Flow nanocytometry devices and methods for nanoparticle detection

Abstract

A flow nanocytometer for measuring extracellular vesicles (EVs) may include a flow cell for receiving a core stream carry at least one EV, at least one excitation source module for outputting a light beam to interrogate a portion of the core stream, a detection module for detecting the light beam, and a processor communicatively coupled to a memory, the at least one excitation source, and the detection module. The processor may execute a method including receiving a plurality of electronic signals from the detection module; detecting an intensity of a polycystic kidney disease (PKD) associated protein expressed by the EVs based on the plurality of electronic signals; determining a quantity of the EVs based on the plurality of electronic signals; and determining a severity or a prognosis of polycystic kidney disease in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs.

Description

[0001] FLOW NANOCYTOMETRY DEVICES AND METHODS FOR NANOPARTICLE DETECTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 562,248, filed March 6, 2024, the contents of which are herein incorporated by reference in their entirety.

[0004] INCORPORATION BY REFERENCE

[0005] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0006] STATEMENT OF GOVERNMENT GRANT

[0007] This invention was made with government support under grant no. 1R43GM155492-01 awarded by National Institutes of Health (NIH) / National Institute of General Medical Sciences (NIGMS). This invention was also made with government support under grant no. R01DK122205 awarded by the NIH. The government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] This disclosure relates generally to the field of precision medicine and, more specifically, to the field of molecular biomarker analysis. Described herein are flow nanocytometry devices and methods for nanoparticle detection, for example in polycystic kidney disease (PKD).

[0010] BACKGROUND

[0011] Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 (85% of total) o PKD2 (15% of total) genes that encode polycystin-1 (PCI) and polycystin- 2 (PC2), respectively. About 1 :800 individuals have ADPKD. In the U.S., 417,000 people have ADPKD. In ADPKD, a subset of nephrons develop fluid filled cysts mainly in the collecting duct which relentlessly expand compromising the function of surrounding nephrons and vasculature ultimately leading to renal failure in the 5thand 7thdecade in PKD1 and PKD2, respectively. Fortunately, ADPKD individuals have a long treatment window and there are several therapies in the pipeline.

[0012] SUMMARY

[0013] In some aspects, the techniques described herein relate to a flow nanocytometer configured to measure extracellular vesicles (EVs), including: a flow cell configured to receive a core stream therethrough, the core stream configured to carry at least one EV in a urine sample from a subject; at least one excitation source module configured to output a light beam to interrogate a portion of the core stream; a detection module configured to detect the light beam; and a processor communicatively coupled to a memory, the at least one excitation source, and the detection module, wherein the memory is configured to store instructions that when executed by the processor, cause the processor to perform a method including: receiving a plurality of electronic signals from the detection module; detecting an intensity of a polycystic kidney disease (PKD) associated protein expressed by the EVs based on the plurality of electronic signals; determining a quantity of the EVs in the urine sample based on the plurality of electronic signals; and determining a severity or a prognosis of polycystic kidney disease in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs in the urine sample.

[0014] In some aspects, the techniques described herein relate to a computer-implemented method, performed by a processor communicatively coupled to a memory, for detecting extracellular vesicles (EVs) in a biological sample from a subject, including: receiving a plurality of electronic signals indicating one or both of: light scattering properties of the EVs and fluorescence emissions of a marker coupled to at least one polycystic kidney disease (PKD) associated protein; detecting an intensity of the at least one PKD associated protein expressed by the EVs based on the plurality of electronic signals; determining a quantity of the EVs in the biological sample based on the plurality of electronic signals; and determining a severity or a prognosis of PKD in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs in the biological sample.

[0015] In some aspects, the techniques described herein relate to a computer-readable medium including computer readable instructions that, when executed by a processor, cause the processor to execute a method including: receiving a plurality of electronic signals indicating one or both of: light scattering properties of a plurality of extracellular vesicles (EVs) and fluorescence emissions of a marker coupled to at least one polycystic kidney disease (PKD) associated protein in a biological sample of a subject; detecting an intensity of the at least one PKD associated protein expressed by the plurality of EVs based on the plurality of electronic signals; determining a quantity of the plurality of EVs in the biological sample based on the plurality of electronic signals; and determining a severity or a prognosis of PKD in the subject, based on the intensity of the at least one PKD associated protein and the quantity of the plurality of EVs in the biological sample.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The foregoing is a summary, and thus, necessarily limited in detail. The above-mentioned aspects, as well as other aspects, features, and advantages of the present technology are described below in connection with various embodiments, with reference made to the accompanying drawings.

[0018] FIG. 1 illustrates schematically a flow nanocytometry device configuration of an embodiment of the present disclosure.

[0019] FIG. 2 shows a flow chart of an embodiment of a method of determining a severity and / or prognosis of polycystic kidney disease (PKD) in a subject.

[0020] FIG. 3 is a schematic illustration of the pneumatic and fluidic control system architecture of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0021] FIG. 4 schematically illustrates a configuration of a sheath pressure control module for a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0022] FIG. 5 schematically illustrates a configuration of a sample pressure control module of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0023] FIG. 6 schematically illustrates several modes of operation of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0024] FIG. 7 illustrates a sample probe wash module of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0025] FIG. 8 illustrates a sheath boost module of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0026] FIGs. 9A-9C schematically illustrate the optical layout architecture of a flow nanocytometry device in accordance with some embodiments of the present disclosure. FIGs. 10A-10B illustrate an embodiment of a flow nanocytometry device where the substantially collimated, substantially Gaussian light beam emitted by one of the excitation light sources passes through beam shaping elements prior to being focused into the flow cell by global focusing lens.

