; arranging the digital signal over the broadband wavelength range; and combining the arranged digital signals together to form a full-spectrum response based on the broadband optical signal having a broadband wavelength range. Attached Figure Description

[0034] Figure 1A This is a conceptual diagram of a flow cytometer system and a sorting flow cytometer (cell sorter) system.

[0035] Figure 1B This is a conceptual diagram of a photoelectric system that uses a single optical fiber and a single array detector to excite and detect the side-scattered light from biological cells and the fluorescence emitted by excited fluorescent dyes (fluorescent pigments).

[0036] Figure 1C yes Figure 1B The diagram shows a magnified view of the objective lens and input end of a single-fiber optical fiber.

[0037] Figure 1D Examples of the same Figure 1C The diagram shown illustrates the chromatic aberration focal shift associated with a single fiber and optical lens that collects full-bandwidth fluorescence and side-scattered light signals.

[0038] Figure 1E A conceptual diagram of a linear array detector with N detection channels and N photodetectors is shown.

[0039] Figure 1F A chart illustrating the properties of a standard anti-reflective (AR) coating used in optical components is provided.

[0040] Figure 2 This is a conceptual diagram of an optoelectronic system that uses multiple beam splitters and multiple array detectors to excite and detect side-scattered light and fluorescence in a separated wavelength range.

[0041] Figure 3A This is a conceptual diagram of an optoelectronic system that uses multiple optical fibers and multiple array detectors to excite and detect side-scattered light and fluorescence in a separated wavelength range.

[0042] Figure 3B This is a graph showing the transmission characteristics of a beam splitter / diffraction mirror.

[0043] Figure 3C This is a block diagram showing the filtering characteristics of a beam splitter detector with overlapping wavelength portions across its wavelength range.

[0044] Figure 3D It is a block diagram of the filtering characteristics of a beam splitter detector with spectral gaps or non-overlapping wavelength portions across the wavelength range.

[0045] Figure 4A This is a block diagram of a pair of linear array detectors packaged together with dual fiber inputs to detect two separate wavelength ranges.

[0046] Figure 4B It is used for Figure 4A The pair of wavelength tables for a pair of linear array detectors are shown.

[0047] Figure 4C This is a conceptual block diagram of a single detector channel used in a linear array detector with multiple detector channels.

[0048] Figure 4D It is a diagram of a pair of linear array detectors housed together and sharing an optical block.

[0049] Figure 5 It is a block diagram of N linear array detectors packaged together with N fiber input terminals to detect N separate wavelength ranges.

[0050] Figure 6A It is a block diagram of a pair of grating detectors packaged together with dual fiber inputs to detect two separate wavelength ranges.

[0051] Figure 6B It is a block diagram of three detectors of a mixed type, packaged together with three optical fiber inputs to detect three separate wavelength ranges.

[0052] Figure 7 Examples of the same Figure 3A The diagram shown illustrates the chromatic aberration focal shift of the two optical fibers associated with each other for collecting separated bandwidth ranges of fluorescence and side-scattered light signals.

[0053] Figures 8A to 8B Examples are shown that can be used Figure 3A The fiber shown is a multimode fiber patch cord.

[0054] Figure 8C A graph illustrating the properties of an anti-reflective coating used in fiber optic patch cords is provided.

[0055] Figure 9A An example of an octagonal spatial detector layout is shown.

[0056] Figure 9B An example of a hexagonal spatial detector layout is shown.

[0057] Figure 9C An example of a triangular spatial detector layout is shown.

[0058] Figures 10A to 10C An example is shown of capturing fluorescence intensity on separate spectra using different detectors and combining the separate spectra to form a full-spectrum signature. Detailed Implementation

[0059] In the following detailed description of the disclosed embodiments, numerous specific details are set forth in order to provide a thorough understanding. However, it will be apparent to those skilled in the art that the disclosed embodiments can be practiced without these specific details. In other instances, well-known methods, processes, components, and subsystems have not been described in detail to avoid unnecessarily obscuring aspects of the disclosed embodiments.

[0060] The disclosed embodiments include a method, apparatus, and system for improving the efficiency of broadband signal collection in flow cytometry / cell sorting systems by dividing a wavelength range in a broadband optical signal into multiple paths, detecting each divided wavelength range separately, and then recombining the spectral results using a central processing unit. Generally, a wide bandwidth (320 nm to 1000 nm – a broadband wavelength range) of the input optical signal to the optoelectronic system can be separated, for example, into two parts (e.g., one part from 320 nm to 550 nm and another from 570 nm to 1000 nm) or (e.g., 320 nm to 480 nm and 480 nm to 1000 nm). Two or more detectors can then be used to detect light in the individual bandwidth portions, and digital signals can be generated separately for each bandwidth portion. Each array detector covers a wavelength range narrower than the overall bandwidth. The individual digital signals are arranged and combined across the wavelength range to provide an overall spectral result in the combined spectral signal.

[0061] Now refer to Figure 1A This diagram illustrates a basic conceptual design of a cell sorting system (sorting flow cytometer) 10. The system 10 comprises five main subsystems: an excitation optics system 12, a flow system 14, an emission optics system 16, a data acquisition system 18, and an analysis system 20. The flow system 14 may include a sample loading system (see [reference]). Figure 1C The sample input stage 130, the inquiry system 28, the cell sorting system 33, and the droplet deposition (droplet receiving) system 29 are shown. Generally, "system" and "subsystem" include (electrical, mechanical, and electromechanical) hardware devices, software devices, or combinations thereof.

[0062] The excitation optics system 12 includes, for example, multiple (e.g., 2 to 5) excitation channels 22A to 22N, each excitation channel having a different laser device 23A to 23N and one or more optical elements 24 to 26 to guide different lasers to spaced optical inquiry regions 30A to 30N along a line in the flow channel 27 of the flow cell 28. One or more of the optical elements 24 to 26 include optical prisms and optical lenses. The excitation optics system 12 illuminates the optical inquiry regions 30 in the flow cell 28. The flow system 14 carries a fluid sample 32 surrounded by sheath fluid through the various optical inquiry regions 30A to 30N in the flow cell / flow channel.

[0063] The emitting optical system 16 includes multiple detector arrays 42A to 42N, each detector array including, for example, one or more optical elements 40, such as optical fibers and one or more lenses, to guide fluorescence and / or (forward, side, and backward) scattered light to various photodetectors (converters). The photodetectors include side-scattered (SSC) channel detectors in each array and multiple (e.g., 16, 32, 48, 64) fluorescence wavelength range optical detectors, such as a first fluorescence optical detector (FL1) receiving fluorescence in a first wavelength range, a second fluorescence optical detector (FL2) receiving fluorescence in a second wavelength range, a third fluorescence optical detector (FL3) receiving fluorescence in a third wavelength range, a fourth fluorescence optical detector (FL4) receiving fluorescence in a fourth wavelength range, a fifth fluorescence optical detector (FL5) receiving fluorescence in a fifth wavelength range, and so on, up to an Nth fluorescence optical detector (FLN) receiving fluorescence in an Nth wavelength range. Each detector array in detector arrays 42A to 42N receives light from cells / particles and / or one or more fluorescent dyes attached to and excited by different lasers generated by various lasers in the interrogation regions / points 30A to 30N along the flow channel 27 of the flow cell 28. An emission optics system 16 collects photons emitted or scattered from the passing cells / particles and / or the fluorescent dyes attached to them. The emission optics system 16 guides and focuses these collected photons onto photodetectors SSC, FL1, FL2, FL3, FL4, and FL5 in the respective detector arrays, for example, via optical fiber cable 39, one or more lenses 40, and one or more mirrors / filters 41. The photodetectors SSC are side-scattering channel detectors used to detect light scattered from the cells / particles. Photodetectors FL1, FL2, FL3, FL4, and FL5 are fluorescence detectors and may include bandpass or longpass filters to detect specific and different fluorescence wavelength ranges derived from different fluorescent dyes excited by different lasers. Each photodetector converts photons into electrical pulses and sends these electrical pulses to the acquisition (electronic) system 18.

[0064] For each detector array 42A to 42N, the acquisition (electronic) system 18 includes one or more analog-to-digital converters 47A to 47N and one or more digital storage devices 48A to 48N, which can provide multiple detector channels (e.g., 16, 32, 48, or 64 channels) of spectral data signals. The spectral data signals can be signal-processed (e.g., digitized by an A / D converter) and timestamped by a packetizer 52, and packaged together into data packets corresponding to individual cells / particles in the sample. These data packets for individual cells / particles can be transmitted by the acquisition (electronic) system 18 to the analysis system 20 for further signal processing (e.g., time-domain conversion / transformation to the wavelength domain) and overall analysis. Alternatively, or in combination, the timestamped digital spectral data signals from the individual detected channels can be directly transmitted to the analysis system 20 for signal processing.

[0065] The analysis system 20 includes a processor, memory, and data storage unit to store data packets of digital spectral data with timestamps associated with cells / particles detected in the sample. The analysis system 20 also includes software with instructions executed by the processor to convert / transform the data from the time domain to wavelength / frequency domain data and to stitch / merge the data together to provide a holistic spectrum of cells / particles / dyes excited by different lasers and sensed by a detector array. Cell / particle counting can be performed in samples processed by flow cytometers and / or cell sorters by detecting the type of cells / particles by one or more fluorescent dyes attached thereto.

[0066] In some cases, it is necessary to use a cell sorter (sorting flow cytometer) to separate cells from a sample for further analysis. Therefore, the spectral data signal can also be processed by the real-time sorting controller 50 in the acquisition (electronic) system 18 and used to control the sorting system 33 to sort cells or particles into one or more tubes 34. In this case, the sorting system 33 communicates with the real-time sorting controller 50 of the acquisition (electronic) system 18 to receive control signals. Alternatively, instead of tubes 34, the spectral data signal can also be processed by the real-time sorting controller 50 of the acquisition (electronic) system 18 and used to control the sorting system 33 and the droplet deposition system 29 to sort cells or particles into the orifices 35 of a moving capture disc / plate. In this case, both the droplet deposition system 29 and the sorting system 33 communicate with the acquisition (electronic) system 18 to receive control signals. In another embodiment, the analysis system 20 can generate these control signals by analyzing spectral data signals to sort out different cells / molecules and control the sorting system 33 and the droplet deposition system 29 to capture sample droplets containing cells / particles into one or more wells 35 in a capture disk / plate.

[0067] Exemplary flow cytometry systems and subsystems are disclosed in U.S. Patent Application No. 15 / 659,610, entitled COMPACT DETECTION MODULE FORFLOW CYTOMETERS, filed July 25, 2017 by Ming Yan et al., and in U.S. Patent Application No. 15 / 942,430, entitled COMPACT MULTI-COLOR FLOW CYTOMETER HAVING COMPACT DETECTION MODULE, filed March 30, 2018 by Ming Yan et al., and these patent applications are incorporated herein by reference for all intents and purposes.

[0068] Now refer to Figure 1B This diagram shows a top view of a conceptual diagram of a photoelectric detection subsystem in a flow cytometer or cell sorter. One or more laser beams 102 are generated by one or more lasers 101 and guided along optical axis 109 into the interrogation region of flow chamber 120 in flow cell 100. Laser and / or forward-scattered light signals 112 are received by a forward-scatter detector (not shown) along optical axis 109. If no cells are present, the laser is received by the forward-scatter detector, and no forward-scattered light is observed.

