Apparatus and methods for circulating flow cytometry using specialized cell recognition

Abstract

A method and apparatus for performing cyclic flow cytometric analysis of a sample population of cellular entities, comprising: marking each cellular entity with an optical marker; for each cellular entity, performing a first pass flow cytometric measurement on the cellular entity in a flow channel with respect to a first set of parameters under conditions that determine identification of the cellular entity that is obtaining values of the first set of parameters, and storing values of the first set in association with the identification; and performing a second pass flow cytometric measurement on the cellular entity in the flow channel with respect to a second set of parameters under conditions that separately determine identification of the cellular entity that is obtaining values of the second set of parameters, and storing values of the second set in association with the identification.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims priority to U.S. Provisional Application No. 62 / 969,380, filed February 3, 2020, entitled “Apparatus and Method for Cyclic Flow Cytometry Using Particularized Cell Identification”. Technical Field

[0003] The present invention relates to apparatus and methods for performing flow cytometry, and more particularly to apparatus and methods that allow the use of specialized cells to identify populations of repeatedly measured cell entities. Background Technology

[0004] Flow cytometry is an analytical technique used to measure the physical and chemical properties of individual cells in a rapidly flowing stream. Modern flow cytometers use fluorescent probes to detect specific molecules or molecular complexes, such as surface membrane proteins, intracellular signaling proteins, and RNA molecules. Flow cytometry can identify and quantify different cell types within a cell population, determine phenotypes and gene expression patterns, and detect disease-related biomarkers. This information is obtained at single-cell resolution and is widely used in diagnostics, treatment monitoring, drug discovery, and basic biological research.

[0005] A typical workflow involves first preparing a cell suspension (typically 10) with a mixture of antibodies conjugated to fluorophores that bind different cell markers. 5 Up to 10 6 Cells are stained one at a time. Next, the cells are injected into a flow cytometer instrument, where they pass one at a time through a laser beam that excites the fluorophores present on each cell. State-of-the-art flow cytometers can perform cell measurements at rates of 10,000 cells per second or higher. Fluorescence emission from each cell is collected and analyzed to reveal the presence or absence of different markers. For research and drug development, flow cytometry is commonly used to analyze cells treated with different drugs or environmental conditions to assess the effects on viability, apoptosis, cell cycle, and proliferation. Blood samples are frequently analyzed by flow cytometry to characterize different cell types; this technique, known as immunophenotyping, is widely used in immunological and immuno-oncology research, disease diagnosis, and treatment monitoring. In addition to cells, emerging applications of flow cytometry include the analysis of multicellular spheroids, bacterial cells, and cellular components such as nuclei and vesicles.

[0006] The ability to detect multiple markers per cell is key to the vast majority of flow cytometry applications. Generally, detecting more markers allows for more detailed and accurate cell analysis. Over the past few decades, flow cytometer users have been measuring an increasing number of markers per cell. For example, a 2001 report found that no clinical application required five or more markers per cell (International Society for Clinical Cell Biology). By 2015, most clinical users were measuring 10 or more markers, particularly for hematological applications such as the diagnosis and monitoring of leukemia and lymphoma (2015 Global Flow Cytometry Trends Survey).

[0007] Recent advancements in sophisticated flow cytometry and the increased availability of diverse fluorophores and high-affinity antibodies have enabled high-parameter flow cytometry with more than 15 labels. High-parameter flow cytometry has accelerated the development of immunotherapy, a new branch of medicine designed to utilize a patient's own immune system to treat disease. As immunotherapy becomes a viable treatment for cancer and genetic abnormalities, comprehensive subclassification and characterization of immune cells have become crucial, making high-parameter flow cytometry a fundamental technique in academic and clinical research.

[0008] However, one of the main limitations of current flow cytometry is the limited number of fluorescent probes that can be used simultaneously. This is because their photoluminescence spectra overlap. The fluorescence emission of fluorescent probes occupies a spectral range of approximately 30-100 nm. Therefore, within the 400-800 nm wavelength range, it is generally easy to distinguish no more than 10 different fluorescent probes. The number of reusable probes can be increased by utilizing multiple excitation lasers with different excitation wavelengths to measure the differences in absorption spectra between probes. To be able to distinguish fluorophores with spectral overlap, a mathematical process called spectral compensation is performed, which also involves optimizing the instrument's acquisition settings to best distinguish the different fluorophores. When spectral overlap is severe, such as when more than 10 fluorophores are used simultaneously, spectral compensation significantly degrades data quality and hinders accurate biological interpretation. Therefore, even for the most complex and state-of-the-art instruments, the number of probes available simultaneously is limited to approximately 30-40.

[0009] Choosing the right set of fluorescent staining reagents (often referred to as a group) is a significant challenge and time-consuming procedure when measuring multiple labels simultaneously. First, distinct fluorophores with minimal spectral overlap should be selected to achieve definitive detection of different labels. Second, the brightness of the fluorophores should match the expected abundance of the protein markers in the cells (brighter fluorophores for less abundant proteins). Finally, steric hindrance between antibodies should be considered when attempting to label multiple labels at once. After the initial panel design, the group's performance should be validated using flow cytometry by analyzing control samples stained separately with each fluorophore-antibody conjugate from the group. This process includes spectral compensation, where instrument settings are optimized to best localize the different fluorophores within the group. Typically, failure to validate performance leads to a partial or complete redesign of the group itself.

[0010] In summary, the iterative design and validation process can take weeks to months to complete, depending on the number of labels required. For applications requiring eight or fewer labels, this process can be simplified (<1 week) due to the use of easily distinguishable combinations of fluorophores. Preparing 15 or more label groups is challenging and requires more than a month of optimization by experienced flow cytometry users. Furthermore, for these highly labeled groups, the limited availability of antibodies with suitable fluorophores can be a major obstacle, typically requiring custom orders with long lead times and further validation.

[0011] Reagent costs are also significant and may be prohibitively expensive for large-scale flow cytometry. Numerous control samples are necessary for optimizing flow cytometry acquisition settings, including single-stain controls for optimizing spectral compensation and fluorescence-minus-one (FMO) controls for optimizing fluorescence signals for each marker. For FMO controls, cells are stained with each antibody in the group, but one antibody serves as a negative control for an antibody / marker not included in the group. Therefore, for a group with 15 markers, there will be at least 15 FMO control samples, each requiring 14 antibodies to target 14 of the 15 markers.

[0012] As mentioned above, when measuring more than 30 labels at a time, spectral overlap becomes difficult to control, and measuring 30-40 labels is very challenging for routine flow cytometry.

[0013] When measuring more than 30-40 labels, other types of molecular analysis tools, such as single-cell mass spectrometry and single-cell sequencing, are used instead of flow cytometry. While these other tools can meet this need, they are generally more expensive and have much lower throughput. Innovations that address the multiplexing challenges of fluorescence-based flow cytometry with low cost and high throughput will have a significant impact on single-cell analysis.

[0014] Implementation Plan Overview

[0015] According to an embodiment of the present invention, a method for performing circulating flow cytometry analysis on a sample population of cell entities is provided. The method includes labeling each cell entity in the population with an optical marker. The method further includes, for each cell entity in the population, performing a first-flow flow cytometry measurement in a flow channel relative to the first set of parameters, and storing the first set of attributes associated with the identification, provided that identification of the cell entity acquiring a first set of parameter attributes is determined. The method further includes, for each cell entity in the population, performing a second-flow flow cytometry measurement in a flow channel relative to the second set of parameters, and storing the second set of attributes associated with the identification, provided that identification of the cell entity acquiring a second set of parameter attributes is determined individually.