[0027] FIG. 11 illustrates an externally adjustable laser fine alignment module of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0028] FIG. 12 illustrates a stray-light blocking module of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0029] FIG. 13 illustrates a spatial filter or stray -light blocking module of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0030] FIG. 14 illustrates a cross-section, perpendicular to the direction of fluid flow, of an embodiment of a light collection configuration of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0031] FIG. 15 illustrates a cross-section, perpendicular to the direction of fluid flow, of an embodiment of a light collection configuration of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0032] FIG. 16 is a flowchart that describes a method of particle analysis that can be performed using a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0033] FIG. 17 shows a block diagram of an exemplary embodiment of a data processing system to provide a particle analysis as described herein.

[0034] FIG. 18 is a mechanical drawing of an optical of a flow nanocytometry device in accordance with some embodiments of the present disclosure.

[0035] FIGs. 19A-19B show two graphs of a flow nanocytometry analysis of reference nanoparticles and illustrate some embodiments of the present disclosure.

[0036] FIGs. 20A-20B show a Western blot image and quantitative ratiometric western blotting, respectively, for polycystin 1 (PCI) and CD133 of urinary exosomes.

[0037] FIGs. 21A-21C show flow nanocytometry data from a normal sample and a PKD patient sample. FIGs. 22A-22B show flow nanocytometry data for urine samples stained with and without an R-phycoerythrin (R-PE)-labeled anti-PCl antibody and a membrane marker CellBrite™ 650 (“FL3 (CellBrite 650)”).

[0038] FIGs. 23A-23B show flow nanocytometry data for samples stained with both a PCI stain (PCl-PE)and a PC2 stain (PC2-AZ488).

[0039] FIGs. 24A-24C show flow nanocytometry data for samples stained with both a PCI stain (PC1-PE) and a CD133 stain (CD133-AF488).

[0040] The illustrated embodiments are merely examples and are not intended to limit the disclosure. The schematics are drawn to illustrate features and concepts and are not necessarily drawn to scale.

[0041] DETAILED DESCRIPTION

[0042] The foregoing is a summary, and thus, necessarily limited in detail. The above-mentioned aspects, as well as other aspects, features, and advantages of the present technology will now be described in connection with various embodiments. The inclusion of the following embodiments is not intended to limit the disclosure to these embodiments, but rather to enable any person skilled in the art to make and use the claimed subject matter. Other embodiments may be utilized, and modifications may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the disclosure, as described and illustrated herein, can be arranged, combined, modified, and designed in a variety of different formulations, all of which are explicitly contemplated and form part of this disclosure.

[0043] Current methods to inform treatment of Autosomal dominant polycystic kidney disease (ADPKD) lack sufficient sensitivity and specificity about the nature and severity of the disease, causing treatment to be delayed, with negative consequences for patients in both morbidity and mortality. The systems and methods described herein provide technical solutions, including sensitive and specific assays for determining a presence of polycystic kidney disease (PKD), a severity of PKD, and / or prognosis of the subject having PKD.

[0044] Conventionally, ADPKD can also be monitored using magnetic resonance imaging (MRI) and by quantification of the height-adjusted total kidney volume (HtTKV), which is in turn adjusted for age to yield a categorical measure of severity, the Mayo / Irazabal score (1 A (mild)-lE (severe)). However, MRI-based methods do not provide actionable clinical information until kidney cysts have already developed, missing the critical window of latency before cysts appear. In addition, MRI-based methods are cumbersome, time-consuming, and relatively expensive.

[0045] Conventionally, ADPKD has been diagnosed using western blot analyses. For example, western blotting of urine-derived extracellular vesicles (PKD-EVs), approximately 100-nm diameter extracellular vehicles (EVs) secreted into the urine, can be used to diagnose individuals with ADPKD. However, the variability inherent in the assay is such that neither disease severity nor prognosis can be determined. As shown in FIG. 20A, quantitative ratiometric western blotting for PCI and CD 133 in urinary exosomes from PKD patients using a palindromic calibration standard loading (STD1, STD2) and two replications of five repeats each (REP3, REP4) for each of PCI (as further described herein, a PKD-associated protein) and CD133 (as further described herein, a PKD-invariant protein) results in within-batch variability of 12% and between-batch variability of 20%, levels too high to be used with confidence for diagnosis, prognosis, and to inform treatment. As shown in FIG. 20B, the ratio of PCI to CD133 in western blots of urinary exosomes shows significant differences between PKD patients (“PKD1”) and healthy controls (wildtype, “WT”). However, even using the PCECD133 ratio in a western blot assay (which is a bulk technique, unable to resolve individual PKD-EVs) does not reduce result variability enough to provide confidently actionable diagnostic / prognostic / therapeutic information. For example, despite the use of multiple technical replicates, the PC1 / CD133 western blot assay cannot be used to determine disease severity or prognosis, which are important factors in clinical treatment decisions.