[0069] Multiple mobile cells 104 in a flowing sample fluid enter the interrogation region of the flow chamber 120 of the flow cell 100 through a flow channel. One or more laser beams 102 impinge on the mobile cells, thereby generating fluorescence and side-scattered light signals 106A along a second optical axis 110. If no fluorescent dye is attached to a label on the mobile biological cells, the side-scattered light signal can be any of the signals formed in the fluorescence and side-scattered light signals 106A. Some biological cells spontaneously fluoresce when excited by the laser, therefore some spontaneous fluorescence can be included in the fluorescence and side-scattered light signals 106A, regardless of any attached fluorescent dye.

[0070] Typically, the objective optics (objective lens) 128 collects fluorescence and side-scattered light signals 106A, which are guided toward its front along a second optical axis 110 at an angle (e.g., 90 degrees or perpendicular) to the first optical axis 109. Therefore, the objective optics (objective lens) 128 is designed and optimized to process the full wavelength bandwidth of the fluorescence and side-scattered light signals 106A.

[0071] Objective lens 128 magnifies the image of the cell and focuses it downwards as a converged fluorescence and side-scattered light signal 106B to the input end 151 of optical fiber (or fiber bundle consisting of multiple fibers) 150. Objective lens 128 and mounting base 155 are coupled to base 140. Mounting base 155 is also coupled to the optical fiber (or fiber bundle) 150 near the input end to keep it aligned with the second optical axis 110. In the case of multiple fibers in the fiber bundle, mounting base 155 can keep the multiple fibers vertically aligned relative to base 140, with each input end aligned with a plane passing through axis 110.

[0072] In one case, objective optics 128 provides 10x (10X) cell magnification. Along optical axis 110, the back focal length (BFL) distance from the back of lens 128 to the input end of fiber optic 150 is selected for the lens to optimize the desired wavelength range (between wavelength minimum (λmin) and wavelength maximum (λmax), including, for example, selecting the midpoint) for detection in fluorescence and side-scattered light signals 106A. Figure 1C An enlarged view of the back focal length (BFL) distance between the back surface of lens 128 and the input end of optical fiber 150 is shown. Mechanical mount 155 is used to maintain the distance between the end of optical fiber 150 and the back surface of objective lens 128, but may be adjustable to allow for small variations in the back focal length.

[0073] The wavelength range of the converged fluorescence and side-scattered light signal 106B exhibits chromatic aberration focal shift 152, thus different wavelengths are converged differently at the input end of the optical fiber 150. Short-wavelength light 106S from the converged fluorescence and side-scattered light signal 106B is converged downwards into the optical fiber 150 closer to its input end 151. Long-wavelength light 106L from the converged fluorescence and side-scattered light signal 106B is further converged downwards into the optical fiber 150 away from its input end 151. The intermediate wavelength of light 106M from the converged fluorescence and side-scattered light signal 106B, converged between short and long wavelengths, enters the optical fiber 150 downwards away from its input end 151.

[0074] exist Figure 1B In the case of the system shown that utilizes the full bandwidth of light collected by optical fiber 150, the position of input 151 and the diameter of optical fiber 150 or the fiber bundle (multiple optical fibers) are set as a compromise to receive the entire wavelength range between the minimum wavelength (λmin) and the maximum wavelength (λmax).

[0075] Now refer to Figure 1D , showing the use of Figure 1B A chromatic aberration and focus shift chart for objective lens 128 of the system. Figure 1BIn the system shown, the converged fluorescence and side-scattered light signal 106B is collected by fiber 150 from short-wavelength light 106S to long-wavelength light 106L across the entire bandwidth. The Y-axis in the graph represents the wavelength of light in micrometers. The X-axis represents the chromatic aberration focal shift from a given center point to the fiber. For shorter wavelengths of light (e.g., 0.38 μm or 380 nm), there is approximately a negative 380 μm chromatic aberration focal shift. For longer wavelengths of light (e.g., 0.90 μm or 900 nm), there is a positive 380 μm focal shift. Therefore, there is approximately 760 μm (0.76 mm) chromatic aberration focal shift across the entire bandwidth. This is a relatively large chromatic aberration shift and can lead to poor performance closer to the ends of the optical wavelength bandwidth. Therefore, the diameter of one or more fibers 150 (or fiber bundle 150 if there are multiple fibers) and the position of their ends should be carefully selected to ensure that the input fiber 150 can receive wavelengths across the entire range of the desired bandwidth. The goal is to reduce the chromatic aberration focal shift entering the optical fiber, thereby enabling more efficient light collection with reduced requirements on the fiber diameter and end position.

[0076] Fluorescence signals in the fluorescence and side-scattered light signals 106B can have a wide wavelength range (λmin to λmax). Side-scattered light signals have a wavelength equal to the wavelength of the laser, which is typically also the minimum wavelength of the detection range (λmin). Autofluorescence from biological cells can have various wavelengths, but is generally higher than the excitation wavelength of the laser from the laser. It is desirable to couple optics (objectives, optical fibers, etc.) and detectors to support the full bandwidth of the desired wavelengths in the fluorescence and side-scattered light signals 106B.

[0077] Recently, there has been a desire to use ultraviolet and infrared lasers in flow cytometry to excite associated fluorescent dyes for cell labeling or staining. Therefore, for more complex analyses of biological cells in a single pass, a large wavelength bandwidth for detecting fluorescence from fluorescent dyes excited by ultraviolet and infrared lasers is desired. For such large wavelength bandwidths, overcoming chromatic aberration in such broadband systems becomes a significant challenge when using optical fibers or fiber bundles to receive and redirect the light. To effectively couple the large-bandwidth fluorescence and side-scattered light signal 106B into the optical fiber 150, it is desirable for the objective lens 128 to produce a small spot at the same location for all wavelengths of light to be detected. However, as... Figure 1DAs shown, it is physically impossible to converge all light at the same spot location. If the expected full spectrum of light wavelengths is divided into multiple wavelength ranges and multiple optical fibers are used, the distance between the ends of each fiber and the diameter of each fiber can be selected for the BFL (Bandwidth), so that the input end of each fiber 150 can better receive all wavelengths in a larger bandwidth for its corresponding wavelength range (bandwidth). An anti-reflective coating can also be selected for the ends of each fiber 150 to support a wider bandwidth and / or optimize it for a bandwidth selected within a wider wavelength range.

[0078] The input end of fiber 150 receives converged fluorescence and side-scattered light signals 106B optimized for a desired wavelength range. Fiber 150 redirects these fluorescence and side-scattered light signals 106B and transmits them from the opposite end to an array detector 160 in a flow cytometry / cell sorting system. Details of an example detector array at the opposite end of fiber 150 are shown and described in U.S. Patent No. 1,102,9243, entitled COMPACT DETECTION MODULE FOR FLOW CYTOMETERS, granted to inventors Ming Yan et al. on June 8, 2021, and are incorporated herein by reference for all intents and purposes.

[0079] exist Figure 1B In the case of the system shown, it is desirable that the optical fiber 150 and optical components within the array detector 160 support the full bandwidth range of wavelengths in the spectrum to be detected (e.g., 320 nm to 590 nm).

[0080] Now refer to Figure 1E An exemplary array detector 160 is shown. The exemplary array detector 160 is a linear array detector (photodiode array detector) in which detector channels and photodetectors are arranged in a row in space. Converged fluorescence and side-scattered light signals 106B are emitted from optical fiber 150 and coupled to the input beam-shaping optics 162 of the array detector 160. The array detector 160 includes a plurality of M micromirrors 163A to 163M, a plurality of N bandpass filters 164A to 164N, and a plurality of N detector channels D1 to Dn 165A to 165N. Figure 4CAs shown, each detector channel D1 to Dn includes photodetectors 466, 477 connected in series, gain-adjustable or selectable amplifiers 468, 478, and analog-to-digital converters (ADCs) 470, 480. The photodetectors 467, 477 in the linear array detector (photodiode array detector) can be PIN photodiodes or avalanche photodiodes (APDs). The gain-adjustable or selectable amplifiers 468, 478 allow tuning of the gain of each detector channel to compensate for noise, such as noise from over-spillage. The plurality of M micromirrors 163A to 163M can be plane mirrors, curved mirrors, or spherical mirrors. In one embodiment, the plurality of M micromirrors 163A to 163M are spherical mirrors bent to have a half-focal length, thereby forming an image on an optical filter at every other detector channel (the odd number of detector channels after the first detector channel).

[0081] Further details of the exemplary array detector are described in U.S. Patent Application No. 15 / 659,610, filed October 5, 2017, entitled "COMPACT DETECTION MODULE FOR FLOW CYTOMETERS," and U.S. Patent Application No. 15 / 942,430, filed March 30, 2018, entitled "COMPACT MULTI-COLOR FLOW CYTOMETER HAVING COMPACTDETECTION MODULE," both of which are incorporated herein by reference for all intents and purposes.

[0082] An input beamforming optics 162 is used to collect fluorescence and side-scattered light signals 106B from the optical fiber and to continue transmitting various wavelengths to different detector channels under appropriate beam conditions. From the input beamforming optics 162, the shaped fluorescence and side-scattered light signals 166 are reflected downwards and between linear rows between the corresponding bandpass filters 164A to 164N and micromirrors 163A to 163M, as shown. Depending on the bandpass filters, discrete wavelength portions of the shaped fluorescence and side-scattered light signals 166 are coupled to individual photodetectors in the respective detector channels. The intensity of the shaped fluorescence and side-scattered light signals 166 may decrease towards the last detector channels Dm and Dn, making the selected wavelengths chosen by the bandpass filters 164M and 164N more difficult to detect. Increasing the number of detector channels in the row (e.g., to 24 detector channels) to accommodate a wider wavelength bandwidth may be problematic for the signal-to-noise ratio in the last few detector channels. In some cases, the final detector channel Dn 165N can be used to detect side-scattered light formed by the laser wavelength, since it is unlikely to require more detailed analysis.

[0083] Now refer to Figure 1F A graph showing the characteristics of a standard antireflective (AR) coating for an 8-degree angle of incidence is presented, where the X-axis represents wavelength and the Y-axis represents the percentage of reflection. It is desired that in the first few passes of the array detector 160, the objective lens 128, along with the input optics 162 and detector optics (including bandpass filters (e.g., 164A to 164D) and dichroic mirrors (e.g., 163A to 163D)), cover the entire bandwidth from the minimum wavelength (λmin) to the maximum wavelength (λmax). That is, it is desired that the optics of the input optics 162 (especially the optical coating) and the first few columns of detector channels in the detector array are designed and fabricated to cover the entire bandwidth. As the initial few detector channels take up some portion of the total bandwidth to be detected, later detector channels in the chain cover a narrower bandwidth range. However, as... Figure 1FAs shown, for larger wavelength bandwidths, such as from 320 nm to 1000 nm, the performance of standard AR coatings 171 to 175 needs improvement. AR coating A 171 has an average reflectance percentage (Ravg) of less than 0.5% in a bandwidth of 350 nm to 700 nm. AR coating AB 172 has an average reflectance percentage (Ravg) of less than 1% in a bandwidth of 400 nm to 1100 nm. AR coating B 173 has an average reflectance percentage (Ravg) of less than 0.5% in a bandwidth of 650 nm to 1050 nm. AR coating C 174 has an average reflectance percentage (Ravg) of less than 0.5% in a bandwidth of 1050 nm to 1700 nm. AR coating D 175 has an average reflectance percentage (Ravg) of less than 1% in a bandwidth of 1650 to 3000 nm. Generally, the wider the bandwidth of the AR coating, the higher the reflectance percentage (Ravg).