[0016] Other related embodiments of the invention include, for each cell entity in the population, using identification to merge attributes of a first set of parameters with attributes of a second set of parameters. In another related embodiment, the method further includes, under the condition of individually determining the identification of a cell entity that is acquiring at least another set of parameter values, performing at least another flow cytometry measurement in the flow channel relative to at least another set of parameters, and storing the values ​​of at least another set associated with the identification.

[0017] Optionally or additionally, the first and second flow cytometry measurements use first and second sets of fluorescent probes that target different sets of parameters, respectively, and the method further includes inactivating the first set of fluorescent probes after performing the first flow cytometry measurement and before performing the second flow cytometry measurement.

[0018] Optionally or additionally, the sample population comprises at least 1,000 cell entities. In another related embodiment, the method further includes collecting the population to be analyzed in a manner that preserves the characteristics of the cell entities, after performing a first-pass flow cytometry measurement and before performing a second-pass flow cytometry measurement.

[0019] Optionally or additionally, collection includes capturing the cell entities of a population at the end of the flow channel using a collection container. Also optionally or additionally, the method includes restoring the captured cell entities prior to performing a second flow cytometry measurement.

[0020] In a relevant embodiment of the invention, the optical marker is a set of microlaser particles. Optionally or additionally, the microlaser particles include semiconductors.

[0021] In other related embodiments of the invention, flow cytometry measurements utilize fluorescence via a fluorophore, and determining the identification of cell entities involves reading optical markers in the emission spectrum using a photoexcitation source. Optionally or additionally, the fluorophore is configured to operate in a fluorescence spectrum different from the emission spectrum. Also optionally or additionally, the fluorophore is configured to be photobleachable after 30 minutes without affecting cell viability.

[0022] In relevant implementations, the first set of parameters is selected from: surface or intracellular protein expression, RNA expression, quantification of organelles (such as mitochondria and lysosomes), cell granularity, cell size, cell shape, and combinations thereof. Optionally or additionally, each of these parameters of the cell entity is measured by phenomena selected from fluorescence, light scattering, and absorption.

[0023] According to an alternative embodiment of the invention, an improved flow cytometry apparatus is of the type having a flow channel coupled to a fluid inlet configured to receive a sample swarm of cell entities to be measured with respect to a set of target parameters. The flow channel is instrumented to obtain and store continuous fluorescence readings from and through a continuous stream of cell entities. The improvement includes an OFID reader associated with the flow channel, wherein each cell entity in the sample swarm is labeled with an optical marker, and the OFID reader is configured to determine the identification of each cell entity as each cell entity is measured in each of a plurality of flows of the sample swarm of cell entities passing through the flow channel, each flow being associated with a corresponding set of target parameters.

[0024] Optionally or additionally, the OFID reader is configured to operate on multiple transmit-collect paths, two of which are defined at an angle of approximately 90 degrees relative to each other.

[0025] In related implementations, the improvement also includes a recirculator configured to repair cell entities passing through the flow channel and position them for further passage through the flow channel.

[0026] According to another embodiment of the invention, an improved flow cytometry apparatus is of the type having a flow channel coupled to a fluid inlet configured to receive a sample swarm of cell entities to be measured with respect to a target parameter set. The flow channel is instrumented to obtain and store continuous fluorescence readings from continuous cell entities passing through it. The improvement includes an OFID reader associated with the flow channel, wherein each cell entity in the sample swarm is labeled with an optical marker, and the OFID reader is configured to determine an identification of each cell entity as each cell entity is measured in each of a plurality of flows of the sample swarm of cell entities passing through the flow channel, each flow being associated with a corresponding target parameter set. The improvement also includes a processor, (i) to associate and store in a storage device the corresponding fluorescence readings with the identification of each cell entity in each flow, and (ii) to use said identification to merge attributes of the corresponding target parameter set.

[0027] Optionally or additionally, the OFID reader is configured to operate on multiple transmit-collect paths, two of which are defined at an angle of approximately 90 degrees relative to each other.

[0028] Also optionally or additionally, the improved flow cytometry device includes a recirculator configured to capture and repair cell entities passing through the flow channel and position them for further passage through the flow channel. Also optionally or additionally, the recirculator has a surface for contacting a population of cell entities configured to have low wettability.

[0029] According to another embodiment of the invention, an improved flow cytometry apparatus is of the type having a flow channel coupled to a fluid inlet configured to receive a sample swarm of cell entities to be measured with respect to a target parameter set. The flow channel is instrumented to obtain and store continuous fluorescence readings from and through a continuous stream of cell entities. The improvement includes an OFID reader associated with the flow channel, wherein each cell entity in the sample swarm is labeled with an optical marker, and the OFID reader is configured to determine an identification of each cell entity as each cell entity is measured to be a cell entity passing through the flow channel, wherein the OFID reader is configured to operate on multiple emission collection paths, two of which are defined at approximately 90 degrees relative to each other. The improvement also includes a processor, (i) to associate and store in a storage device the corresponding fluorescence readings with the identification of each cell entity flowing through, and (ii) to use the identification to merge attributes of the corresponding target parameter set.

[0030] According to another embodiment of the invention, a kit is provided for converting a flow cytometry device of the type having a flow channel coupled to a fluid inlet configured to receive a sample swarm of cell entities to be measured relative to a target parameter set (the flow channel is instrumented to obtain and store continuous fluorescence readings from and through continuous cell entities) into an improved flow cytometry device. The kit includes an OFID reader associated with the flow channel, wherein each cell entity in the sample swarm is labeled with an optical marker, and the OFID reader is configured to determine an identification of each cell entity during measurement of each of multiple flows of the sample swarm of cell entities passing through the flow channel, each flow being associated with a corresponding target parameter set. The kit also includes a recirculator configured to capture and repair cell entities passing through the flow channel and position them for further passage through the flow channel. The kit further includes a processor, (i) to associate and store in a storage device the corresponding fluorescence readings with the identification of each cell entity in each flow, and (ii) to use the identification to merge attributes of the corresponding target parameter set.

[0031] Brief description of the attached figures

[0032] The aforementioned features of the implementation scheme will be more readily understood by referring to the following detailed description and the accompanying drawings.

[0033] Figure 1 depicts a conventional flow cytometry instrument.

[0034] Figures 2A to 2E Different strategies for labeling cells with microparticles according to embodiments of the present invention are described. Figure 2A The embodiment of the invention demonstrates the delivery of microparticles into cells via endocytosis. Figure 2B This illustrates another embodiment of the invention, which describes the delivery of microparticles into cells via a liposome carrier. Figure 2C This illustrates another embodiment of the invention, which demonstrates the delivery of microparticles into cells via gene gun particle bombardment. Figure 2D This illustrates the attachment of microparticles to the cell surface according to another embodiment of the invention. Figure 2E Optical images of microlaser particles (LPs) labeled onto the cell surface (left) and inside the cell (right) according to an embodiment of the present invention are shown.

[0035] Figure 3 An improved flow cytometry instrument according to an embodiment of the invention is described, which combines conventional fluorescence measurement and spectrometry to read optical markers.