[0046] Conventionally for immunofluorescence, the target (urine or tissue on slides) is incubated with the primary antibody then detected with a secondary antibody which is linked to a fluorescent dye. When working with tissue sections, the protocol includes a washing step between the two applications. This is not practical in EV work as the vesicles do not ‘pellet’ unless very high g- forces are used (an ultracentrifuge run). Furthermore, the ultracentrifugation step is not efficient, as most of the vesicles are lost on the sides of the tube or remain in suspension.

[0047] The devices and methods described herein solve the above technical problems with technical solutions. For example, the devices and methods described herein can evaluate single EVs using flow cytometric approaches tailored to the detection of nanoparticles, i.e. flow nanocytometry, to detect EVs, analyze EVs, and determine a severity and / or prognosis. Although extracellular vesicles (EVs) are described herein, the systems, devices, and methods described herein may be applied to, extended to, or otherwise encompass other nanoparticles. The systems, devices, and methods described herein may be used to detect, quantify, or otherwise measure extracellular vesicles (EVs), oncosomes, exosomes, exosome vesicles, exosome-like vesicles, exomeres, ectosomes, microparticles or microvesicles, blebbisomes, apoptotic bodies, and the like. The systems, devices, and methods described herein may be used to detect, quantify, or otherwise measure particles ranging in size from about 20 nm to about 150 nm; about 100 nm to about 1,000 nm; greater than about 50 nm; less than about 50 nm; etc. The systems, devices, and methods described herein may be used to detect particles involved in cell communication, RNA transfer, protein transfer, inflammation, coagulation, metabolism, and cancer or disease progression. The systems, devices, and methods described herein may be used to detect particles that are released from a plasma membrane, an endosomal system, immune cells, tumor cells, neurons, glial cells, endothelial cells, stem cells, and the like.

[0048] For example, the devices and methods described herein provide a simple and inexpensive biochemical methodology for determining a severity and / or prognosis of an ADPKD individual based on the analysis of single EVs in a substantially unprocessed biological sample. For example, the devices and methods described herein use one or more of: single-particle detection (i.e., not bulk / averages); and multiparametric detection (e.g., allowing simultaneous detection of multiple biomarkers). Further, the devices described herein have high throughput for greater accuracy; increased flexibility for measuring a wide range of particle sizes; increased stability to increase confidence in the results; increased ease of use to allow focusing on measuring, not tweaking; and increased sensitivity for detecting smaller particles.

[0049] The methods described herein overcome the above technical problems with technical solutions that include analyzing the sample in a substantially unprocessed state (e g., urine in a tube) and / or with fast (e.g., about 100,000g), low g-force centrifugation (e.g., about 4000g); titrating the primary and secondary antibodies to reduce secondary immune complex formation that mimics EVs and crosslinking of vesicles into clumps; and / or diluting the biological sample. As a further example, the methods described herein may include diluting the biological sample (e.g., urine, blood, lymph, interstitial fluid, spinal fluid, sweat, marrow, and the like) in phosphate buffered saline (PBS) or 2-(N-morpholino)ethanesulfonic acid (MES) at about pH 6.0 to about pH 8.0 for antibody addition. This avoids the problems inherent with ultracentrifugation which entails long spins and, in the case of urine, co-preci pitati on of abundant Tamm Horsfall protein (THP). THP causes clumping of EVs and can interfere with assays as THP binds IgG.

[0050] The conventional approach to flow cytometry uses forward scattering channel (FSC) and side scattering channel (SSC) signals to assess cellular diameter and granularity of a cell, respectively. EVs present a problem as EVs are orders of magnitude smaller than cells, with PKD- EVs having typical electron microscopy (EM)-derived diameters of about 100 nm — far below the resolution of lasers commonly used on flow cytometers (e.g., 488 nm) to detect cells, which have typical diameters of several microns to tens of microns. It has been shown that conventional flow cytometry is only capable of detecting single EVs down to about 250 nm. Conventionally, SSC is better than FSC due to its greater sensitivity. Using conventional flow cytometric analysis, urine EVs less than about 250 nm (based on EM) in diameter could not be efficiently detected even by SSC. In conventional flow cytometry, EVs and other nanoparticles smaller than about 250 nm can generally be detected only in the “swarm regime,” where multiple particles are coincidentally present in the light path. In one example of the prior art, when conventional flow cytometric counts were compared to the known number of microspheres or EVs in a sample, “at least 77 silica microspheres or 155 EVs had to be simultaneously present in the laser beam to generate a detectable signal”. Thus, conventional flow cytometry is incapable of detecting individual EVs.

[0051] The devices described herein solve the above technical problems of EV detection through flow cytometry with technical solutions. For example, the devices described herein employ one or more of: highly stable, pressure-driven fluidics for more precise measurements; a flow microchannel with dimensions tailored for optical interrogation of nanoparticles; high-power lasers for higher signal-to-noise ratios; ultraviolet / violet lasers for higher scattering signals; a collinear excitation architecture for improved scattering sensitivity; a seven-element, high-numerical- aperture (NA), broadband-transmission objective for improved light collection; a stray-light rejection architecture for reduced background levels; three scattering detection channels for improved scattering discrimination; a photomultiplier tube (PMT)-based detection system for high sensitivity, low dark current, and high dynamic range; and signal-processing algorithms for reduced background events. In some embodiments, the devices described herein allow detection and interrogation of fluorescent intensity for individual PKD-EVs.