[0084] It is desirable to apply antireflective coatings optimized to cover all wavelengths to be measured (e.g., from 320 nm to 1000 nm) to some optics in flow cytometers. For example, objective optics (objectives) 128 are designed and optimized to handle the full wavelength bandwidth of fluorescence and side-scattered light signals 106A. Therefore, it is desirable to have an antireflective coating that can be applied to objective 128 to cover the full wavelength bandwidth to improve optical efficiency.

[0085] By combining more than standard coatings together (e.g., coatings A, B, and C together), novel broadband AR coatings with varying reflectance / transmission properties across the entire wavelength range from 300 nm to 1000 nm have been developed. For example, the new broadband AR coatings exhibit a lower bandwidth average reflectance percentage (Ravg) of less than 2% in the wavelength bandwidth between 330 nm and 400 nm, a middle bandwidth average reflectance percentage (Ravg) of less than 1.5% in the wavelength bandwidth between 400 nm and 860 nm, and an upper bandwidth average reflectance percentage (Ravg) of less than 2.0% in the wavelength bandwidth between 860 nm and 1000 nm. Some transmission performance is sacrificed (Ravg of 0.5% to 1.5% or 2%) to obtain an antireflective coating with a wider wavelength range.

[0086] Standard industrial coatings cannot provide good performance over such a wide range. For conventional flow cytometers, the AB-combined AR coating 172 has an average reflectance percentage (Ravg) of less than 1% over a bandwidth of 400 nm to 1100 nm, and may be sufficient even as its reflectance increases from 0.5% to 1%. However, this wavelength range is insufficient for broadband flow cytometers with bandwidths extending beyond, for example, from 320 nm to 1000 nm. A new, custom-designed antireflective coating has been developed that combines more than two (three or more) antireflective coatings to cover the full bandwidth from 320 nm to 1000 nm. The objective lens 128 near the flow cell will be coated with this new combined antireflective coating, consisting of three or more antireflective coatings, to support the full bandwidth of light. Optics in the input channels of the individual array detectors, as well as mirrors and optical filters for the first to third detection channels of the respective array detectors, can be coated with antireflective coatings based on the corresponding wavelength range of light detected by the respective array detector. Therefore, components with novel, custom-designed anti-reflective coatings for the first few detector channels, mirrors, and bandpass filters may be more expensive than those in more conventional systems. Furthermore, anti-reflective coatings can be selected for the desired bandwidth range and applied to the ends of the fiber, thereby improving optical efficiency.

[0087] Now refer to Figure 2 To improve detection of higher bandwidths, the objective output (side scattering and fluorescence signal 106B) from objective lens 128 can be split into two or more beams. Typically, optoelectronic system 200 may include one or more coupling devices (e.g., beam splitters, dichroic mirrors, or optical fibers) to capture a portion of the bandwidth or a portion of the light intensity and redirect it to other detectors, thereby using multiple detectors associated with all lasers to detect the full bandwidth.

[0088] exist Figure 2 In this process, one or more lasers 101 are used to generate one or more laser beams 102. When a moving biological cell in the interrogation region of the flow chamber 102 of the flow cell 100 is struck, one or more laser beams are used to excite different fluorescent dyes to different wavelength bands to form side-scattered light and fluorescence signals 106A, as well as forward-scattered light and laser 112. Instead of attempting to use a single detector, multiple detectors 260A to 260C are used to detect a larger full bandwidth. The spectral results from each of the multiple detectors 260A to 260C are fused together by a central processing unit 299 executing firmware instructions to represent the full bandwidth spectral results.

[0089] According to one embodiment, an optoelectronic system 200 in a flow cytometer is shown. The optoelectronic detection subsystem 200 includes an objective lens 128, one or more beam splitters 220A to 220B, multiple array detectors 260A to 260C, and a processor 299. When moving cells and their fluorescent dyes are struck by various laser beams 102, the objective lens 128 collects side-scattered light signals and fluorescence signals 106A. The objective lens 128 converges the side-scattered light signals and fluorescence signals 106A into the array detectors 260C as converged side-scattered light signals and fluorescence signals 106B having the full bandwidth (e.g., λ0 to λ4).

[0090] One or more beam splitters (or dichroic mirrors) 220A to 220B split the output 106B from objective lens 128 into two or more beams 106C to 106D, while the output 106B continues to enter array detector 206C. Each beam in the two or more beams 106C to 106D is coupled to a different array detector assembly 260A to 260B. Beam splitter (or dichroic mirror) 220A also redirects the beam of side-scattered light signal and fluorescence signal 106C in the first wavelength range along optical axis 111A into array detector 260A. Beam splitter (or dichroic mirror) 220B also redirects the beam of side-scattered light signal and fluorescence signal 106D in the second wavelength range along optical axis 111B into array detector 260B. The remaining portion of the beam 106B in the third wavelength range enters array detector 206C through one or more beam splitters (or dichroic mirrors). Each detector assembly 260A to 260C processes portions of the side-scattered light signal and fluorescence signal 106B with different wavelength ranges. The different wavelength ranges separated into detector assemblies 260A to 260B are smaller than the original wavelength range of the side-scattered light signal and fluorescence signal 106B output from lens 128.

[0091] The coupling optics in the detector assembly can be lenses, optical fibers, gratings (Bragg gratings), dichroic mirrors, or other types of optics. Different optics can be selected for each detector assembly to resolve and detect the wavelength of the light it receives. For example, a grating can be used in array detector 260A, a filter can be used in detector 260B, and a prism can be used in detector 260C.

[0092] Each array detector component 260A to 260C generates a digital signal for its respective wavelength range to be detected. These digital signals can be combined in a processor to provide a response across the entire wavelength range of the side-scattered light signal and the fluorescence signal 106B. The processor 299 and digital-to-analog converter within the array detectors can be synchronized with the same clock signal and a given cell detection event. The processor 299 executes instructions to combine the results from the multiple array detectors 260A to 260C. The individual spectral results from each array detector are arranged together according to the wavelength bandwidth received by each array detector.

[0093] The spectral result from array detector 260A, which receives a lower wavelength bandwidth (e.g., from λ0 to λ1), first provides the next spectral result along the spectrum. The spectral result from the next array detector 260B, which can receive wavelengths with intermediate bandwidths (e.g., from λ1 to λ2), provides the next spectral result along the spectrum. The spectral result from array detector 260C, which receives higher bandwidth wavelengths (e.g., from λ3 to λ4), can be the final spectral result along the spectrum. In the spectral results, gaps may exist in the wavelengths of light to be detected by adjacent detectors (e.g., see [reference needed]). Figure 3D This is to avoid unwanted optical signals, such as those from lasers. There can also be overlapping wavelengths of light between adjacent detectors (e.g., see...). Figure 3C (e.g., light from the transition wavelength range, which is a characteristic of spectroscopic optics). In any case, the spectral results from the array detector are in digital form, so the processor 299 can relatively easily arrange them along the wavelength spectrum.

[0094] For now, please refer to... Figure 3B A graph illustrating the transmission characteristics 502 of an example beam-splitter dichroic mirror is shown. The transmitted wavelength range (e.g., above λt2) and the reflected wavelength range (e.g., below λt1) can be altered by the design of the beam-splitter dichroic mirror. In the transmission characteristics 502 of the beam-splitter dichroic mirror, there is typically a transition region 504 with wavelengths (λt1 to λt2) of a beam-splitting optics (e.g., a beam splitter or dichroic mirror) used to separate optical signals into individual different wavelength bandwidths. The transition region 504 is typically around the center wavelength 506 of the laser light, which can be ignored by the detector. However, the transition region 504 can cause overlapping wavelengths of light between adjacent detectors. For example, there can be overlapping wavelengths between wavelength ranges from λ1 to λ2 and from λ3 to λ4, where the wavelength of λ2 is greater than the wavelength of λ3, as shown below. Figure 3B As shown (see also) Figure 3CIn this case, wavelengths in the transition wavelength range between λ2 and λ3 can appear with partial intensity in both array detectors 260B and 260C. The separated wavelength 506 can be set at a laser wavelength that does not need to be detected, such as the center wavelength of the laser 101 that generates laser 102. Sometimes a gap may exist between wavelengths (e.g., between wavelengths λ2 and λ3, where λ2 is less than λ3 – see...). Figure 3D In any case, the spectral results from the array detectors are digital, making the processor 299 relatively easy to program to address overlapping wavelengths between detectors and wavelength gaps between detectors, so as to arrange all the results together along the spectrum of light.

[0095] Now return to the reference. Figure 2 The optoelectronic system 200 extends the spectral range of a flow cytometer to the ultraviolet (UV) and near-infrared (NIR) wavelength ranges. It is very difficult to make optics cover a large wavelength range including visible, UV, and NIR wavelengths. The disclosed embodiments provide a way to divide a large wavelength range (spectral range) into several smaller wavelength ranges and process them in parallel using several different detector assemblies. By separating the wavelength ranges, good spectral performance can be achieved over a larger wavelength range.

[0096] Now refer to Figure 3A The diagram illustrates an optoelectronic system 300 that uses one or more dichroic mirrors 320 and multiple different optical fibers (or multiple fiber bundles) 350A to 350B as coupling devices to multiple array detectors (optical detection devices) 360A to 360B. The extended wavelength range of the side-scattered and fluorescence signals 106B is separated into two or more ranges by one or more dichroic mirrors (beam splitters) 320 and coupled to multiple array detectors via multiple optical fibers 350A to 350B. Although in Figure 3A Only one dichroic mirror (beam splitter), two optical fibers, and two array detectors are shown in the diagram, but the optoelectronic system 300 can be expanded to include more dichroic mirrors, more optical fibers (or fiber bundles), and more array detectors to further divide the desired spectrum into multiple wavelength ranges (bandwidths) for detection by flow cytometers or cell sorters. One or more dichroic mirrors (beam splitters) 320 have their planes at a 45-degree angle to the optical axis 110 to guide reflected light along the optical axis 111A, which is at a 45-degree angle to the optical axis 110.

[0097] Mechanical mount 155B, connected to optical fiber 350A, is used to maintain the BFL distance between the input end of optical fiber 350B and the back surface of objective lens 128 via dichroic mirror 320, but may be adjustable to allow for small variations in back focal distance. Objective lens 128 focuses light into a spot on the input end of the optical fiber. In one embodiment, the spot size matches the core size of the optical fiber at the BFL distance. Mechanical mount 155A, connected to optical fiber 350A, is used to maintain the total BFL distance between the end of the optical fiber and dichroic mirror 320 and between dichroic mirror 320 and the back surface of objective lens 128, but may be adjustable to allow for small variations in back focal distance. Mechanical mount 155C is connected to dichroic mirror 320 and base 140 to maintain their position and angle relative to optical axis 110. Mechanical mounts 155A to 155B are connected together with objective lens 128 to base 140, such as an optical bench, to also maintain alignment of fibers 350A to 350B with their respective optical axes 110, 111A. The selection of fiber 350A (e.g., core diameter) and its back focal length (BFL) distance can be optimized for a first sub-wavelength range, which is a subset of the overall wavelengths expected to be generated at the flow cell by exciting the fluorophores, particles, and cells flowing through the flow cytometer. The back focal length (BFL) distance of fiber 350B can be optimized for a second sub-wavelength range that is expected to be received, different from the first sub-wavelength range. The anti-reflective coating for the optics in the input of detector 360A and the initial detector channel, as well as for at least the input of fiber 350A, can be selected based on the first sub-wavelength range to be received. The anti-reflective coating for the optics in the input of detector 360B and the initial detector channel, as well as for at least the input of fiber 350B, can be selected based on the second sub-wavelength range to be received.