[0036] Figure 4A to Figure 4D The procedures in conventional and circulating flow cytometry are shown. Figure 4A shows the procedure in conventional flow cytometry. Figure 4B The process of initial operation of a circulating flow cytometry analysis according to an embodiment of the present invention is shown. Figure 4C The procedure in circulating flow cytometry according to an embodiment of the present invention is shown, which uses optical markers and destaining and staining with fluorescent probes. Figure 4D The procedure in cyclic flow cytometry according to another embodiment of the invention is shown, which uses optical markers and sequential fluorescence readings using DNA barcode probes.

[0037] Figures 5A to 5B An instrument for circulating flow cytometry according to an embodiment of the present invention is described. The instrument for circulating flow cytometry includes a flow cytometry system for reading fluorescent and optical markers, such as... Figure 3 As shown, a recirculator device for automatically processing cells between cycles of cyclic flow cytometry is also included.

[0038] Figures 6A to 6D Exemplary optical markers are shown, which can be used to uniquely label cells for use in embodiments of the invention. Although such optical markers have recently become known in the art, their use in the current context is novel. Figure 6AElectron microscopy images of semiconductor microlaser particles coated with silicon dioxide and their typical stimulated emission spectra are shown, with a peak at 1392 nm and a full width at half maximum (FWHM) of 0.35 nm. Figure 6B Fluorescence microscopy images of cells containing three microlaser particles with different laser emission peaks are shown. Figure 6C An optical image of an optical marker including three microdisk lasers is shown. Figure 6D The emission spectrum from the triplet laser particle is shown. The three narrow spectral peaks define the unique OFID assigned by the optical identifier.

[0039] Figures 7A to 7D An optical system for collecting light from micro-laser particles according to an embodiment of the present invention is described. Figure 7A A system using multiple optical fibers to collect light from microlaser particles is shown. Figure 7B An alternative optical configuration using only two collection paths and free-space optics is depicted. Figure 7C An alternative optical configuration using three collection paths is depicted relative to a typical flow cell. Figure 7D Depicting Figure 7C A 3D illustration of the optical configuration.

[0040] Figure 8 An optical system for collecting light from microlaser particles in a microfluidic device, according to an embodiment of the invention, is described.

[0041] Figures 9A to 9B The comparison of flow cytometry data of cells obtained with and without microlaser particles is shown. Figure 9A The data shown are from two samples: one composed of human Jurkat T cells without microlaser particles (left) and the other labeled with microlaser particles (right). Figure 9B Data are shown for two samples: one composed of human peripheral blood mononuclear cells (PBMCs) without microlaser particles (top) and the other labeled with microlaser particles (bottom).

[0042] Figures 10A to 10B Simulated and experimental data of reading optically labeled cells from an improved flow cytometer prototype according to embodiments of the present invention are shown respectively. Figure 10A The cumulative percentage of the detection spectrum for a given collection efficiency is shown in a simulation of 1000 randomly oriented microdisk laser particles. Figure 10B Exemplary data obtained using an improved flow cytometer prototype according to an embodiment of the present invention are shown.

[0043] Figures 11A to 11BExemplary data obtained by measuring optically labeled cells in multiple cycles using a prototype circulating flow cytometry instrument according to an embodiment of the invention are shown. Figure 11A An exemplary cell associated with the generation of microlaser particles that produce OFID consisting of three spectral peaks, which were measured and identified in eight consecutive flow cycles measuring 1100–1114. Figure 11B Proof-of-concept flow cytometry data are shown, in which THP1 cells labeled with microlaser particles were analyzed in three flow cycles 1120–1124.

[0044] Figure 12 Exemplary data are shown, illustrating the restoration of human PBMCs by photobleaching through multiple measurement cycles according to an embodiment of the present invention.

[0045] Figure 13 Exemplary data obtained by characterizing different immune cell populations of human PBMCs using a prototype circulating flow cytometry instrument according to an embodiment of the present invention are shown.

[0046] Detailed description of the specific implementation plan

[0047] Definitions. As used in this specification and the appended claims, the following terms shall have the meaning indicated unless the context requires otherwise.

[0048] A group has at least one member.

[0049] An "optical identifier" is a set of identifying particles that can be optically read and physically associated with a cellular entity as a unique identifier defining the cellular entity. For example, this set of identifying particles could be a collection of three microlaser particles, each emitting coherent light at a unique frequency based on its specific geometry and composition when properly excited, preferably by a laser, and the particles having a size of less than 100 micrometers. A particle with a size of 10 nm is still considered a "particle" in this context because it has a size less than 100 micrometers.

[0050] "Cellular entities" include cells or parts of cells, such as the nucleus, vesicles, or organelles, or coherent tissues of cells, such as multicellular spheroids. Cellular entities can also be living or chemically fixed.

[0051] “Tag” a cellular entity means to link an optical marker to the physical structure of the cellular entity.

[0052] "Parameters" are characteristics of a cellular entity measured by flow cytometry, such as surface or intracellular protein expression, RNA expression, quantification of organelles (such as mitochondria and lysosomes), cell granularity or size, or cell shape. Each such parameter of a cellular entity is measured by fluorescence, light scattering, or absorption. Each parameter measurement has a value determined by calculation using raw data obtained from the flow cytometry system.

[0053] An "OFID reader" is a combination of (i) an optical excitation source, such as a laser, used to excite an optical marker to generate a corresponding signal and (ii) a spectrometer used to read the corresponding signal to determine identification based on the optical marker. The result is optical frequency identification (OFID).

[0054] "Inactivating" a group of fluorescent probes means removing or altering the fluorescence associated with that group of probes, or altering the ability of a probe to interact with a specified target, or physically removing a probe from a sample.

[0055] A "recirculator" is a device that collects cell bodies after measurement in a flow channel, repairs the cells, and positions them for further measurement in the flow channel.

[0056] "Repair" refers to a population of cell entities that has undergone a first flow cytometry measurement cycle targeting a set of parameters. This means processing the population so that it can withstand the next flow cytometry measurement cycle targeting a different set of parameters. Typically, repairing a population involves inactivating the probes used in the first flow and re-staining the population.

[0057] A “collection path” is defined as a series of optical elements used to guide light emitted within a solid angle into a detector or spectrometer. These optical elements may include free-space components such as lenses and mirrors or fiber optic assemblies. Light from different collection paths is typically guided to different spatial regions of the detector or spectrometer, or combined via a 50 / 50 beam splitter or beam combiner before reaching the detector or spectrometer.

[0058] The fabrication and characterization of the microparticles are described in published PCT application WO2017 / 210675. This application describes a novel use and background for identifying microparticles in a class of flow cytometry we call "circulatory flow cytometry".

[0059] Figure 1 illustrates a conventional flow cytometry instrument. Cells in sample 100 are stained with fluorescent probes. Commonly used probes include fluorescent dyes, fluorescent dyes, fluorophores or fluorescent dyes conjugated with antibodies or nucleic acids, and reporter proteins encoded by genes. Flow cell 110 receives cells 100 via a cannula, tube, or motorized pipette 114. The flow cell is also connected to a tube to receive sheath fluid 118. The flow cell generates single-cell droplets or cell flows in flow channels 120. In most cases, the flow channels are vertical and fall by gravity, but microfluidic channels are also used for more precise control of flow rate and pressure. The device employs an optical arrangement to measure optical parameters from individual cells. It includes one or more laser sources 130 that deliver excitation light to the flowing cells in a row. Forward or side-scattered light 132 from each cell is measured using a laser line suppression filter 140 and a photodetector, such as a single-photon counter, avalanche photodiode (APD), or photomultiplier tube (PMT) 142. Fluorescence emission is collected by dichroic mirrors 140, 152, and 156, directing different spectral components to different photodetectors or PMTs 150. Prisms and PMT arrays (1x4 to 1x16) can be used instead of dichroic mirrors and PMTs. Electrical signals from photodetectors 142 and 156 are connected to a computer 160. The computer determines the size of each fluorescent probe from the electrical signals. When there is significant overlap between the spectra from the co-stained fluorescent probes, spectral separation based on linear or nonlinear fitting or machine learning will be applied.