[0052] In some embodiments, the devices described herein use collinear excitation and a combination of multiple short- wavelength lasers including in the blue / violet / ultraviolet to maximize scattering and fluorescence signals. For example, the devices herein may include a 375- nm laser, a 405-nm laser, and a 488-nm laser, and a plurality of detection channels (e.g., a forward scatter channel, two side scatter channels, one or more fluorescence channels, etc.).

[0053] In some embodiments, the devices and methods described herein function to monitor the levels of ADPKD associated proteins with reference to a control protein to determine a disease severity and / or prognosis of an ADPKD individual. The devices and methods described herein provide a new technical approach for monitoring, diagnosing, and / or treating an individual having ADPKD. The devices and methods described herein use an assay designed to run on a flow cytometer tailored for the detection, analysis, characterization, and quantification of nanoparticles, i.e., a flow nanocytometer, to measure amounts, concentrations, and / or ratios, of EVs expressing one or more ADPKD-associated proteins, relative to one or more other ADPKD-associated proteins and / or to one or more control proteins, in a biological sample; and to measure amounts of one or more ADPKD-associated proteins, either absolute or relative to amounts of one or more other ADPKD-associated proteins and / or of one or more control proteins, expressed on EVs in a biological sample. For example, the amounts and / or ratios may be measured in urine. ADPKD associated proteins may include, but may not be limited to, polycystin-1 (PCI), poly cystin-2 (PC2), fibrocystin (FC), exosomal polycystin- 1 interacting protein (CU062, EPCIP, C21orf62 gene product), C16orf89, Clorf95, and cell surface hyaluronidase CEMIP2(TMEM2), and the like. The one or more control proteins may include a co-expressed invariant protein. For example, an invariant protein may include, but may not be limited to, prominin 1 (CD133), tetraspanin-4 (TSN3), tetraspanin-4 (TSN4), tetraspanin-7 (TSN7) and tetraspanin-8 (TSN8) etc. For example, polycystin-1 (PCI) and / or poly cystin-2 (PC2) may be measured relative to the control or invariant protein to determine a disease severity, prognosis, and / or treatment path for an ADPKD individual. As an exemplary relationship, the PC 1 / CD 133 ratio is significantly lower in individuals with PKD / mutations versus those with normal kidneys. Further, mass spectrometry shows that the PC1 / CD133 ratio correlates inversely with the HtTKV with an R2=0.63, suggesting that PKD-EVs may be used as a proxy for cyst development.

[0054] In some embodiments, the devices and methods described herein may be used to measure or detect PCI-negative PKD-EVs (CD I 33 / PC I ) indicating an onset of PKD or presence of PKD in an individual. In some embodiments, the devices and methods described herein may be used to measure or detect an absolute number of PKD-EVs with substantially unaltered PC1 / CD133 ratio (as compared to a control group). For example, the methods and devices described herein may measure or detect a decrease in a total number of PC1+, PC2+, and / or CD133+PKD-EVs present in a biological sample. In some clinical presentations of PKD, PKD-EVs may tend to lose PCI and PC2 expression as compared to expression of an invariant or control protein, and this may be detected or measured as an alteration of the mean fluorescence per particle for PCI and / or PC2 in relationship to the mean fluorescence per particle for invariant or control protein. The disease severity may be measured, for example, in terms of percentage of CD133+, PC I PKD-EVs, a general reduction in the number of CD133+, PC1+PKD-EVs in the biological sample, or the mean amount of PC1+or PC2+signal per CD133+PKD-EV. For example, individuals with low levels of PCI or PC2 on PKD-EVs, or with a low number of total PC1 / PC2+ PKD-EVs may have ADPKD and the degree of the reduction of PCI per PKD-EV or the degree to which the number of PC1 / PC2+ PKD-EVs are reduced may correlate with disease severity and thus prognosis (low PCI per PKD-EV or low numbers of PKD-EVs correlates with high height adjusted total kidney volume and high Mayo score).

[0055] Described herein is a flow nanocytometry device structured to detect EVs and / or measure proteins associated with EVs. In some embodiments, a multicolor flow nanocytometric assay may be performed by the devices described herein to detect PCI, CD 133, FC, EPCIP, C16orf89, CEMIP2(TMEM2), and / or PC2 for PKD-EV flow nanocytometry. The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of PCI and / or PC2 versus a control protein that does not change in ADPKD, for example CD133.

[0056] The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of CEMIP2(TMEM2), which may increase in PKD-EVs. The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of EPCIP, which may decrease in PKD-EVs. The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of EPCIP, which may decrease in PKD-EVs. The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of fibrocystin, which may decrease in PKD-EVs. The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of PCI, which may decrease in PKD-EVs. The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of PC2, which may decrease in PKD-EVs. The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of C16orf89, which may decrease in PKD-EVs. The devices and methods described herein may be used to interrogate individual PKD-EVs in a biological sample for a level of Clorf95 (stum), which may decrease in PKD-EVs.