[0098] In one aspect, the extended wavelength range in the side scattering and fluorescence signal 106B is from 320 nanometers (nm) to 1000 nanometers (nm). A single or first dichroic mirror 320 may be arranged at an angle along the optical axis 110. Figure 3B An exemplary transmission characteristic of the dichroic mirror 320 is illustrated. The first dichroic mirror 320 is a key component in the system 300. Similar to an objective lens, the first dichroic mirror 320 receives the entire spectral range (including the extended wavelength range spectral range) of light from side-scattered and fluorescence signals 106B and divides its wavelength range into two parts through transmission and reflection. Due to inherent characteristics in the dichroic mirror design, a transition region 504 exists that allows the dichroic mirror to change from high reflectivity to high transmission (see [link to dichroic mirror design]). Figure 3B The transition region 504 can be positioned around the center wavelength of the laser, as shown by laser line 506, for example. The wavelength range (λt1 to λt2) within the transition region 504 is typically not used by flow cytometers to avoid noise signals from the laser. For example, in... Figure 3BIn this configuration, the dichroic mirror has a transition region 504 around the laser line 506 (e.g., 561 nm) in its transmission characteristic 502. Light signals with wavelengths below wavelength λt1 (e.g., 550 nm) are reflected by the dichroic mirror into the first optical fiber used for the first array detector. Light signals with wavelengths above wavelength λt2 (e.g., 570 nm) are transmitted by the dichroic mirror and received by other different optical fibers / detectors and beam splitters (dichroic mirrors). Light signals with wavelengths from (λt1 to λt2) in the transition region 504 are partially reflected / transmitted by the dichroic mirror into both paths (two optical fibers). Optical filters in the detector array can provide gaps in the spectrum to prevent the detector from detecting light in the transition region. Alternatively, the optical filters in the detector array can have some overlap, such that all data is captured, and if necessary, data in or around the laser line 506 in the transition region 504 can be ignored by the processor.

[0099] Now refer to Figure 3C A block diagram illustrating the characteristics of an optical filter for the wavelength range of two separate detectors with overlapping wavelength portions is shown. The wavelength range of the linear array detector module is defined by a series of optical filters (e.g., bandpass, low-pass, high-pass) 164A to 164N for each detector channel. In the case of an optical bandpass filter, two suppression bands R1 and R2 exist around the passband PB of the light wavelength in its transmission characteristics. The two suppression bands R1 and R2 can be reflective or absorptive. In the series of bandpass optical filters in the linear array detector module, the two suppression bands are reflective to reflect the suppressed light onto the next optical filter in the series. In the transmission characteristics of the optical bandpass filters in this series, each optical bandpass filter can have a sharp rising edge to accurately capture the starting wavelength, while the falling edge slope is looser and overlaps into the passband of the next optical filter in the series. The overall transmission characteristics of the optical filter series 370A and 370B result in substantially continuous detection by the first detector module in the wavelength range of λ1 to λ2, and substantially continuous detection by the second detector module in the wavelength range of λ3 to λ4.

[0100] exist Figure 3CIn the optical wavelength spectra of the overall transmission characteristics 370A to 370B used for the first detector module 360A and the second detector module 360B respectively, there is an overlapping wavelength range 372 between wavelength λ3 and wavelength λ2 (where wavelength λ2 is greater than wavelength λ3) to ensure that all spectral data is captured between adjacent detector modules. Data related to laser lines can be ignored by the processor. The last optical filter in the first detector module has the optical filter transmission characteristic 374N in the overall transmission characteristic 370A. The first optical filter in the second or adjacent detector module has the optical filter transmission characteristic 374A in the overall transmission characteristic 370N. The wavelength portion 372 in the passband PB1 of the optical filter transmission characteristic 374A overlaps with the wavelength portion 372 in the passband PBN of the optical filter transmission characteristic 374N.

[0101] Now refer to Figure 3D A block diagram illustrating the filtering characteristics of a beam splitter detector with a spectral gap or non-overlapping wavelength range 373 is shown. The gap or non-overlapping wavelength range 373 lies between wavelengths λ2 and λ3 (where wavelength λ2 is less than wavelength λ3) in the optical wavelength spectrum of the overall transmission characteristics 370A to 370B used for the first detector module 360A and the second detector module 360B, respectively. Typically, the laser line associated with the wavelength of the excitation laser is within the gap 373, so the laser line can be ignored by adjacent detector modules and their corresponding array detectors.

[0102] exist Figure 3D In the first detector module, the last optical filter has optical filter transmission characteristic 374N in the overall transmission characteristic 370A. The first optical filter in the second or adjacent detector module has optical filter transmission characteristic 374A in the overall transmission characteristic 370N. The passband PB1 of optical filter transmission characteristic 374A does not overlap with the passband PBN of optical filter transmission characteristic 374N. In the transmission characteristics of the optical bandpass filters in this series, each transmission characteristic can have a sharp rising edge to accurately capture the starting wavelength. Typically, the falling edge of the transmission characteristic of each optical bandpass filter is relatively loose in slope and overlaps into the passband of the next optical filter in the series. However, the falling edge of the passband PBN of optical filter transmission characteristic 374N can also have a sharp falling edge to better define and reduce the wavelength range in the gap 373 around the laser line.

[0103] Refer again Figure 3AAccording to one embodiment, the dichroic mirror 320 separates the wavelengths of the side-scattered and fluorescent light signals 106B from 320 nanometers (nm) to 550 nanometers (nm) into a lower bandwidth optical signal 106C. The lower bandwidth optical signal 106C is redirected along the optical axis 111A at an angle to the optical axis 110 toward the input end 352A of the optical fiber 350A or an equivalent fiber bundle. The lower bandwidth optical signal 106C is redirected and emitted from the opposite end of the optical fiber 350B into the array detector 360A.

[0104] The remaining wavelengths (570 nm to 1000 nm) in the high-bandwidth optical signal 106D from the side-scattered and fluorescence signals 106B are allowed to pass through the dichroic mirror 320 and coupled to the input end 352B of the optical fiber 350B or an equivalent fiber bundle. The high-bandwidth optical signal 106D is redirected by the optical fiber 350B and emitted from its opposite end to the array detector 360B. If more dichroic mirrors, more optical fibers, and more array detectors are used, the wavelength range of the optical signal from 570 nm to 1000 nm can be further subdivided for individual analysis.

[0105] Due to the transmission characteristics 502 of the dichroic mirror, the optical signal in the wavelength range (e.g., 550 nm to 570 nm) of the transition region 504 is coupled to the two fiber bundles with only partial intensity. The wavelength range in the transition region 504 is designed not to be measured because it is close to the wavelength of the laser line at 561 nm.

[0106] In a flow cytometer, an optical objective (e.g., a lens) 128 is used to collect lateral scattered and fluorescence signals 106A from the flow chamber 120 of the flow cell along the optical axis 110. The optical objective (e.g., a lens) 128 focuses the lateral scattered and fluorescence signals 106A into small spots that are desired to be coupled into optical fibers 350A to 350B at their respective input ends 352A to 352B. Preferably, the diameter of the small spot matches the core diameter of the optical fiber, but the various wavelengths in signals 106A, 106B make it difficult for all wavelengths to work in the same way, as discussed here regarding chromatic aberration and focal shift. This can be mitigated to some extent by dividing the range of wavelengths to be detected and selecting different core diameters and different back focal lengths (BFL) distances for different optical fibers based on the range of wavelengths to be received.

[0107] like Figure 1C As shown, different wavelengths are typically focused at different locations at the fiber input. A single fiber is often placed at a damaged location in an attempt to collect as many wavelengths as possible across the entire bandwidth. Figure 3AIn the illustrated case, the objective light output 106B is split into two or more beams and coupled into two or more different optical fibers 350A to 350B. With the wavelength ranges separated, it is easier to optimize the BFL distance from one end of each fiber to the output surface of the objective lens 128. For example, the position of the input end 352A of fiber 350A can be optimized using the total distance along optical axes 111A and 110 to the output surface of lens 128 to collect light in the wavelength range of 320 nm to 550 nm. The position of the input end 352B of fiber 350B can be optimized using the total distance along optical axis 110 to the output surface of lens 128 to collect light in the wavelength range of 570 nm to 1000 nm. The gap between 550 nm and 570 nm is due to the need to avoid the 561 nm center laser beam wavelength. Furthermore, the optics receiving light after objective lens 128 and mirror 320 can be adjusted for the correspondingly separated wavelength ranges they receive and further transmit to improve efficiency and reduce cost.

[0108] For now, refer to Figure 7 The chromatic aberration focus shift graph in the figure shows wavelengths in micrometers along the Y-axis, while the relative focus shift, starting from zero, is positioned along the X-axis. Fiber 350A supports wavelengths from 320 nm to 550 nm, as indicated by the height of the relevant box, the width of which is defined by the maximum / minimum points on the curve within the wavelength range. For fiber 350A, there is a relative chromatic aberration focus shift of approximately 0.35 mm. Fiber 350B supports wavelengths from 570 nm to 1000 nm, as indicated by the height of the relevant box, the width of which is defined by the maximum / minimum points on the curve within the wavelength range. For fiber 350B, there is a relative chromatic aberration focus shift of approximately 0.32 mm. Figure 1D An example of chromatic aberration focus shift is shown for a single fiber 150. In Figure 1D In this study, the relative chromatic aberration focus shift of a single fiber 150 is approximately 0.76 mm, significantly greater than that of fibers with... Figure 7 The relative chromatic aberration focus shifts of any one of the separated wavelength ranges of optical fibers 350A to 350B are shown. Therefore, a dual-fiber configuration with separated wavelength ranges will provide much better optical coupling efficiency than a single fiber with light across the full bandwidth.

[0109] Return to reference Figure 3AWhen the wavelength range of 320 nm to 550 nm in the side-scattered and fluorescence signals 106B is emitted, redirected, and transmitted to the array detector 360A, the array detector 360A can be optimized to process this wavelength range of the optical signal. When the remaining wavelength range of 550 nm to 1000 nm in the side-scattered and fluorescence signals 106B is allowed to pass, redirected, and transmitted to the array detector 360B, the array detector 360B can be optimized to process this wavelength range of the optical signal. Not only can the array detectors 360A to 360B be optimized for the corresponding wavelength range of the optical signals they receive, but the optical fibers 350A to 350C can also be optimized for the corresponding wavelength range of the optical signals they will redirect and transmit to the respective array detectors.

[0110] Figure 8A An example is shown: a multimode fiber patch cord (cable) 800 with anti-reflective coating, customizable for use with fiber optics 350A to 350B. For example, cable 800 could be a THORLABS type M200L02S-B cable. Cable 800 has an input end 801 and an output end 802, with connectors around them. Figure 8B Enlarged views of the various ends 801, 801 are shown. The optical cable 800 may have a multimode core 804 with various core diameters based on the cable's intended operating wavelength range. For example, core diameters of 50 micrometers, 100 micrometers, or 200 micrometers can be selected to optimize the collection of wavelengths separating side scattering and fluorescence signal 106B. The input end 801 and output end 802 of the optical cable may also have various anti-reflective coatings based on the cable's intended operating wavelength range.