[0060] Cells after fluorescence measurement are collected in chamber 170. The device may also include a sorting arrangement, typically including a deflector 180, which is switched to guide specific target cells to individual chambers 174. Typically, the deflector includes one or a pair of electrodes to which a high voltage is applied as the target cells pass. This technique is called fluorescence-activated cell sorting (FACS).

[0061] Figures 2A to 2E Different strategies for labeling cells with microparticles according to embodiments of the present invention are described. Figure 2A The invention illustrates the delivery of microparticles 200 into cells 210 via endocytosis or macropinocytosis according to an embodiment of the invention. Figure 2B The illustration shows an embodiment of the invention in which microparticles 220 are delivered into cells via a carrier-mediated mechanism, such as by using a liposome carrier 224.

[0062] Another strategy involves delivery within the cell via physical means across the cell membrane, including ballistic particle bombardment delivery (“biolistics”) or electroporation to create transient membrane pores. Figure 2CThe invention illustrates another embodiment of delivering microparticles 240 into cells via gene gun particle bombardment. Microparticles 240 are delivered at a speed 244 sufficient to allow them to penetrate the cell membrane 248.

[0063] Further strategies involve the biochemical conjugation of microparticles to the cell surface, where microparticles are attached to the cell membrane via antibodies or chemical binding.

[0064] Figure 2D A method for attaching microparticles 250 to the cell surface is shown according to another embodiment of the invention. Figure 2E Two exemplary methods for labeling microparticles onto cells are shown. It displays optical images of a semiconductor microlaser particle 260 labeled onto the outer surface of a cell and another semiconductor microlaser particle 270 labeled inside the cell (right), according to an embodiment of the invention.

[0065] The best method for labeling cells with microparticles depends on the cell type and the type of microparticles used.

[0066] Figure 3 An improved flow cytometry instrument according to an embodiment of the invention is described, which combines conventional fluorescence measurement and spectrometry to read optical markers. Identifying particles 300 and 302 are bound to the cells. As in conventional flow cytometry, cells in sample 310 are loaded into flow cell 314 using pipette 312 through fluid inlet 315, and fluorescence emission and light scattering from the cells are measured as the cells flow in flow channel 316. Cells are measured using a measurement device employing excitation laser source 318, dichroic filter 322, and PMT 324. The flow cell and accompanying jet system are optimized for maximizing cell collection after optical measurements.

[0067] To read optical markers 300 and 302, the system further employs a pump laser 330, which provides activation or pump light 332 to the cells. A preferred embodiment includes a pulsed laser 330 that generates nanosecond pulses with a repetition rate greater than 1 MHz; however, a continuous-wave laser can be used depending on the microparticle being identified. For microlaser particles, the peak optical power of the pump beam 332 should be sufficient to reach a threshold laser intensity.

[0068] Emissions 340 from optical markers associated with each cell are received and measured in a spectrometer 342. To achieve high spectral resolution better than 1 nm, the spectrometer typically includes at least one diffraction grating 344 and a charge-coupled device (CCD) camera 346. The camera's output is connected to a computer 350. The computer 350 is used to identify the light emission spectra of the particles and thereby determine the cell's optical frequency identification (OFID).

[0069] The excitation and emission wavelengths of optical markers can differ spectrally from those of fluorescent probes. For example, a 1064 nm laser is used to excite (or pump) microlaser particles that may emit in the 1100–1600 nm range, while fluorescent probes are typically excited by visible lasers between 350–700 nm and emit in the 400–800 nm range. Furthermore, the emission spectral shape of microlaser particles is significantly narrower than that of fluorescent probes, typically with a linewidth <1 nm, in contrast to the >50 nm linewidth of fluorescent probes. The FACS control signal 360 to deflector 380 can be based on fluorescence measurements. Optionally, if desired, signal 360 can be based on cell-based OFID measurements.

[0070] In some cases, the spectral region of OFID emission can overlap with the spectral region of fluorescence emission from stained cells. In such cases, dichroic filters can no longer adequately separate OFID emission from cellular fluorescence. In these situations, OFID emission and fluorescence are measured sequentially at different spatial locations (e.g., upstream and downstream of the flow channel), and the timestamp of each measurement is recorded. The OFID and fluorescence of each cell are then synchronized using the individually measured timestamps and cell velocity. Depending on the specific fluid system implementation, cell velocity measurements can be performed in various ways, such as using flow rate sensors or calibration beads.

[0071] In the conventional and modified cytometry shown in Figures 1 and 2, cells pass through the flow channel once. In principle, cells collected in bins 170 or 174 can be reloaded into the flow channel for repeated measurements to improve accuracy. However, without the use of optical markers, this repeated measurement does not improve the multiplexing capability of each individual cell measured in flow cytometry.

[0072] Figure 4A to Figure 4D The procedures in conventional and circulating flow cytometry are shown. Figure 4A shows the procedure in conventional flow cytometry. Figure 4B The process of initial pass (run) of circulating flow cytometry analysis according to an embodiment of the present invention is shown. Figure 4C The procedure in circulating flow cytometry according to an embodiment of the present invention is shown, which uses optical markers and destaining and staining with fluorescent probes. Figure 4D The procedure in cyclic flow cytometry according to another embodiment of the invention is shown, which uses optical markers and sequential fluorescence readings using DNA barcode probes.

[0073] According to one embodiment of the invention, the circulating flow cytometry method allows for multiple measurements of cells in the same or different channels, and allows for the combination of measurements from the same cells.

[0074] In circulating flow cytometry, according to embodiments of the invention, cells are collected after each cycle to enable repeatable measurements. Cells can also be washed between cycles during processing steps such as cell repair and staining. To maximize collection efficiency for each process, the surfaces of the equipment used for cell collection, including components such as tubing, containers, vials, and pipettes, can be made of suitable materials with low wettability and / or configured to have minimal surface roughness to reduce unwanted cell retention and adhesion. Suitable materials include polytetrafluoroethylene, polypropylene, polycarbonate, polyethylene, and polyetheretherketone. Surfaces can also be treated with chemical surfactants to reduce surface tension and prevent cell adhesion, for example, using commercially available solutions (Anti-Adherence Rinsing Solution, STEMCell Technologies).

[0075] Microfluidic devices can also be used to process and wash cells, including using laminar flow to wash cells with minimal disturbance and maximum retention. Cell collection can also be performed at 4°C to minimize cell adhesion and maintain cell viability.

[0076] According to an embodiment of the invention, a unique procedure in circulating flow cytometry is cell repair after each measurement and cell collection. The cells are inactivated and stained with a separate set of several easily distinguishable fluorescent probes. This process allows for the measurement of different molecular markers in each cycle.

[0077] The repair or destaining process 400 may involve one of several methods. One method involves inactivating or removing the fluorescent probe. This can be achieved by photobleaching the fluorophore, for example, using white or blue light from a light source at a sufficient intensity to inactivate the fluorophore without impairing cell viability. Another method involves using a compound to cleave the fluorophore or the probe itself, for example, using a commercially available chemically releaseable antibody (REAlease, Miltenyi Biotec).