[0057] In some embodiments, the devices described herein may use a plurality of light sources, for example, a plurality of lasers to measure PKD-associated proteins (e.g., using antibodies specific for the PKD-associated proteins and labeled directly or indirectly with a fhiorophore), such as PC 1, PC2, FC, EPCIP, C16orf89, and / or CEMIP2(TMEM2) in an assay. The devices and methods described herein may be used to detect individual PKD-EVs and measure the amount of these proteins per PKD-EV.

[0058] The devices and methods described herein may be used to examine the urine of ADPKD individuals for a population of CD133+PC I PKD-EVs not present in normal individuals, count the number of PKD-EVs per unit of urine, and / or examine the samples for a decrease in mean PCI or PC2 intensity versus CD 133 level per PKD-EVs. In some embodiments, the devices and methods may be used to detect a CD133+, PC I population. For example, microcysts may stop producing PCI or PC2 such that their PKD-EVs lack these proteins. The devices and methods may be used to detect or determine a decrease in the total number of CD133+, PC1+PKD-EVs without substantial alteration in mean PCI signal per EV. For example, a decrease in mean PC1+intensity per CD133+PKD-EV as PCI is silenced may result in the disease development. In some embodiments, the devices and methods may generate a prognostic score, or prognostic indication based on the detected EV populations, the level (i.e., intensity) of PKD associated proteins, and / or a total number of EVs having a particular PKD associated protein expression profile. The prognostic score or indication may correlate with HtTKV or Mayo scores. For example, a prognostic score or indication may be generated from one or more of the following measured parameters: (1) PC1+PKD-EV absolute count; (2) PC2+PKD-EV absolute count; (3) FC+PKD- EV absolute count; (4) EPCIP+PKD-EV absolute count; (5) C16orf89+PKD-EV absolute count; (6) CEMIP2(TMEM2)+PKD-EV absolute count; (7) CD133+PKD-EV absolute count; (8) Clorf95+(stum) PKD-EV absolute count; (9)— (16) the corresponding concentration in the sample of any of the preceding parameters; (17)-(72) the ratio of any one of the parameters (1) (8) to any one of the others; (73)-(128) the ratio of any one of the parameters (9)-(16) to any one of the others; (129) a linear combination of two or more of the parameters ( 1)— (128); (130) a polynomial formula of two or more of the parameters (1 )-(l 28); and (131) other mathematical formulae as are known to those skilled in the art to provide parametric dependence from multiple variables. The prognostic score or indication may be chosen (e.g., from the list above), by linear or nonlinear regression or other fitting means known to those skilled in the art, to have correlation with HtTKV or Mayo scores, so as to provide actionable predictive power.

[0059] FIG. 1 illustrates schematically a flow nanocytometry device configuration of an embodiment of the present disclosure, which provides an apparatus for sensitive particle analysis in a sample. One or more light source modules 150, e.g., a laser, produce one or more optical energy (light) beams 122 with desired wavelength, power, dimensions, and cross-sectional characteristics. The beam(s) are optionally directed to a set of relay optics 154 (which can include, without limitation, lenses, mirrors, prisms, or optical fibers), which may additionally, and / or optionally, perform a beam-shaping function. Here relay optics will be intended to represent means to transmit one or more beams from one point in the system to another, and will also be intended to represent means to shape one or more beams in terms of dimensions and convergence, divergence, or collimation. The output beam(s) 132 from the beam-shaping relay optics are optionally directed to another optional set of relay optics 158 (which can include, without limitation, lenses, mirrors, prisms, or optical fibers), which may additionally, and / or optionally, perform a focusing function. The set of relay optics 154, the optional set of relay optics 158, or both, may alternatively be incorporated into the light source module 150, or may alternatively be made unnecessary by suitably prescribed beam characteristics from the light source module 150, such as, e.g., without limitation, suitable beam dimensions, a suitably elliptical beam cross-section profde, and / or suitably convergent beam properties, as further described herein. The combined effect of the two sets of relay optics 154, 158 (or corresponding elements in alternative embodiments as described herein) upon the input beam(s) from the light source(s) is to impart upon the beam(s) the desired output beam propagation characteristics suitable for interrogating particles. The second set of relay optics 158 then directs the beam(s) 108 to the flow cell 100. The flow cell 100 provides for the passage of particles to be analyzed (which can include, without limitation, cells, blebbisomes, exosomes, exomeres, ectosomes, extracellular vesicles, extracellular vesicles, liposomes, microvesicles, microparticles, nanoparticles, and natural and synthetic microspheres and nanospheres; collectively, herein, “particles”) by conveying a sample stream 140 containing the particles as a suspension, and a stream of sheath fluid 142 that surrounds and confines the sample stream, as further described herein. An input portion of the flow cell focuses, e.g., without limitation, by hydrodynamic means, by mechanical means, by inertial means, by acoustic means, by electroosmotic means, by di electrophoretic means, and / or by microfluidic means as are known in the art, the sample stream and the surrounding sheath stream to result in a sample core stream flowing through a microchannel portion of the flow cell, surrounded by sheath fluid. The sheath- fluid-surrounded sample core stream flowing past the interrogation region of the flow cell typically exposes, on average, less than one particle at a time to the beam or beams for interrogation (this is sometimes referred to in the art as “cell-by-single-cell” or “particle-by-single-particle” interrogation). In the example shown in FIG. 1, the sheath fluid and the sample core stream are directed to a single outlet 144 (and generally discarded as waste) after passage through the interrogation portion of the flow cell. As the interrogating beam(s) of optical energy (light) interact with particles in the sample core stream by scattering, absorption, fluorescence, and other means, optical signals 110 are generated. These optical signals can be collected by relay optics in module 160 (which can include, without limitation, single lenses, doublet lenses, multi-lens elements, mirrors, prisms, optical fibers, or waveguides) positioned around the flow cell. The relay optics can then convey the optical signals to filtering optics in module 160 (which can include, without limitation, colored filters, dichroic filters, dichroic beamsplitters, bandpass filters, longpass filters, shortpass filters, multiband filters, diffraction gratings, prisms, holographic optical elements, pinholes, apertures, slits, windows, and spatial filters). The filtered light signals 112 can be conveyed by optional further relay optics in module 160 to one or more detectors 170 (which can include, without limitation, photodiodes, avalanche photodiodes, photomultiplier tubes, silicon photomultipliers, or avalanche photodiode microcell arrays). The one or more detectors 170 convert the optical signals 112 into electronic signals 172, which are optionally further amplified and processed to reduce the impact of unwanted noise. The electronic signals 172 are sent to an electronic signal processing unit 190 which executes further processing steps upon the electronic signals 172. The electronic signal processing unit 190 may include a digitization front end with an analog-to-digital converter for each signal stream, as well as discrete analog and digital filter units, and also may include one or more of a Field-Programmable Gate Array (FPGA) chip or module; a Digital Signal Processing (DSP) chip or module; an Application-Specific Integrated Circuit (ASIC) chip or module; a single-core or multi-core Central Processing Unit (CPU); a Graphics Processing Unit (GPU); a microprocessor; a microcontroller; a standalone computer; and a remote processor located on a “digital cloud” -based server and accessed through data network or wired or cellular telephony means. The processed signals 174 can be sent to a data storage unit 192 (which can include, without limitation, a read-only memory unit, a flash memory unit, a hard-disk drive, an optical storage unit, an external storage unit, or a remote or virtual storage unit connected to the instrument by means of a wired data or telecommunication network, a Wi-Fi link, an infrared communication link, or a cellular telephony network link). The stored or preliminarily processed data, or both, can also be made available for optional inspection of results.