[0111] exist Figure 8C In the diagram, various anti-reflective coatings (ARCs) illustrate the different wavelength operating ranges of the optical cable 800. A first anti-reflective coating 811 is used to optimize, for example, the operating wavelength range between 250 nm and 370 nm. A second anti-reflective coating 812 is used to optimize, for example, the operating wavelength range between 400 nm and 700 nm. A third anti-reflective coating 813 is used to optimize, for example, the operating wavelength range between 650 nm and 1100 nm. Therefore, each of the multiple optical fibers 350A to 350B used with the system 300 can be customized to desired separate wavelength ranges, such as 320 nm to 550 nm and 570 nm to 1000 nm, by their respective core diameters and anti-reflective coatings.

[0112] Figure 4AA conceptual block diagram of two array detectors 360A to 360B is illustrated. The two array detectors 360A to 360B can be packaged together as a single dual array detector module 400 in a single package, wherein two fiber optic inputs 450A to 450B are coupled to two input channels having input optics 462A to 462B, respectively. Array detector 360A in the package is optimized to process optical signals in the wavelength range from 320 nm to 550 nm, and the second array detector 360B in the package is optimized to process optical signals in the wavelength range from 570 nm to 1000 nm. Therefore, the optics and optoelectronic devices (e.g., mirrors, bandpass filters, photodetectors) within each array detector 360A to 360B can be optimized to support their narrower bandwidth wavelength range. For example, the photodetectors in the input optics 462A, dichroic mirrors 463A-463L, bandpass filters 464A-464M, and detection channels 465A to 465M of the array detector 360A can all be optimized for a bandwidth range of light from 320 nm to 550 nm. Similarly, the photodetectors in the input optics 462B, dichroic mirrors 473A to 473M, bandpass filters 474A-474N, and detection channels 475A to 475N of the array detector 360B can all be optimized for a bandwidth range of light from 570 nm to 1000 nm. Each of the multiple dichroic micromirrors 463A to 463L, 473A to 473M can be a plane mirror, a curved mirror, or a spherical mirror. In one embodiment, the plurality of dichroic micromirrors 463A to 463L, 473A to 473M are spherical mirrors having a radius of curvature to have half a focal length, such that an image is formed on an optical filter at every other detector channel (an odd number of detector channels after the first detector channel).

[0113] Now refer to Figure 4BThe array detectors 360A and 360B may contain different numbers of detection channels 409A and 409B and photodetectors, which are packaged together for supporting different bandwidths of light. Center wavelengths 410A and 410B and bandwidths 411A and 411B around these center wavelengths can be assigned to the respective photodetectors in each detection channel number 409A and 409B for supporting different bandwidths of light. The first center wavelength of the detector typically begins above the center wavelength of the laser that excites the fluorescent dye. For example, for a 300 nm wavelength UV laser, the center wavelength of the first detector in the array can be set to, for example, 320 nm. For each detection channel 409A and 409B, the bandwidths 411A and 411B around the center wavelengths 410A and 410B can be different. For example, bandwidths 411A and 411B can range from a small number of wavelengths such as 15 nm (plus or minus 7.5 nm) to a large number of wavelengths such as 34 nm (plus or minus 17 nm).

[0114] Furthermore, the number of detectors supported for different bandwidths can vary. For example, array detector 360A can have M detection channels 409A to support light in the 320 nm to 550 nm range. Array detector 360B can have N detection channels 409B to support light in the 570 nm to 1000 nm range. The bandwidths of each array detector can be different, and the bandwidths around the center wavelength can be different, such that in different array detectors, the number of detection channels N is greater than M, or the number of detection channels M is greater than N.

[0115] By utilizing light with varying bandwidths supported by array detectors, the optics used in each array detector can differ, providing better performance at a lower cost. The input optics 462A of array detector 360A can be optimized to support light in the wavelength range of 320 nm to 550 nm. The input optics 462B of array detector 360B can be optimized to support light in the wavelength range of 570 nm to 1000 nm. For example, the anti-reflective coatings used for each input optics can be optimized to support light in the corresponding wavelength range. The optics of the different photodetectors in the different array detectors can be optimized for their respective center wavelengths and corresponding bandwidths around those center wavelengths along the detector channel chain. For example, the anti-reflective coating for a corresponding photodetector can be optimized for its center wavelength and its bandwidth around that center wavelength. The bandpass filters in the earlier channels of the chain, such as bandpass filters 464A and 474A for detector channel 1 in each array detector 360A and 360B, are optimized to support their respective bandwidths and substantially reflect more of their respective bandwidths compared to bandpass filters 464M and 474N in the respective last detector channels M and N in each array detector 360A and 360B.

[0116] In the case where each detector array 360A to 360B is a linear photodetector array, it includes one or more input optics 462A to 462B to receive light from one or more optical fibers; multiple detector channels 465A to 465M, 475A-475N; multiple micromirrors 463A to 463L, 473A to 473M; and multiple optical filters 464A to 464M, 474A to 474N. Figure 4C As shown, each detector channel 465A to 465M (collectively referred to as 465) and 475A to 475N (collectively referred to as 475) includes one of a plurality of filters 464 and 474; optical devices (e.g., lenses) 466 and 476; photodetectors (PDs) 467 and 477; gain-selectively adjustable amplifiers 468 and 478; and analog-to-digital converters (ADCs) 469 and 479. Bandpass filters 464 and 474 receive an incident light beam 406I at an angle to the incident optical axis. Bandpass filters 464 and 474 remove or filter out discrete wavelength ranges from the incident light beam 406I and allow it to pass as light 406P, which is passed through or filtered out in the passband around the center wavelength. The passed or filtered light 406P with discrete wavelength ranges is intended to be detected by a given photodetector 467 or 477. Bandpass filters 464 and 474 will also reflect the reflected light 406R within the remaining wavelength in the suppression band to the next mirror in the serial chain at the exit angle. The incident and exit angles are similar (equivalent) to the planes of bandpass filters 464 and 474.

[0117] Optical devices 466 and 476 converge the filtered light 406P to photodetectors 467 and 477. The optical devices can be integrated with photodetectors 467 and 477. Photodetectors 467 and 477 convert the light 406P into an analog electrical signal, which is coupled to gain-selectively adjustable amplifiers 468 and 478. Gain-selectively adjustable amplifiers 468 and 478 amplify the analog electrical signal as needed to provide a better signal-to-noise ratio based on a gain selection signal. The amplified analog signal is coupled to ADCs 469 and 479. ADCs 469 and 479 are clocked and convert the amplified analog signal into a digital signal output for a given detection channel on a portion of the clock cycle (the first phase on the edge), and then retain the digital signal during the next portion of the clock cycle (the second phase on the opposite edge). An ADC may include registers, memory, or other storage devices 470, 480 to store one or more digital signals generated by a processor for collection into a larger storage device, such as a computer's solid-state storage device or hard disk drive. The digital signals represent discrete portions of the wavelength of light (e.g., 50 nanometers) around the center wavelength of the passband of an optical filter.

[0118] Each array detector 360A to 360B generates multiple digital signals for its respective wavelength range of detection. Array detector 360A generates a first plurality of digital signals representing discrete wavelength portions within a first wavelength range. Array detector 360B generates a second plurality of digital signals representing discrete wavelength portions within a second wavelength range. If there are N detection channels in array detector 360A and M detection channels in array detector 360B, there are N plus M digital signals, which can be combined by processor 299 into a combined detection spectral signal covering the entire wavelength (full bandwidth) range of the side-scattered light signal and fluorescence signals 106A, 106B. Clock signal generator 399 can generate a clock signal coupled to the processor and array detectors 360A to 360B to provide a synchronized timing signal. The processor 299 and digital-to-analog converter in the array detector can be synchronized with the same clock signal and timestamped for the digital signals and the number of cell detection events, thus allowing signals to be easily grouped together for each cell detection event. Processor 299 executes instructions to combine results from multiple array detectors 360A to 360B. Individual spectral results from each array detector are arranged together based on the corresponding wavelength bandwidth received by each array detector for each event (excitation of fluorescent dye / detection of cells).

[0119] Now refer to Figures 10A to 10CSubsequently, a digital microprocessor or digital signal processor can be used to group the results from various detector types together for a full-spectral response over the desired full bandwidth. Figure 3A A central processing unit 299 is illustrated, which is used to stitch together the results from the individual linear detector arrays 360A and 360B to obtain a full-spectral response over the desired full bandwidth range. Figure 2 An example of a central processing unit 299 is shown, which is used to arrange and combine the results from the individual detector modules 260A, 260B, and 260C to obtain a full-spectral response over the desired full bandwidth. Two or more detector modules, each having a series of multiple optical filters, a series of multiple mirrors, and a series of multiple photodetectors (PMTs, photodiodes, or avalanche photodiodes), can be used to detect and form corresponding portions of the fluorescence spectrum (or autofluorescence spectrum) generated by excited fluorescent pigments attached to cells or particles.

[0120] exist Figure 10A In this configuration, the first fluorescence spectral portion 1000A is detected and captured by a first linear detector array and a series of multiple photodetectors (e.g., in detector module 360A). Figure 10B In this process, the second fluorescence spectral portion 1000B is detected and captured by a second linear detector array and a series of multiple photodetectors (e.g., in detector module 360B). Central processing unit (signal processor) 299 (see...) Figure 3A The detector modules 360A and 360B are synchronized with clock signals generated by clock signal generator 399 and coupled to the respective clock signals. Data generated by detector modules 360A and 360B can be additionally tagged with the same event number, allowing appropriate data to be merged and stitched together. Software instructions executed by the processor convert / transform the data from the individual detector modules from the time domain to the wavelength / frequency domain. The wavelength / frequency domain spectral data 1000A and 1000B of the individual detector modules can then be arranged based on wavelength, stitched together, and merged to provide a holistic spectrum 1000C of cells / particles / dyes excited by different lasers and sensed by the detector array. Any overlap in the spectrum between detector arrays due to beam splitters can be arranged and stitched together, their corresponding intensities added together to provide overall detection in the overlapping spectra. One or more laser gaps 1073 can be formed by removing data around the center wavelengths of two or more excitation lasers using the holistic spectrum 1000C, or otherwise formed by gaps between detector modules.

[0121] Multiple detectors packaged together

[0122] Now refer to Figure 4D The diagram illustrates a dual compact wavelength detection module 400. The dual compact wavelength detection module 400 includes dual array detectors 360A to 360B, which share a transparent optical block 480 within a detector module portion 414 mounted on a base 410. For array detector 360A, light is received from a first optical fiber 450A and propagates from left to right along a series of optical filters 464A to 464M and a series of reflectors 463A to 463L, respectively, and enters a series of detector channels 465A to 465M. For array detector 360B, light is received from a second optical fiber 450B and propagates from right to left along a series of optical filters 474A to 474N and a series of reflectors 473A to 473L, respectively, and enters a series of detector channels 475A to 475N. Although a dual 8-bit detector array is shown, other even-numbered combinations of detector channels (e.g., 2, 4, 6, 10, 12, 14) or other odd-numbered combinations of detector channels (e.g., 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, etc.) available in the package can be used to form dual detector arrays for different wavelength bandwidths. One or more photodetectors in the package (equipped with optical filters to receive (allow transmission) laser light of one or more center wavelengths) can be used as side-scattering detectors for one or more lasers.