[0078] After the destaining process 400, the restaining process 402 involves labeling the cells with another set of fluorescent probes, which are typically different from the previous fluorescent probes.

[0079] Alternative methods for destaining and restaining cells using fluorescent probes involve staining cells with oligonucleotide barcode probes that target all target molecular markers at once 410. One example is a commercially available DNA barcode antibody (CODEX, Akoya Biosciences). As previously described, cells are measured in multiple consecutive flow cytometry measurements. At each measurement, a fluorophore 414 binding to a subset of the oligonucleotide barcode probe is added, and chemicals are added to remove the fluorophore from previous measurements 412 (except the first measurement).

[0080] Circulating flow cytometry involves measuring the properties of each cell multiple times, typically 2-4 times or more as needed. Five or more cycles should also be possible. Circulating flow cytometry also includes a computational process to assign fluorescence measurements obtained across multiple cycles to the same cells based on measurements of the cells' unique OFIDs in each cycle.

[0081] Figures 5A to 5B An instrument for circulating flow cytometry according to an embodiment of the present invention is described. The instrument for circulating flow cytometry includes a flow cytometry system for reading fluorescent and optical markers, such as... Figure 3 As shown, a recirculator device for automatically processing cells between cycles of cyclic flow cytometry is also included.

[0082] Figure 5A An instrument for circulating flow cytometry according to an embodiment of the present invention is shown, comprising: Figure 3The system and recirculator device 550 for cell processing are depicted. The recirculator device uses mechanical components including a motorized stage, a sample collection container, a motorized stage, and a motorized pipette to perform processes including cell collection, cell remediation, and cell positioning for each measurement cycle. Cells from a sample after a flow measurement in one cycle are collected into a tube or well plate 500. The well plate 500 can be mounted on a motorized translation stage 502 to collect cells from different samples in different wells. The collected cells are then moved to a remediation process 510. Remediation may involve destaining using a light-emitting diode 520 that provides light 522 of sufficient intensity to photobleach fluorophores in fluorescent probes. Optionally, chemicals 530 for separating fluorophores or fluorescent probes from target molecules in the cells may be provided to the sample. Moreover, a laser 524 may be incorporated to provide bleaching light 526 to the cells in the flow channel. The restaining process 530 involves introducing another set of fluorescent probes 540 into the cells, targeting a different set of molecules than the previous target set. These new probe sets may have the same set of fluorophores conjugated to different antibodies targeting different targets, or they may use different fluorophores. The stained samples were loaded into the same flow cell 314, enabling measurements using the new fluorescent probe sets.

[0083] Figure 5BAn improved instrument for circulating flow cytometry according to an embodiment of the invention is depicted. The instrument includes a system 552 depicted in FIG. 5, and a fluid-based recirculation device 554 to automate the circulating flow cytometry process, including cell repair. The instrument employs one or more repair chambers 556-560 connected in series via conduits 568, in which cells undergo different treatments, including chemical, thermal, or light. The repair chambers may also be temperature-controlled and include sensors to detect the status of the repair process. Each repair chamber may be exposed to atmospheric pressure via valves 562-566. The pressure in each chamber may be additionally controlled via a pressurized gas line connected to a fluid controller. In the latter case, the pressure in each chamber is also monitored by the controller. The recirculator may also include one or more storage chambers 590 in which cells reside while awaiting further processing. Multi-repair chamber configurations utilize multiple valves and pumps 570-578 to transfer samples between chambers. Multiple pump chambers are used, including but not limited to peristaltic pumps, volumetric pumps, or pressure pumps. One or more additional sets of pumps and valves 580-586 allow the introduction of one or more types of external fluids into each chamber, which can mix with or interact with the cell sample. These ports also allow the introduction of cleaning agents to sterilize the chambers after sample removal or to extract samples from the system. The various pumps and valves are electronically controlled by a fluid controller 588. In cases where pressure pumps are used for sample transfer, additional pressurized gas lines can be connected between the fluid controller and each recovery chamber. At the end of each recovery cycle, the controller transports the sample to the storage chamber 590 before the flow cytometer 552 can begin a new measurement cycle.

[0084] Figures 6A to 6D Exemplary optical markers are shown, which can be used to uniquely label cells for use in embodiments of the invention. Although such optical markers have recently become known in the art, their use in the current context is novel. Figure 6A Electron microscopy images of semiconductor microlaser particles coated with silicon dioxide and their typical stimulated emission spectra are shown, with a peak at 1392 nm and a full width at half maximum (FWHM) of 0.35 nm. Figure 6B Fluorescence microscopy images of cells containing three microlaser particles with different laser emission peaks are shown. Figure 6C An optical image of an optical marker including three microdisk lasers is shown. Figure 6D The emission spectrum from the triplet laser particle is shown. The three narrow spectral peaks define the unique OFID assigned by the optical identifier.

[0085] Recently, various schemes for labeling cells with optical markers have been demonstrated, including fluorescent polystyrene particles, upconversion nanoparticle microlasers, perovskite nanowires, and plasmonic nanoparticles. In particular, semiconductor microdisk lasers with diameters of 0.5–3 μm are especially suitable for uniquely labeling large numbers of cells (>1,000) without affecting cell viability. Upon photoexcitation, these microlaser particles emit narrowband lasers with linewidths less than 1 nm, and their center wavelength can be tuned over a wide spectral range.

[0086] Cyclic cytometry requires reproducible and stable optical markers throughout multiple cycles of the cytometry procedure. For example, the optical markers should be stable after a photobleaching process of the fluorophores. The photobleaching process inactivates the vast majority of fluorophores on the internal cell surface. Typically, it is desirable for more than 99% of the fluorophores to be bleached or inactivated. Almost any type of fluorophore can be photobleached by prolonged, repeated light excitation. However, ideally, fluorophores that can be bleached at practically achievable light intensities for a practical duration, such as 30 minutes, are used, and the photobleaching process does not significantly affect cell viability due to photothermal and photochemical effects. In a simple model, the probability of inactivation can be described as… Where τ B (I B ) indicates that the fluorophore is at an intensity of I B The half-life under illumination. For example, for a given I B Bleaching 99.9% of the fluorophore within 30 minutes will require τ B >4.3 minutes.

[0087] It is also essential to use optical markers with a sufficient number of OFIDs to essentially uniquely label cells in a sample.

[0088] Let N represent the total number of OFIDs, and M represent the number of OFIDs used. The probability P of having a unique OFID in a given set of M OFIDs is... It equals e -M / N Because N, M >> 1. When M = N, P is 36.7%. Generally, N >> M, for example, when M = 10. 6 When N = 10 7 Given P = 90.5% and N = 10 8 The given value is P = 99.0%.

[0089] In this calculation, M is also equal to the number of labeled cells. Depending on the application, M equals the total number of cells in the sample, or it may represent a small portion of the entire cell population in the sample. Typically, M ranges from 1,000 to 1,000,000.

[0090] In the case of micro-laser particles, N can be 10 3 -10 12 Or on a larger scale, depending on the number of microlaser particles in each cell. Assuming each microlaser particle can emit one of n = 1000 distinguishable wavelengths, Where r is the number of microlaser particles per cell. For n = 1000 and r = 1, N = 1000. For n = 1000 and r = 3, N = 1.67 × 10⁻⁶. 8 For n = 1000 and r = 5, N = 8.42 × 10⁻⁶. 12 .