[0060] The methods described herein may use flow nanocytometric analysis to assess the levels of EPCIP, PCI, PC2, and / or a control protein such as CD133 or a protein that is increased in ADPKD such as CEMIP2(TMEM2). The technical effect is that a flow nanocytometric assay can be applied to an unprocessed or minimally processed biological sample, for example a urine sample.

[0061] As shown in FIG. 2, an embodiment of a method 200 for diagnosing or treating PKD includes receiving a plurality of electronic signals from a detection module at block S210; detecting a level of a PKD associated protein expressed by the EVs based on the plurality of electronic signals in block S220; determining a quantity of the EVs in the urine sample based on the plurality of electronic signals at block S230; and determining a severity and / or a prognosis of the PKD in the subject, based on the intensity of the PKD associated protein and / or the quantity of the EVs in the biological sample (e.g., urine, blood, lymph, interstitial fluid, spinal fluid, sweat, marrow, and the like) at block S240. The method may function to diagnose PKD in a subject. The method may function to determine a severity of PKD in a subject. The method may function to determine a prognosis of a subject having PKD. The method may function to determine a treatment path for a subject having PKD. The treatment path may be based on a severity and / or prognosis of the subject in need thereof. In some embodiments, the method functions to use flow nanocytometry for diagnostic methods for PKD, but can additionally, or alternatively, be used for any suitable applications, clinical or otherwise where detection of nano-sized particles is desirable. Any of the flow nanocytometry devices, or portions thereof, of FIGs. 1, 3-8, 9A-9C, 10A-10B, 11-15, 17- 18 may perform the method 200 of FIG. 2.

[0062] Although extracellular vesicles (EVs) are described herein, the methods described herein may be applied to, extended to, or otherwise encompass other, similar nanoparticles. The methods described herein may be used to detect, quantify, or otherwise measure extracellular vesicles (EVs), oncosomes, exosomes, exosome vesicles, exosome-like vesicles, exomeres, ectosomes, microparticles or microvesicles, blebbisomes, apoptotic bodies, and the like. The methods described herein may be used to detect, quantify, or otherwise measure particles ranging in size from about 20 nm to about 150 nm; about 100 nm to about 1,000 nm; greater than about 50 nm; less than about 50 nm; etc. The methods described herein may be used to detect particles involved in cell communication, RNA transfer, protein transfer, inflammation, coagulation, metabolism, and cancer or disease progression. The methods described herein may be used to detect particles that are released from a plasma membrane, an endosomal system, immune cells, tumor cells, neurons, glial cells, endothelial cells, stem cells, and the like.