[0123] The dual compact wavelength detection module 400 includes a first input stage (head or input channel) 401A, a second input stage 401B, and a detection module 414 mounted to a base 410 to keep them aligned together. Light from a first wavelength range from the flow cell is coupled into the first input stage (head or input channel) 401A via optical fiber 450A. Light from a second wavelength range from the flow cell is coupled into the second input stage (head or input channel) 401B via optical fiber 450B. The first input stage (head or input channel) 401A includes input optics 462A, and the second input stage (head or input channel) 401B includes input optics 462B. The input optics 462A to 462B can be customized to best receive light of each wavelength from the respective optical fibers 450A to 450B. Typically, each input optics in input optics 462A to 462B may include a collimating lens 402, a long-pass filter 403, a clearing optical blocker 404, and a converging lens 405 mounted in the housing or optical bench of input stages (heads or input channels) 401A to 401B. Input stages (heads or input channels) 401A to 401B set the magnification M1 to M2 of the initial spot size image A(1) for each array detector on the first optical filters 464A to 474A. The corresponding laser is output from the input stage and coupled to the detection module 414 of the dual array detector.

[0124] The ends of each input stage (head or input channel) 401A to 401B are coupled to corresponding transparent wedges 407A to 407B to receive corresponding laser light from the converging lenses 605 of the input optics 462A to 462B. The detection module 414 includes a dual 1f image array 408 for the corresponding dual array detectors 360A to 360B. The image array 408 includes a transparent block 480 with wedges 407A to 407B and micromirrors 463A to 463L, 473A to 473L on one side. On the opposite side of the transparent block 480, optical filters 464A to 464M, 474A to 474N are present.

[0125] According to one embodiment, optical filters 464A to 464M, 474A to 474N are bandpass optical filters having a transmission band surrounded by a pair of reflection suppression bands. In another embodiment, optical filters 464A to 464M, 474A to 474N may be low-pass optical filters having both a transmission band and a reflection suppression band. In yet another embodiment, optical filters 464A to 464M, 474A to 474N may be high-pass optical filters having both a transmission band and a reflection suppression band.

[0126] Light coupled to the image array 408 from the first input stage 401A is wavelength demultiplexed into photodetectors in detector channels 465A to 465M. Light coupled to the dual 1f image array 408 from the second input stage 401B is wavelength demultiplexed into photodetectors in detector channels 475A to 475N. The dual detection module can analyze a pair of wavelength ranges of fluorescence from the flow cell and side-scattered light from one or more lasers.

[0127] Wavelength demultiplexers in each linear array detector are formed by an optical block to receive light from at least one optical fiber; multiple optical filters are arranged in a row on one side of the optical block to transmit different discrete portions of light and reflect the remaining portions; and multiple micromirrors are arranged in a row on the opposite side of the optical block to receive the remaining portions of light reflected from each of the optical filters except for the last optical filter. The multiple micromirrors reflect the remaining portions of light received from the respective optical filters toward the next optical filter in the row. The first optical filter in this series is somewhat unique because it directly receives light from at least one optical fiber through optics in the optical head (input channel). The last optical filter in this series may be slightly different because it no longer needs to reflect light, but can instead absorb only the light in the stopband instead of reflecting it. Multiple photodetectors are arranged in series below the multiple optical filters to receive different discrete portions of light and convert them into electrical signals.

[0128] The detector channel series D1 to D8 in the array detector 360A and 465A to 465M include multiple photodetectors (e.g., Figure 4C Photodetectors 466, 467), each photodetector has a lens (e.g., Figure 4C Optical devices 466, 474 are used to focus the demultiplexed light into a photodetector. Similarly, the detector channel series D1 to D8 475A to 475N in the array detector 360B includes multiple photodetectors (e.g., Figure 4C Photodetectors 466, 467), each photodetector has a lens (e.g., Figure 4C Optical devices 466, 474 are used to focus the demultiplexed light onto a photodetector. Each detector channel in array detectors 360A to 360B also includes adjustable or selectable gain amplifiers 468, 478; and analog-to-digital converters (ADCs) 469, 479, which, together with… Figure 4C The photodetectors 466 and 467 shown are coupled together in series.

[0129] like Figure 4A and Figure 4DAs shown, dual array detectors 360A to 360B are packaged together in a single package as single array detectors 360 and 400 with two fiber optic inputs 450A to 450B. However, the overall bandwidth of the fluorescence and side-scattered signals can be further separated into more than two paths and two signals. Therefore, in situations such as Figure 5 and Figure 6B The system shown can use more than two array detectors. If space permits, more than two array detectors can be arranged in a row. Alternatively, two array detector pairs in a row can be arranged vertically and packaged back-to-back (front to front, or back to front), possibly sharing a base or optical plate with the input channel and optical block mounted on the opposite side, to provide three or four array detectors in a single compact package. Arranging photodetectors together on one side of the package simplifies electrical connections. Arranging optical connections on the opposite sides of the photodetectors within the package (e.g., on the left and right sides of the top side) simplifies the location for optical connections. Furthermore, the cooling location of the avalanche photodiode photodetector on one side of the package can be centralized to further compress the flow cytometer. More than one package with multiple detector arrays can be provided in a flow cytometer with supporting optics to achieve desired efficiency improvements in detection with increased bandwidth of full fluorescence spectra.

[0130] Now refer to Figure 5 This diagram illustrates the integration of multiple N array detectors 360A to 360N into a single package within an array detector 500 having N fiber optic inputs 450A to 450N. Combining multiple detectors into a single integrated package saves space and cost compared to using separate packages for each detector. The multiple N array detectors 360A to 360N divide the overall bandwidth range into N smaller, distinct bandwidth ranges (sub-wavelength ranges or sub-bandwidths). Within the overall bandwidth range from wavelength λ0 to λn, the first array detector 360A detects the wavelength range from λ0 to λ1. The second array detector 360B detects the wavelength range from λ1 to λ2. The nth array detector 360N detects the wavelength range from λm to λn. Each of the N array detectors 360A to 360N receiving different wavelength ranges can have customized input optics 462A to 462N to receive the transmitted signal from each fiber coupled to the N fiber optic inputs 450A to 450N. Each fiber can be selected to be coupled to one of the N fiber inputs from 450A to 450N, and the inputs coated with an anti-reflective coating are optically effective for the different wavelength ranges received and transmitted in the various array detectors.

[0131] Multiple N array detectors 360A to 360N are combined to provide Z detector channels with Z photodetectors for the overall bandwidth range. For example, Z could be 24 detector channels with 24 photodetectors. The total number of detectors in each array detector can vary. For example, assume three array detectors 360A, 360B, and 360C have a total of 24 detector channels. For example, the first array detector 360A could have 10 detector channels, while the second array detector 360B and the third array detector 360C could each have 6 detector channels. Although details of the 8-bit detector array are shown and described, detector arrays for different wavelength bandwidths can be formed using other even-numbered combinations (e.g., 2, 4, 6, 10, 12, 14) of detector channels (e.g., 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, etc.) to obtain the total number of photodetectors available in the package (e.g., 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, etc.). Furthermore, the N array detectors 360A to 360N in the package 500 can be of the same type, such as a linear array detector with photodiodes, mirrors, and dichroic mirrors / filters arranged in a linear row, as shown in the figure. Other types of optics and detectors can be used as array detectors in the system.

[0132] Now refer to Figure 6A This diagram illustrates a pair of grating-based array detectors 610A to 610B, which can be packaged together within a packaged grating detector 610. This stack of grating-based array detectors 610A to 610B can replace, for example... Figure 3A The pair of array detectors 360A to 360B are described. Each grating-based array detector 610A to 610B includes input optics 662A to 662B, a first grating 664A to 664B, a second grating 665A to 665B, and a detector array 660A to 660B. Each detector array 660A to 660B may be based on a photomultiplier tube (PMT) or multiple photodetectors arranged in a row with different optical filters.

[0133] Input optics 662A to 662B converge the input beam along a first optical axis into the spot of first gratings 664A to 664B. The first gratings 664A to 664B reflect the beam at a certain angle into a second optical axis, initially extending its wavelength into first extended light incident on second gratings 665A to 665B. Further wavelength extension is required within a small package; the second gratings 665A to 665B receive the first extended light along the second optical axis and reflect it into a third optical axis, further extending its wavelength as second extended light incident on array detectors 660A to 660B.

[0134] Grating-based array detectors can provide optimal results over certain wavelength ranges, such as from 494 nm to 844 nm for a given laser. Typically, grating-based detectors exhibit poor performance for optical signals with wavelengths below 494 nm. However, different types of detectors can be used to detect optical signals below 494 nm. For example, the first array detector in the package could be a linear photodetector array for its corresponding bandwidth, while the second array detector could be a grating-based type detector for its corresponding bandwidth.

[0135] Now refer to Figure 6B Different types of detectors can be used to achieve optimal performance in various wavelength bands. Figure 6B A diagram illustrating a hybrid array detector 620 packaged together is shown. Detector 620 includes a first linear array detector 360A, a grating-based array detector 610B, and a second linear array detector 360B packaged together with three fiber optic inputs. The first linear array detector 360A receives light in the lower wavelength range from λ1 to λ2 from a first beamsplitter and a first fiber coupled to a first optical input. The grating-based array detector 610B receives light in the intermediate wavelength range from λ2 to λ3 from a second beamsplitter and a second fiber coupled to a second optical input. The second linear array detector 360B receives light in the higher wavelength range from λ3 to λ4 from a third beamsplitter and a third fiber coupled to a third optical input. In this way, the grating-based detector 610B can be used in the intermediate wavelength range (e.g., from 500 nm to 800 nm), where it can be effective; the first linear array detector 360A can be used in the lower wavelength range (e.g., from 300 nm to 500 nm), where it can be effective; and the second linear array detector 360B can be used in the higher wavelength range (e.g., from 800 nm to 1000 nm), where it can be effective.

[0136] like Figure 4A and Figure 5 The linear array detector shown, with its rows of linearly arranged photodetectors and channels, can be used as an array detection device in a flow cytometry / cell sorting system. Alternatively, in a system with one or more optical coupling devices, different types of detectors and different spatial arrangements of the detectors can be used to divide the detected optical bandwidth, which is then combined by a processor to form a total spectral response for the overall bandwidth.

[0137] Now refer to Figures 9A to 9C The array detector type is not limited in space to, for example, Figure 4A and Figure 5 A to Figure 5B shows a linear array detector with a series of photodetectors arranged in rows. Additional optical elements can be used to create and utilize other spatial layouts to divide the overall bandwidth into discrete parts, and then the results are assembled together using a processor if it is desired to detect a broadband wavelength.

[0138] Figure 9A An octagonal spatial detector layout 900A is illustrated, comprising eight photomultiplier tubes (PMTs), each supported by a long-pass (LP) filter and a band-pass (BP) filter to select the passband for detection and reflect the remaining bandwidth to the next PMT detector in the octagon. An optical fiber X couples light to an input optics device starting with a first LP filter having a lower bandwidth range, a first BP filter, and a first PMT detector. The light then passes through the octagon to the next seven LP filters, BP filters, and PMT detectors for higher bandwidth ranges. Two or more octagonal spatial detector arrangements 900A can replace linear detectors and be coupled with, for example,... Figure 3A One or more beam splitters in the optical system shown are used together to combine / stitch together two or more spectra using a central processing unit.