[0091] Matching OFIDs involves using an algorithm to assign a set of measured OFIDs to unique cellular entities with a high probability confidence level (>95%). The algorithm includes a method for extracting features from the measured OFIDs, including but not limited to spectral peak positions, widths, and how these properties vary with pump laser intensity. The algorithm also includes a method for calculating a distance metric or score between feature pairs, allowing a composite numerical score to be assigned between two OFIDs based on all pairwise scores acquired from a set of features from one OFID and a second set of features from another OFID. Finally, the algorithm includes a method to determine the equivalence of two OFIDs based on this composite numerical score, since they originate from the same physical set of optical identifiers.

[0092] In some cases, cells measured across different cycles may have missing, mismatched, or duplicated OFIDs. These errors are typically resolved by excluding these mismatched OFIDs and their respective cells from the computer analysis. However, by determining the most probable match based on the measured optical markers and / or the light scattering and fluorescence patterns of the cells, cells with mismatched OFIDs in one cycle can still be matched with cells from another cycle to a reasonable statistical confidence level.

[0093] To reduce the number of duplicate OFIDs to less than 10%, the possible number of OFIDs (M) should be 10 times larger than the number of cells (N) in the sample. One strategy is to include redundancy in the OFIDs by labeling each cell with more optical markers than necessary.

[0094] Figure 6 illustrates an exemplary semiconductor laser particle that meets the requirements. A single microlaser particle 600 can generate narrowband laser emission 610. Cells are allowed to absorb more than one microlaser particle 620, 622, and 624 with different emission wavelengths. Optionally, the microlaser particles can be combined together in multiples 630 prior to intracellular delivery. The combined laser emission 640 from this group of microdisks constitutes the cell's OFID 650. With an average of three microdisks per cell, this scheme can produce N > 10,000,000.

[0095] Figures 7A to 7D An optical system for collecting light from micro-laser particles according to an embodiment of the present invention is described. For laser particles with substantially isotropic or omnidirectional radiation modes, a single collection path is suitable. However, laser particles have optical cavities, and the intensity of the output laser emission is related to the coupling between the cavity and its surrounding environment. As a result, the emission modes from laser particles such as micro-laser particles tend to be spatially non-uniform. This direction-dependent emission intensity can cause problems with dynamic range and signal-to-noise ratio in the detection of the laser output spectrum. Collecting the emission with the largest possible numerical aperture or solid angle can minimize these problems.

[0096] To further mitigate this problem, a preferred implementation can employ multiple collection paths instead of a single path. Two paths may be sufficient when the angle dependence of laser particle emission is moderate. If the direction dependence of laser emission is strong, three paths may be necessary. Four paths can also be used. Figure 7A A system using multiple optical fibers to collect light from microlaser particles is shown. In this design, emission from cells 700 within water droplets 710 is detected via three paths 720, 722, and 724. These three paths are approximately 90, 60, or 120 degrees relative to each other. The emitted beams 720, 722, and 724 are collected by optical fibers 730, 732, and 734.

[0097] At the distal end of the optical fiber, lenses are placed to collimate the output beams and guide them to grating 752 in spectrometer 750. Collimators 740, 742, and 744 are arranged parallel to the grating pattern of diffraction grating 342, such that the collected beams undergo the same diffraction. The diffracted beams are focused onto camera 754. For example, when a laser particle emits a single laser line, the three beams form an elongated pattern 780 in the camera. When the CCD has a linear array with a high aspect ratio (e.g., 500 μm x 10 μm) of pixel size, the entire pattern can fall on a single pixel, or, if a two-dimensional CCD array is used, on different pixels along a vertical dimension perpendicular to the diffraction plane. In the latter case, the pixel data is integrated along the vertical dimension to produce the output spectrum. Various different optical schemes known to those skilled in the art for multipath beam collection can be used, including free-space setups with beam combiners.

[0098] Figure 7B An alternative optical configuration using only two collection paths and free-space optics is depicted. A pump laser 330 excites an optical marker contained in a cell 700 that flows into the page plane. The light collected by the two collection paths is approximately collimated by lenses 760 and 762, positioned at 90 degrees to each other. The light is then redirected by mirrors 764 and 766 before being combined by a beam splitter 768. The combined light is then focused by a lens 770 into a spectrometer 750.

[0099] Figure 7C Another optical configuration using three collection paths is depicted. A flow cell 780 provides the flow channel. A pump laser 332 excites optical markers contained within cells flowing continuously along the channel. Light emitted from the microlaser particles in approximately the forward direction 722 is collected by lens 790 and collimated or focused for measurement. Light emitted at angles φ1 and φ2 relative to the pump direction is collected by lenses 792 and 794, respectively. Approximately, φ1 and φ2 are 60 degrees to minimize the angle dependence of emission from the microlaser particles. To reduce optical aberrations of the emitted beam at the flow cell and air interface, a solid immersion lens, such as hemispherical glass 796, can be used. Collimated light following lenses 790, 792, and 794 can be emitted into an optical fiber and guided to a spectrometer. Figure 7D Depicting Figure 7C The 3D map configured in the middle.

[0100] When a cell contains multiple microlaser particles, the pump light 332 is partially absorbed and partially reflected by each laser particle. When the laser particles are positioned such that one particle is behind another along the path of the pump light, the next particle may receive less pump energy than the preceding particle. This results in emission from the two laser particles of different sizes. If the attenuation of the pump light by each laser particle is significant, the next particle may not receive enough pump energy to generate laser light. To minimize this problem, additional pump light 798 can be arranged so that the cell is illuminated with two pump beams in approximately two orthogonal directions. This reduces the pump shadowing effect. More than two pump beams can be used to minimize the difference in pump energy received by different laser particles.

[0101] Figure 8 An optical system for collecting light from micro-laser particles in a microfluidic device, according to an embodiment of the present invention, is depicted. The system employs a microfluidic chip 800 providing a flow 802. Three collecting optics, identical to those shown in Figure 7, are depicted. To avoid beam aberrations caused by refractive index mismatch between the microfluidic chip material and the surrounding air, a glass or plastic hemispherical lens 810 can be used.

[0102] In this configuration, the distal ends of optical fibers 730, 732, and 734 are stacked into a linear array. Beams 740, 742, and 744 exiting the fibers are collimated by a single lens 820 and diffracted by a diffraction grating 830 having grating lines parallel to the direction of the fiber stack. The diffracted beams are focused by a compound lens 840 onto a camera 860, where a CCD array is arranged approximately orthogonal to the fiber array and grating lines to receive the diffraction pattern 870. The optical fibers can be multimode fibers with an aperture of 50–105 μm and a cladding size of 125 μm, with a numerical aperture of approximately 0.22.

[0103] Figure 9A and Figure 9B The comparison of flow cytometry data of cells obtained with and without microlaser particles is shown. Figure 9A Data for human Jurkat T cells without microlaser particles (900) and those labeled with microlaser particles (950) are shown. The data indicate that the presence of microlaser particles did not cause significant changes in scattering distribution (902 and 952), cell viability (904 and 954), CD3 staining (906 and 956), CD19 staining (908 and 958), CD4 staining (910 and 960), and CD20 staining (912 and 962). Figure 9BData for human primary peripheral blood mononuclear cells (PBMCs) without microlaser particles (920) and labeled with microlaser particles (970) are presented. The data show that the presence of microlaser particles did not cause significant changes in CD3 versus CD19 staining (922 and 972), CD8 versus CD4 staining (924 and 974), CD16 versus CD56 staining (926 and 976), or CD16 versus CD14 staining (928 and 978).