[0063] As shown in FIG. 2, an embodiment of a method 200 for diagnosing or treating PKD includes block S210, which recites receiving a plurality of electronic signals from a detection module. Block S210 functions to receive electronic signals generated from interaction between at least one excitation module and at least one particle in the flow cell for further processing steps. As described elsewhere herein, optical signals are generated when the interrogating beam(s) of optical energy (light) interact with particles in the sample core stream by scattering, absorption, fluorescence (e.g., where a fluorescently labeled marker is coupled to a protein of the particle), and other means. In some embodiments, the optical signals can be collected by relay optics (e.g., single lenses, doublet lenses, multi-lens elements, mirrors, prisms, optical fibers, or waveguides) positioned around the flow cell. The relay optics can convey the optical signals to filtering optics (e.g., colored filters, dichroic filters, dichroic beamsplitters, bandpass filters, longpass filters, shortpass filters, multiband filters, diffraction gratings, prisms, holographic optical elements, pinholes, apertures, slits, windows, and spatial filters). The filtered light signals can be conveyed by optional further relay optics to one or more detection modules (e.g., photodiodes, avalanche photodiodes, photomultiplier tubes, silicon photomultipliers, or avalanche photodiode microcell arrays). The one or more detection modules convert the optical signals into electronic signals, which are optionally further amplified and processed to reduce the impact of unwanted noise. The electronic signals are transmitted to the processor which executes further processing steps upon the electronic signals.

[0064] As shown in FIG. 2, an embodiment of a method 200 for diagnosing or treating PKD includes block S20, which recites detecting a level of a PKD associated protein expressed by the EVs based on the plurality of electronic signals. Block S220 functions to determine which protein(s) are expressed on the EVs. Further, block S220 functions to determine a level or intensity of the expressed proteins by the EVs. An amplitude, optionally an area under the curve, and further optionally a width, of the received electronic signals (based on conversion of the emitted scatter signals and / or fluorescence signals to analog electronic signals, and on further conversion of the analog electronic signals to digital electronics signals) may be used to determine a presence (expression) or absence (lack of expression) of the protein, a level of expression (intensity) of the protein, and / or a size and / or granularity of the EV (e.g., using side scatter). Each event (e.g., EV detection event), produced by excitation by a light source and sensed by detectors in the devices disclosed herein, has an associated light scattering property, which through gating, may be used to identify a population of EVs of interest and / or distinguish EVs from other particles (e.g., cells) and / or debris. Each event has an associated fluorescence intensity, which may be expressed, e.g., without limitation, as a detector output value (e.g., without limitation, in millivolts), as an equivalent number of fluorophore molecules, or using other value scales as are known in the art. The aggregate signal intensity for an EV population may be expressed, e.g., as a mean fluorescence intensity or a geometric mean, in arbitrary fluorescence units on a logarithmic scale, as shown for example in FIGs. 21A-21C, 22A-22B, 23A-23B, and 24A-24C. The expressed protein may be one or more of: PCI, PC2, FC, EPCIP, C16orf89, Clorf95, and / or CEMIP2(TMEM2). The method may further include normalizing an intensity of the expressed protein to a co-expressed, invariant protein. For example, the co-expressed invariant protein may be CD133, tetraspanin-4 (TSN3), tetraspanin-4 (TSN4), tetraspanin-7 (TSN7) and tetraspanin-8 (TSN8). In some embodiments, block S220 includes detecting an increase in an intensity of CEMIP2(TMEM2). In some embodiments, additionally or alternatively, block S220 includes detecting a decrease in an intensity of EPCIP. In some embodiments, additionally or alternatively, block S220 includes detecting a decrease in an intensity of fibrocystin. In some embodiments, additionally or alternatively, block S220 includes detecting a decrease in an intensity of PCI. In some embodiments, additionally or alternatively, block S220 includes detecting a decrease in an intensity of PC2. In some embodiments, additionally or alternatively, block S220 includes detecting a decrease in an intensity of C16orf89. In some embodiments, additionally or alternatively, block S220 includes detecting a decrease in an intensity of Clorf95. In some embodiments, additionally or alternatively, block S220 includes detecting an increase in an intensity of TMEM2 (CEMIP2).

[0065] As shown in FIG. 2, an embodiment of a method 200 for diagnosing or treating PKD includes block S230, which recites determining a quantity of the EVs in the biological sample based on the plurality of electronic signals. A quantity of EVs in a biological sample may indicate the presence of PKD and / or the disease severity. A quantity of the EVs may be determined using an event count (indicating a number of detected particles) for a biological sample from the flow cytometer, relative to, in some embodiments, a counting bead control to determine an absolute number of EVs. Alternatively, when a volume of the biological sample is known, a concentration of the EVs may be determined by dividing the number of events by the analyzed volume. The number of events may be determined based on gating for the EV population of interest, an example of which is shown in FIG. 19A. In some embodiments, FSC and SSC gating may be used to exclude debris, aggregates or doublets of EVs, or non-target populations.

[0066] As shown in FIG. 2, an embodiment of a method 200 for diagnosing or treating PKD includes block S240, which recites determining a severity and / or a prognosis of the PKD in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs in the biological sample. Block S240 functions to diagnose a subject suspected of having PKD. In some embodiments, block S240 functions to treat a subject having PKD. A severity of PKD may be determined by the number of PC1 / PC2 / CD133+ PKD-EVs per unit volume urine, or the intensity of PCI or PC2 on the PKD-EVs. A prognosis of PKD may be determined by either number of PC1 / PC2+ PKD-EVs and / or the amount of PCI or PC2 present per PKD-EV.