[0139] Figure 9B An example of a hexagonal spatial detector layout 900B with six detectors and six mirrors is illustrated. Optical fiber Z couples light to the input optics and the first detector, starting with the first mirror representing the lower bandwidth range. The light then passes through the hexagon to the next five mirrors and the five detectors for the higher bandwidth range. The hexagonal spatial layout detector 900B can individually detect wavelengths from 355 nm to 860 nm. If used alone, the mirrors and other optics used in the hexagonal spatial layout detector 900B must support such a wide bandwidth. Therefore, the signal performance using a single detector may be compromised. Furthermore, with a single detector, it is difficult to extend the wavelength bandwidth to a wider range from 330 nm to 1000 nm, becoming more extensive in the UV and infrared. Therefore, it is desirable to use two or more hexagonal detectors 900B, utilizing beam splitters and optimized optical fibers to divide the bandwidth discussed herein into two or more hexagonal detectors 900B. The central processing unit can be used to merge (merge / stitch) separate spectral results from two or more hexagonal detectors 900B into a complete spectral result.

[0140] Figure 9CA triangular spatial detector arrangement 900C with three PMT detectors is illustrated, each supported by a long-pass (LP) filter and a band-pass (BP) filter, respectively. An optical fiber Z couples light to the input optics, starting with the first LP filter for a lower bandwidth range, the BP filter, and the PMT detector. The light then passes through the triangle to the next two LP filters, the BP filter, and the PMT detector for a higher bandwidth range. The bandwidth detected by three PMT detectors is very limited. To broaden the bandwidth detected in a flow cytometry system, it is desirable to use two or more triangular spatial layout detectors 900C, with the bandwidth divided among the two or more triangular spatial layout detectors 900C using a beam splitter and optimized optical fiber, as discussed here. A central processing unit can be used to combine (merge / stitch) the separate spectral results from the two or more triangular spatial layout detectors 900C into a complete spectral result.

[0141] While two or more detectors of the same type can be used to improve wavelength bandwidth, a hybrid approach (mixing and matching) can be employed, where two or more detectors of different types are selected to match a specific wavelength bandwidth, where each wavelength bandwidth can be more efficient in detection. For example, linear array detectors 360A and 360B can be used for one wavelength bandwidth range, while a triangular spatial layout detector 900C with three PMT detectors can be used for a different wavelength bandwidth range, a hexagonal spatial layout detector 900B for another different wavelength bandwidth range, an octagonal spatial detector array 900A for yet another different wavelength bandwidth range, and a grating detector array 610A and 610B for yet yet another different wavelength bandwidth range. In any case, one or more beam splitters and one or more optical fibers can be used to separate the bandwidth, allowing more than one of multiple detector types to detect the corresponding bandwidth of light separately and generate digital signals from it. A central processing unit can be used to combine (merge / stitch) the separated spectral results from two or more detectors of different types into a complete spectral result.

[0142] advantage

[0143] The disclosed embodiments offer numerous advantages. They enable the detection of excited cell characteristics in flow cytometers or cell sorting laboratory instruments using ultrawideband light. Even with pre-existing bandwidth, the disclosed embodiments improve the overall system performance of existing bandwidths used to capture light from excited cells in flow cytometers or cell sorting laboratory instruments. Dispersion exists due to the optical devices used to collect and detect the light. Separating the wavelength range to utilize different optics with different wavelength ranges, such as using two or more optical cables optimized for different wavelength ranges, can increase collection efficiency across the entire wavelength range. Dividing the wavelength range can also reduce the number of detectors used in an array detector, thereby increasing the signal transmitted to the last detector in the detector array. Furthermore, space and cost can be saved while performance is improved by using a single package with two separate detectors.

[0144] Therefore, embodiments of the invention have been described. Although embodiments of the invention have been specifically described, they should not be construed as limiting them, but rather should be interpreted in accordance with the following claims.

[0145] Although certain exemplary embodiments have been described and illustrated in the accompanying drawings, it should be understood that these embodiments are merely illustrative and not intended to limit the disclosed embodiments, and that the disclosed embodiments are not limited to the specific constructions and arrangements shown and described, as various other modifications will be apparent to those skilled in the art.

[0146] When implemented in software, the elements of embodiments of the present invention are essentially code segments that perform necessary tasks. The program or code segment can be stored in a processor-readable medium for execution by a processor. The code segment can be downloaded to the processor-readable medium via a computer network such as the Internet, intranet, etc. Alternatively, the code segment can be transmitted from the processor-readable medium to the processor for execution via a transmission medium or communication link using computer data signals carried on a carrier wave. The processor-readable medium can include any medium capable of storing information. Examples of processor-readable storage media include electronic circuits, semiconductor memory devices, random access memory (RAM), read-only memory (ROM), flash memory, erasable programmable read-only memory (EPROM), floppy disks, CD-ROMs, optical disks, hard disks, etc.

[0147] Although this specification includes numerous details, these details should not be construed as limiting the scope of this disclosure or the scope that may be claimed, but rather as descriptions of features characteristic of a particular implementation of this disclosure. Certain features described in this specification in the context of separate implementations may also be implemented in combination in a single implementation. Conversely, various features described in the context of a single implementation may also be implemented individually or in sub-combinations in multiple implementations. Furthermore, although features may be described above as functioning in certain combinations, and even initially claimed in this way, one or more features from a claimed combination may be removed from that combination in certain circumstances, and the claimed combination may involve sub-combinations or variations thereof. Therefore, the claimed invention is defined only by the following patent claims.

Claims

1. A method for use in a flow cytometer, the method comprising the following steps: The objective lens is used to collect fluorescence from one or more moving cells and lateral scattered light exiting from the one or more moving cells over the full bandwidth range between the minimum and maximum wavelengths to be detected; The fluorescence and the side-scattered light over the entire bandwidth are converged along the first optical axis outside the objective lens toward the focal point to form a spot size; The first light in the first wavelength range from the minimum wavelength to the first wavelength in the full bandwidth range is coupled to the first optical detection device using the first optical coupling device, and the remaining light in the full bandwidth range is allowed to pass toward the last optical detection device; The first light is converted into a first plurality of digital signals representing discrete wavelength portions of the first light within the first wavelength range using the first light detection device. The remaining light is converted into a plurality of digital signals representing discrete wavelength portions of the remaining light over the full bandwidth using the final light detection device. as well as The signal processor receives the first plurality of digital signals and the last plurality of digital signals, and processes the first plurality of digital signals and the last plurality of digital signals into a combined spectral signal over the full bandwidth range.

2. The method according to claim 1, further comprising: Before conversion using the final optical detection device: A second coupling device is used to couple a second light from one or more nanometers above the maximum wavelength of the first light to a second wavelength within the full bandwidth range to a second light detection device, and to allow the remaining light within the full bandwidth range to pass toward the final light detection device; The second light is converted into a second plurality of digital signals representing discrete wavelength portions of the second light within the second wavelength range using the second light detection device. as well as The signal processor receives the second plurality of digital signals and processes the first plurality of digital signals, the second plurality of digital signals, and the last plurality of digital signals into a combined spectral signal over the full bandwidth range.

3. The method according to claim 2, further comprising: After conversion using the second optical detection device and before conversion using the final optical detection device: A third light, ranging from one or more nanometers above the maximum wavelength of the second light to the third wavelength within the full bandwidth range, is coupled to a third light detection device using a third coupling device, and the remaining light within the full bandwidth range is allowed to pass toward the final light detection device. The third light is converted into a third plurality of digital signals representing discrete wavelength portions of the third light within the third wavelength range using the third light detection device. as well as The signal processor receives the third plurality of digital signals and processes the first plurality of digital signals, the second plurality of digital signals, the third plurality of digital signals, and the last plurality of digital signals into a combined spectral signal over the full bandwidth range.

4. The method according to claim 1, wherein: The first optical coupling device is selected from one or more of the group consisting of a lens, an optical fiber, a beam splitter, a grating and / or a mirror.

5. The method according to claim 2, wherein: Each second optical coupling device is selected from one or more of the group consisting of lenses, optical fibers, beam splitters, gratings and / or mirrors.

6. The method according to claim 1, wherein, The first optical coupling device is a first beam splitter, and the coupling includes: The first beam splitter separates the first light within the first wavelength range from the minimum wavelength to the first wavelength within the full bandwidth, while allowing the remaining light within the full bandwidth to pass through; and The first beam splitter is used to redirect the first light in the first wavelength range along the second optical axis toward the first optical detection device at a 45-degree angle.

7. The method according to claim 2, wherein: The first optical coupling device is a first beam splitter, and the first coupling includes: The first beam splitter separates the first light within the first wavelength range from the minimum wavelength to the first wavelength within the full bandwidth, while allowing the remaining light within the full bandwidth to pass through; and The first beam splitter is used to redirect the first light in the first wavelength range along the second optical axis toward the first optical detection device at an angle. as well as, The second optical coupling device is a second beam splitter, and the second coupling includes: The second beam splitter separates the second light from one or more nanometers above the maximum wavelength of the first light to the second wavelength within the full bandwidth, and allows the remaining light within the full bandwidth to pass toward the final light detection device; and The second beam splitter is used to redirect the second light in the second wavelength range along the third optical axis toward the second optical detection device at a 45-degree angle.

8. The method according to claim 1, wherein, The first optical coupling device is a first dichroic mirror and a first optical fiber, and the coupling includes: The first light in the first wavelength range from the minimum wavelength to the first wavelength in the full bandwidth range is separated by the first dichroic mirror, while the remaining light in the full bandwidth range is allowed to pass through; Using the first dichroic mirror, the first light in the first wavelength range is redirected along the second optical axis toward the first end of the first optical fiber at a 45-degree angle; and The first light is received at the first end of the first optical fiber and emitted from the second end of the first optical fiber into the first optical detection device.

9. The method according to claim 2, wherein: The first optical coupling device is a first dichroic mirror and a first optical fiber, and the first coupling includes: The first light in the first wavelength range from the minimum wavelength to the first wavelength in the full bandwidth range is separated by the first dichroic mirror, while the remaining light in the full bandwidth range is allowed to pass through; Using the first dichroic mirror, the first light in the first wavelength range is redirected along the second optical axis toward the first end of the first optical fiber at a first angle; and The first light is received at the first end of the first optical fiber, and the first light is emitted from the second end of the first optical fiber to the first optical detection device. as well as, The second optical coupling device is a second dichroic mirror and a second optical fiber, and the second coupling includes: The second dichroic mirror is used to separate the second light in the second wavelength range from the minimum wavelength to the first wavelength in the full bandwidth range, while allowing the remaining light in the full bandwidth range to pass through; Using the second dichroic mirror, the second light in the second wavelength range is redirected along the second optical axis toward the first end of the second optical fiber at a second angle; and The second light is received at the first end of the second optical fiber, and the second light is emitted from the second end of the second optical fiber into the second optical detection device.

10. The method according to claim 1, wherein: Each optical detection device is a photodiode array detector, which has multiple mirrors in a first row on one side and multiple wavelength filters and multiple photodetectors arranged in rows on the opposite side to detect discrete wavelength portions of the optical bandwidth.

11. The method of claim 10, wherein: Each of the plurality of photodetectors is an avalanche photodiode.

12. The method according to claim 1, wherein: Each optical detection device includes: A first grating receives light coupled to a light detection device along a first axis and reflects and first spreads the light along a second axis; A second grating receives the first extension of the light along the second axis and reflects and second extends the light along the third axis; and A photodiode array detector having multiple mirrors in a first row on one side and multiple wavelength filters and multiple photodetectors arranged in rows on the opposite side to detect discrete wavelength portions over an optical bandwidth.

13. The method according to claim 12, wherein: Each of the multiple photodetectors in the photodiode array detector is an avalanche photodiode.