[0104] Figures 10A to 10B The invention illustrates a modified flow cytometer prototype for reading simulated and experimental data from cells labeled with optical markers. Figure 10A The figures show the cumulative percentage of the detected spectrum for a given collection efficiency in simulations of randomly oriented microdisk laser particles 1000. Dashed line 1010 shows the results for a system with a single optical collection path, while solid line 1020 shows the results for a system with dual optical collection paths. In this simulation, the total percentage of collected light is 4% for the single-axis configuration and 8% for the dual-axis configuration when the microdisk is optimally oriented towards the collection lens (assuming a numerical aperture of 0.4). The blue arrows depict the minimum dynamic range required to detect all spectral peaks: ~180,000 for single-axis 1030 and ~10 for dual-axis 1040. Considering readout noise, sensitivity, and dynamic range (typically <10,000), the dynamic range required for single-axis simulations far exceeds the capabilities of typical CCDs, PMTs, and photodetectors. In contrast, the significantly reduced signal variation in dual-axis detection can be easily adapted to spectrometers employing CCD cameras and analog-to-digital converters (ADCs).

[0105] Figure 10B Exemplary data obtained using a modified flow cytometer prototype according to an embodiment of the invention are shown. The OFID reader consists of a 1064-nm pump laser with a repetition rate of 10 MHz and a spectrometer, and is used in conjunction with a capillary-based flow cytometer. The system was tested using uniaxial and biaxial collection paths. A white light image 1040 of cells labeled with microlaser particles flowing in a flow cell is shown. As the cells flow through the flow cell, OFID spectra are acquired and recorded in a computer 1042. Each row, defined by black lines, corresponds to the OFID of a specific cell 1044 (depicted with lines of different colors for different laser emission peaks). The system was tested using uniaxial and biaxial collection paths. A representative OFID spectrum collected using the uniaxial configuration 1050 shows microlaser particle emission peaks 1052-1058 with intensity variations of several orders of magnitude. In contrast, a representative OFID spectrum collected using the biaxial configuration 1060 shows microlaser particle emission peaks 1062-1066 with intensity variations of less than one order of magnitude.

[0106] Figures 11A to 11B Exemplary data obtained by measuring optically labeled cells over multiple cycles using a prototype circulating flow cytometry instrument according to an embodiment of the invention are shown. A biaxial collection configuration is used to read the emission of the microlaser particles. The prototype has a total of 5 PMTs for measuring forward scattering, side scattering, and 3 fluorescence spectra. The pump laser of the OFID reader is coupled such that both the fluorescence excitation light and the pump light illuminate the cells in the flow channel through a common objective. A dichroic beam splitter is used to separate the NIR emission of the laser particles from the fluorescence emission of the fluorescent probes.

[0107] Figure 11A An exemplary cell associated with microlaser particles generating OFID consisting of three spectral peaks measured and identified over eight consecutive flow cycles ranging from 1100 to 1114. Minor variations in the intensity of each peak do not affect the algorithm's ability to match OFIDs between cycles.

[0108] Figure 11B Proof-of-concept flow cytometry data are shown, in which THP1 cells labeled with microlaser particles were analyzed in three flow cycles 1120–1124. Staining and recovery were performed to target three different markers in each cycle. Cells were collected after the first flow measurement. Existing fluorescent antibodies in the cells were removed (using chemically released antibodies), and the cells were stained with a new set of three fluorescent antibodies. The stained cells were loaded into the instrument for a second flow measurement. After the measurement, a third set of fluorescent antibodies was applied to the collected cells. A third flow measurement was then performed. A personal computer was used to store fluorescence data from the PMT and spectrophotometer data for each cell. Custom software was used to correlate the fluorescence data with the OFID of each cell in each of the three measurements, and the properties of the corresponding target parameter set were combined using the OFID. OFID matching between cycles enabled the measurement of a total of nine different markers per cell.

[0109] Figure 12Proof-of-concept data demonstrating the repair of human PBMCs for multiple measurement cycles were presented. High-power white light-emitting diodes (LEDs) with an output power >3W were used to photobleach the fluorophore-conjugated antibodies attached to the cells between measurements. Typically, complete photobleaching of stained cells required 30 minutes of white light irradiation, also performed at 4 degrees Celsius to maintain cell viability. Cells were first stained with CD56-phycoerythrin (PE) and measured on a flow cytometer 1200. Next, cells were bleached to completely inactivate the fluorescence signal from the PE antibody 1210. Cells were then stained with CCR7-PE and measured again on a flow cytometer 1220. Next, cells were bleached to completely inactivate the fluorescence signal from the PE antibody 1230. Finally, cells were stained with CD21-PE and measured again on a flow cytometer 1240. The data showed no significant difference in the proportion of different cell types in the photobleached cell samples compared to the control samples without photobleaching.

[0110] Figure 13 This demonstrates proof-of-concept flow cytometry data in which human PBMCs labeled with microlaser particles were analyzed in two flow cycles to measure the proportions of different immune cell populations. Freshly thawed PBMCs were labeled with microlaser particles such that most cells contained three or more microlaser particles to generate a unique OFID. Approximately 500,000 cells in a 100 μL volume were measured at a rate of 30 μL / min. In the first flow, the labeled cells were stained with fluorophore-conjugated antibodies, including CD3-APCA750, CD19-ECD, CD56-PE, CD14-PE-Cy7, CD8-APCA700, and CD4-APC, and measured using a circulating flow cytometry prototype instrument equipped with OFID. Fluorophores were chosen in part because they allow for photobleaching with minimal loss of cell viability. After photobleaching the labeled cells with a white LED for 30 minutes (similar to...), the cells were... Figure 12 (As described in the original text), cells were washed and restained with a second set of fluorophore-conjugated antibodies, including PD1-PECy5.5, CD57-PacBlue, CD45RA-FITC, CCR7-PE, CD28-ECD, and CD27-PECy7, and measured on a circulating flow cytometer. Fluorescence data were matched between cycles using OFID for each cell to generate a dataset with 12 different labels per cell. This data was used to characterize the frequencies of different lymphocyte subsets 1300, natural killer cells 1310, monocytes 1320, T cells 1330, and CD4+ T cell subsets 1340, 1340, 1360, and 1370.

[0111] This invention describes a system and method for performing cyclic flow cytometry. This invention overcomes current challenges in cost, complexity, and accuracy associated with highly multiplexed flow cytometry. This invention eliminates the need for complex and expensive spectral separation processes, or significantly reduces the burden of selecting, optimizing, and testing panels. This invention enables highly multiplexed cell counting with lower cost and higher accuracy. The instrument is significantly less expensive than state-of-the-art flow cytometers because the complex fluorescence detection scheme involving numerous lasers and detectors does not require the measurement of 30 labels at once. Reagent costs are also reduced because fewer antibodies are needed in the workflow for preparing flow cytometry panels, especially for fluorescence minus one (FMO) controls, as previously described.

[0112] Furthermore, through multiple flow cycles, the number of molecular contents from each cell that can be analyzed can be far greater than that possible with a state-of-the-art flow cytometer in a single flow or single run (e.g., >30 targets). For example, by measuring 10 markers in each run, four runs would allow for the measurement of a total of 40 markers.