14. The method according to claim 1, wherein: Each light detection device is a photomultiplier tube (PMT).

15. A flow cytometer, the flow cytometer comprising: A flow cell that receives mobile biological cells in a sample fluid and a sheath fluid encapsulating the biological cells, the flow cell having a questioning region that receives one or more laser beams from one or more lasers to strike the mobile biological cells and generate fluorescence, side-scattered light and forward-scattered light; An objective lens, located near the flow cell, collects the fluorescence and side-scattered light across the full bandwidth range between the minimum and maximum wavelengths to be detected, and converges the fluorescence and side-scattered light across the full bandwidth range towards the focal point along a first optical axis outside the objective lens to form a spot size. A first optical coupling device is provided, which is positioned along the first optical axis prior to the focal point, and along the second optical axis, redirects the first light within a first wavelength range from the minimum wavelength to the full bandwidth range of the first wavelength. The first optical coupling device also allows the remaining light within the full bandwidth range to pass along the first optical axis. A first optical detection device receives the first light along the second optical axis and converts the first light into a first plurality of digital signals representing discrete wavelength portions of the first light within the first wavelength range. The final light detection device receives the remaining light along the first optical axis and converts the remaining light into a last plurality of digital signals representing discrete wavelength portions of the remaining light over the full bandwidth. as well as The processor is communicatively connected to the first optical detection device and the last optical detection device. The signal processor receives the first plurality of digital signals and the last plurality of digital signals, and processes the digital signals into a combined spectral signal over the full bandwidth range.

16. The flow cytometer according to claim 15, further comprising: A second optical coupling device is located along the first optical axis between the first optical coupling device and the focal point. The first optical coupling device, along a third optical axis, redirects the second light in a second wavelength range from one or more nanometers above the maximum wavelength of the first light to the second wavelength in the full bandwidth range. The second optical coupling device allows the remaining light in the full bandwidth range to pass along the first optical axis. as well as A second optical detection device receives the second light along the third optical axis and converts the second light into a second plurality of digital signals representing discrete wavelength portions of the second light within the second wavelength range. The signal processor is also communicatively connected to the second optical detection device to receive the second plurality of digital signals, and the signal processor processes the first plurality of digital signals, the second plurality of digital signals and the last plurality of digital signals into the combined spectral signal over the full bandwidth range.

17. The flow cytometer according to claim 16, further comprising: A third optical coupling device is located along the first optical axis between the second optical coupling device and the focal point. The third optical coupling device is located along the fourth optical axis to redirect the third light from one or more nanometers above the maximum wavelength of the second light to the third wavelength range within the full bandwidth. The third optical coupling device allows the remaining light in the full bandwidth range to pass along the first optical axis toward the last optical coupling device. as well as A third optical detection device receives the third light along the fourth optical axis and converts the third light into a third plurality of digital signals representing discrete wavelength portions of the third light within the third wavelength range. The signal processor is also communicatively connected to the third optical detection device to receive the third plurality of digital signals, and the signal processor processes the first plurality of digital signals, the second plurality of digital signals, the third plurality of digital signals and the last plurality of digital signals into the combined spectral signal over the full bandwidth range.

18. The flow cytometer according to claim 15, wherein: The first optical coupling device is selected from one or more of the group consisting of lenses, Bragg gratings and / or dichroic mirrors and optical fibers.

19. The flow cytometer according to claim 16, wherein: Each optical coupling device in the optical coupling device is selected from one or more of the group consisting of lenses, Bragg gratings and / or dichroic mirrors and optical fibers.

20. The flow cytometer according to claim 17, wherein: Each optical coupling device in the optical coupling device is selected from one or more of the group consisting of lenses, Bragg gratings and / or dichroic mirrors and optical fibers.

21. A method for improving a flow cytometer or cell sorter, the method comprising the following steps: Multiple different fluorescent pigments labeled with multiple moving cells are excited to fluoresce using one or more laser beams; Broadband optical signals with a wide wavelength range are collected from the fluorescence of the plurality of different fluorescent dyes; The wavelength range of the collected broadband optical signal is divided into multiple smaller wavelength ranges in multiple optical signal paths; The optical signals in each smaller wavelength range within the multiple smaller wavelength ranges in the multiple optical signal paths are detected by using multiple different photodetectors. Multiple analog-to-digital converters are used to generate multiple digital signals based on the detected optical signals in each of the multiple smaller wavelength ranges in the multiple optical signal paths; The plurality of digital signals are arranged over the broadband wavelength range; and Based on the broadband optical signal having the broadband wavelength range, the multiple digital signals arranged together are combined to form a full-spectrum response.

22. The method according to claim 21, wherein: The collected broadband optical signals have a broadband wavelength range of 320 nanometers to 1,000 nanometers.

23. The method according to claim 21, wherein: The broadband wavelength range of the collected broadband optical signal is separated into two wavelength ranges in the two optical signal paths.

24. The method according to claim 23, wherein: The first wavelength range in the first optical signal path is from 320 nanometers to 480 nanometers; and The second wavelength range in the second optical signal path is from 480 nanometers to 1,000 nanometers.

25. The method according to claim 21, wherein: Separating the wavelength range of the collected broadband optical signal into multiple smaller wavelength ranges in multiple optical signal paths also includes: The optical signal is collected from at least one of the plurality of different smaller wavelength ranges using the first end of the optical fiber, and the optical signal is redirected toward the first photodetector; and The optical signal is transmitted from the second end of the optical fiber to the first photodetector.

26. The method of claim 25, wherein: The first photodetector is a linear array detector having multiple photodetectors arranged in a row.

27. A flow cytometer, the flow cytometer comprising: A flow cell that receives mobile biological cells in a sample fluid and a sheath fluid encapsulating the biological cells, the flow cell having a questioning region that receives one or more laser beams from one or more lasers to strike the mobile biological cells and generate fluorescence, side-scattered light and forward-scattered light; An objective lens, located near the flow cell, collects the fluorescence and side-scattered light across the full bandwidth range between the minimum and maximum wavelengths to be detected, and converges the fluorescence and side-scattered light across the full bandwidth range towards the focal point along a first optical axis outside the objective lens to form a spot size. A first beam splitter, which is located along the first optical axis before the focal point, redirects light of a first wavelength range along a second optical axis different from the first optical axis, and allows the remaining light of the full bandwidth range to pass along the first optical axis. A first optical fiber has an input end aligned with the second optical axis to receive light in the first wavelength range, and redirects the light in the first wavelength range toward an output end and emits the light in the first wavelength range out of the output end. A first array detector has an input channel coupled to the output end of the first optical fiber to receive light in the first wavelength range, and the first array detector converts the light in the first wavelength range into a first plurality of digital signals representing discrete wavelength portions of the light in the first wavelength range. The last optical fiber has an input end aligned with the first optical axis to receive the remaining light across the full bandwidth, and redirects the remaining light to an output end and emits the remaining light from the output end. The final array detector has an input channel coupled to the output of the final optical fiber to receive the remaining light across the full bandwidth range, and the final array detector converts the remaining light into a plurality of digital signals representing discrete wavelength portions of the remaining light across the full bandwidth range. as well as A signal processor communicatively connected to the first array detector and the last array detector, the signal processor receiving the first plurality of digital signals and the last plurality of digital signals, and processing the digital signals into a combined spectral signal over the full bandwidth range.

28. The flow cytometer according to claim 27, further comprising: A second beam splitter is located between the first beam splitter and the focal point along the first optical axis. The second beam splitter redirects light of a second wavelength range different from the first wavelength range along a third optical axis different from the first optical axis, and allows the remaining light of the full bandwidth range to pass along the first optical axis. The second optical fiber has an input end aligned with the third optical axis to receive light in the second wavelength range, and redirects the light in the second wavelength range toward the output end and emits the light in the second wavelength range out of the output end; as well as A second array detector, having an input channel coupled to the output of the second optical fiber to receive light in the second wavelength range, converts the light in the second wavelength range into a second plurality of digital signals representing discrete wavelength portions of the light in the second wavelength range. The signal processor is also communicatively connected to the second array detector to receive the second plurality of digital signals, and the signal processor processes the first plurality of digital signals, the second plurality of digital signals and the last plurality of digital signals into the combined spectral signal over the full bandwidth range.

29. The flow cytometer according to claim 28, further comprising: A third beam splitter is located between the second beam splitter and the focal point along the first optical axis. The third beam splitter redirects light along a fourth optical axis different from the first optical axis to a third wavelength range different from the first and second wavelength ranges, and allows the remaining light of the full bandwidth range to pass along the first optical axis. A third optical fiber has an input end aligned with the fourth optical axis to receive light in the third wavelength range, and redirects the light in the third wavelength range toward an output end and emits the light in the third wavelength range out of the output end. as well as A third array detector has an input channel coupled to the output of the third optical fiber to receive light in the third wavelength range. The third array detector converts the light in the third wavelength range into a third plurality of digital signals representing discrete wavelength portions of the light in the third wavelength range. The signal processor is also communicatively connected to the third array detector to receive the third plurality of digital signals, and the signal processor processes the first plurality of digital signals, the second plurality of digital signals, the third plurality of digital signals, and the last plurality of digital signals into the combined spectral signal over the full bandwidth range.

30. The flow cytometer according to claim 29, wherein: The objective lens near the flow cell is coated with a combined antireflective coating, which is formed by three or more antireflective coatings based on the full bandwidth of light.

31. The flow cytometer according to claim 30, wherein: The optics in the first input channel of each array detector, as well as the mirrors and optical filters for the first to third detection channels of each array detector, are coated with an anti-reflective coating based on the corresponding wavelength range of the detected light.

32. The flow cytometer according to claim 29, wherein: The diameters of the first optical fiber, the second optical fiber, the third optical fiber, and the last optical fiber are selected based on the corresponding wavelength range of light received from the respective beam splitters.

33. The flow cytometer according to claim 32, wherein: Based on the corresponding wavelength range of the light received from the respective beam splitters, the respective input ends of the first optical fiber, the second optical fiber, the third optical fiber, and the last optical fiber are positioned at a back focal distance along the respective optical axis from the back of the objective lens.

34. The flow cytometer according to claim 33, wherein: The respective input ends of the first optical fiber, the second optical fiber, the third optical fiber, and the last optical fiber are coated with an anti-reflection coating based on the respective wavelength range of the light received from the respective beam splitter.

35. The flow cytometer according to claim 27, further comprising: A clock generator is connected to the signal processor and each array detector to synchronize the capture of multiple digital signals by each array detector.

36. The flow cytometer according to claim 27, wherein: Each array detector is a linear array detector, which includes: An optical block that receives light from at least one optical fiber; Multiple optical filters are arranged in rows on one side of the optical block to transmit different discrete portions of light and reflect the remainder of light. A first optical filter receives light from the at least one optical fiber through at least one input channel. Multiple micromirrors, arranged in a row on opposite sides of the optical block, receive the remaining portion of reflected light from each corresponding optical filter (excluding the last optical filter), and reflect the remaining portion of light from each optical filter toward the next corresponding optical filter in the row of optical filters; and Multiple photodetectors are arranged in a row below the multiple optical filters to receive different discrete portions of light and convert them into electrical signals.

37. The flow cytometer according to claim 36, wherein: At least two linear array detectors are packaged together as a package that shares the optical block.

38. The flow cytometer according to claim 36, wherein: At least two linear array detectors are packaged together back to back, sharing a base on which the various optical blocks and input channels are mounted.

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