[0113] The preferred embodiments described herein are used for cell analysis. However, instead of whole cells, the apparatus and method can also be used to analyze a portion of a cell, such as the nucleus, other organelles, or cell extracts such as exosomes. For example, the nuclei of a group of cells are extracted and labeled with an optical marker. The embodiment shown in Figure 5 receives the nuclei instead of cells 300 as the sample and processes the sample in the same manner as described.

[0114] The embodiments of the present invention described above are intended to be exemplary only; many variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to fall within the scope of the invention as defined in any of the appended claims.

Claims

1. A method of performing cyclic flow cytometry analysis on a sample population of cellular entities, the method comprising: causing each cellular entity in the sample population to be labeled with an optical marker; for each cellular entity in the sample population, under a condition that determines identification of a cellular entity that is obtaining a value of a first set of parameters via the optical marker, performing a first pass flow cytometry measurement on the flow stream with respect to the first set of parameters, and storing the value of the first set of parameters in relation to the identification; and under a condition that separately determines identification of a cellular entity that is obtaining a value of a second set of parameters via the optical marker, performing a second pass flow cytometry measurement on the flow stream with respect to the second set of parameters, and storing the value of the second set of parameters in relation to the identification.

2. The method of claim 1, further comprising, for each cellular entity in the sample population, using the identification to merge attributes of the first set of parameters with attributes of the second set of parameters.

3. The method of claim 1, further comprising: under a condition that separately determines identification of a cellular entity that is obtaining a value of at least another set of parameters via the optical marker, performing at least another pass flow cytometry measurement on the flow stream with respect to the at least another set of parameters, and storing the value of the at least another set of parameters in relation to the identification.

4. The method of claim 1, wherein the first and second pass flow cytometry measurements use a first and second set of fluorescent probes that target different sets of parameters, respectively, and the method further comprises deactivating the first set of fluorescent probes after performing the first pass flow cytometry measurement and before performing the second pass flow cytometry measurement.

5. The method of claim 1, wherein the sample population comprises at least 1,000 cellular entities.

6. The method of claim 1, further comprising, after performing the first pass flow cytometry measurement and before performing the second pass flow cytometry measurement, collecting a sample population that is the subject of analysis in a manner that preserves characteristics of cellular entities in the sample population.

7. The method of claim 6, wherein collecting comprises using a collection vessel to capture cellular entities of the sample population at an end of the flow stream.

8. The method of claim 7, further comprising, before performing the second pass flow cytometry measurement, rehabilitating the captured cellular entities.

9. The method of claim 1, wherein the optical marker is a set of micro-laser particles.

10. The method of claim 9, wherein the micro-laser particles comprise a semiconductor.

11. The method of claim 1, wherein the flow cytometry measurements utilize fluorescence by fluorophores, and determining identification of the cellular entity comprises using a light excitation source to read the optical marker in an emission spectrum.

12. The method of claim 11, wherein the fluorophores are configured to be photo-bleachable within 30 minutes without affecting cell viability.

13. The method of claim 11, wherein the fluorophore is configured to operate in a fluorescence spectrum separate from the emission spectrum.

14. The method of claim 1, wherein the first set of parameters is selected from the group consisting of surface or intracellular protein expression, RNA expression, quantification of organelles, cell granularity, cell size, cell shape, and combinations thereof.

15. The method of claim 14, wherein the first set of parameters is quantification of mitochondria or lysosomes.

16. The method of claim 14 or 15, wherein each parameter of the cellular entity is measured by a phenomenon selected from the group consisting of fluorescence, light scattering, and absorption.

17. An improved flow cytometry apparatus of the type having a flow channel coupled to a fluidic input port configured to receive a sample population of cellular entities to be measured relative to a target set of parameters, the flow channel instrumented for obtaining and storing successive fluorescence readings from successive cellular entities passing therethrough, wherein the improvement comprises: an OFID reader associated with the flow channel, wherein each cellular entity in the sample population has been labeled with an optical identifier, and the OFID reader is configured to determine identification of each cellular entity via the optical identifier as each cellular entity is measured in each of a plurality of flow passes of the sample population of cellular entities through the flow channel, each flow pass being associated with a respective target set of parameters.

18. The improved flow cytometry apparatus of claim 17, wherein the OFID reader is configured to operate on a plurality of emission collection paths, two of which define an angle of approximately 90 degrees relative to each other.

19. The improved flow cytometry apparatus of claim 17, further comprising a recycler configured to repair cellular entities passing through the flow channel and position them for further passage through the flow channel.

20. An improved flow cytometry apparatus of the type having a flow channel coupled to a fluidic input port configured to receive a sample population of cellular entities to be measured relative to a target set of parameters, the flow channel instrumented for obtaining successive fluorescence readings from successive cellular entities passing therethrough, wherein the improvement comprises: an OFID reader associated with the flow channel, wherein each cellular entity in the sample population has been labeled with an optical identifier, and the OFID reader is configured to determine identification of each cellular entity via the optical identifier as each cellular entity is measured in each of a plurality of flow passes of the sample population of cellular entities through the flow channel, each flow pass being associated with a respective target set of parameters; and a processor to (i) associate and store in a storage device respective fluorescence readings with the identification of each cellular entity in each of the flow passes via the optical identifier, and (ii) to use the identification to consolidate attributes of the respective target sets of parameters.

21. The improved flow cytometry apparatus of claim 20, wherein the OFID reader is configured to operate on a plurality of emission collection paths, two of which define an angle of approximately 90 degrees relative to each other.

22. The improved flow cytometry apparatus of claim 20, wherein the improvement further comprises a recycler configured to capture and repair cellular entities passing through the flow channel and position them for further passage through the flow channel.

23. The improved flow cytometry apparatus of claim 22, wherein the recycler has a surface for contacting a sample population of cellular entities configured to have low wetness.

24. An improved flow cytometry apparatus of the type having a flow channel coupled to a fluid input port configured to receive a sample population of cellular entities to be measured relative to a set of target parameters, the flow channel instrumented for obtaining successive fluorescence readings from successive cellular entities passing therethrough, wherein the improvement comprises: an OFID reader associated with the flow channel, wherein each cellular entity in the sample population has been labeled with an optical identifier, and the OFID reader is configured to determine identification of each cellular entity via the optical identifier as each cellular entity is measured in the flow channel, wherein the OFID reader is configured to operate on a plurality of emission collection paths, two of which define an angle of approximately 90 degrees relative to each other; and a processor to (i) associate and store respective fluorescence readings with the identification of each cellular entity via the optical identifier in a storage device, and (ii) use the identification to consolidate attributes of the set of target parameters.

25. A kit for converting a flow cytometry apparatus of the type having a flow channel coupled to a fluid input port configured to receive a sample population of cellular entities to be measured relative to a set of target parameters, the flow channel instrumented for obtaining and storing successive fluorescence readings from successive cellular entities passing therethrough, into an improved flow cytometry apparatus, the kit comprising: an OFID reader associated with the flow channel, wherein each cellular entity in the sample population has been labeled with an optical identifier, and the OFID reader is configured to determine identification of each cellular entity via the optical identifier as each cellular entity is measured in each of a plurality of flow passes of the sample population of cellular entities through the flow channel, each flow pass associated with a respective set of target parameters; a recycler configured to capture and repair cellular entities passing through the flow channel and position them for further passage through the flow channel; and a processor to (i) associate and store respective fluorescence readings with the identification of each cellular entity via the optical identifier in each of the flow passes in a storage device, and (ii) use the identification to consolidate attributes of the respective sets of target parameters.

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