[0067] Optionally, the biological sample may be gently centrifuged at about 2000g for about 10 minutes and be buffered to about pH 7.0 to about pH 8.0 using TRIS or about pH 6.0 to about pH 8.0 using MES. Monoclonal antibodies (mAb) may be added in a single pipetting maneuver, incubation may be for about 30 minutes at room temperature followed by nanocytometry analysis. As such, no ultracentrifugation step, Western loading, transfer and two antibody probing steps, etc. are needed as is conventional with Western blotting analyses. The devices and methods described herein generate a distribution of intensities for each interrogated protein and one or more ratios, for example, PC1 / CEMIP2(TMEM2), PC2 / CEMIP2(TMEM2), EPCIP / CEMIP2(TMEM2), PC1 / CD133, PC2 / CD133, and / or EPCIP / CD133. The methods may include internally calibrating the devices described herein with standard fluorescent microbeads, calibrated to bind 0, 200, 1000 mAb molecules (e.g., Cellarcus vCal™ antibody capture nanobeads).

[0068] FIG. 3 is a schematic illustration of the pneumatic and fluidic control system architecture of a flow nanocytometry device in accordance with some embodiments of the present disclosure. A purpose of the pneumatic and fluidic control modules depicted in FIG. 3 is to provide hydrodynamic focusing of the sample stream so as to present, generally, only single particles to the laser beams for interrogation, and to prevent the sample suspension from contacting the flow cell walls. Hydrodynamic focusing involves injecting sample and sheath fluids substantially concentrically and forcing them through a constriction in the two transversal dimensions to produce a narrow sample stream having, on average, one sample particle in the interrogation region. Although an interrogation region is described, one of skill in the art will appreciate that more than one interrogation region is possible and within the scope of the present disclosure. The architecture illustrated in FIG. 3 achieves hydrodynamic focusing by providing two separate hydrostatic pressure control modules — one for the sheath fluid and one for the sample. Each module is described in greater detail below.

[0069] Referring to the embodiment illustrated in FIG. 3, a pneumatic and fluidic control module 300 includes: pressurizable tank 311; fluid container 312; pressure vessel 313; tank 314; pump 321; pump 322; pump 323; pressure regulator 331; pressure regulator 332; pressure regulator 333; pressure regulator 334; sheath pressure gauge 341; pressure gauge 342; optional valve 350; valve 351; valve 352; valve 353; valve 354; valve 355; valve 356; valve 357; valve 358; valve 359; check valve 360; flow restrictor 361; flow restrictor 362; sample probe 371; probe wash port 372; sample pressure port 373; fluid ports 374; flow cell 375; fluid port 376; filter 381; and filter 382. Intake air is filtered by filter 381 and provided to pumps 321, 322. As described further herein in reference to FIG. 4, the output of pump 321 is regulated by pressure regulators 331, 332, optionally monitored by gauge 341, delivers pressurized air to valve 351, and pressurizes pressurizable tank 311 (which is provided with a safety relief valve set at an appropriate pressure level to prevent malfunction or injury); fluid container 312 delivers pressurized fluid through filter 382 and valve 350 to valves 352, 356, 357. As described further herein with reference to FIG. 5, the output of pump 322 is regulated by pressure regulators 333, 334, pressurizes pressure vessel 313, is optionally monitored by gauge 342, and delivers pressurized air through to valve 353.

[0070] As described further herein with reference to FIG. 6, in “Run” mode, valve 356 opens to deliver pressurized fluid, optionally monitored by a flow sensor, to valve 358, which routes the pressurized fluid to the flow cell module through one or more fluid ports 374. Valve 353 connects the pressurized air through check valve 360 to the flow cell module through pressure port 373, through which it pressurizes a sample tube positioned in the aspiration port. Valve 359 routes the waste fluid, optionally monitored by a flow sensor, from flow cell port 376 to valve 354, which routes it to tank 314. Valve 353 is controlled in part by a switch connected to a mechanical arm, or flip arm. The flip arm is positioned near the sample probe and is configured to be moveable from a position under the sample probe (with a sample tube inserted in the aspiration port of the flow cell module so that the sample probe can aspirate the sample) to one or more positions not under the sample probe. Valve 353 functions to route sample pressure from the sample pressure vessel to the flow cell module in “Run mode”, and when the flip arm is positioned under the sample probe.

[0071] As also described further herein with reference to FIG. 6, in “Sample Boost” mode, valve 358 switches from flowing fluid substantially unimpeded to flowing fluid through flow restrictor 361

[0072] As described further herein with reference to FIG. 7, in “Probe Wash” mode, valve 357 opens to deliver pressurized fluid to the flow cell module 720 through port 372, through which the fluid runs down probe 371 and into rinse cup 315. Pump 323 drains rinse fluid from cup 315 and routes fluid to tank 314.

[0073] As described further herein with reference to FIG. 8, in “Sheath Boost” mode, valve 359 switches from flowing fluid through flow restrictor 362 to flowing fluid substantially unimpeded.

[0074] In “Backflush” mode, valve 352 opens to run fluid through fluid port 376 of the flow cell, and valve 355 opens to route fluid through to tank 314.

[0075] In “Drain” mode, valve 351 opens to run pressurized air through fluid port 376 of the flow cell, and valve 355 opens to route fluid through to tank 314.

[0076] In “Standby” mode, valves 351, 352, 354, 355, 356, 357 close, and valve 353 prevents pressurized air from being routed to the flow cell module.

[0077] In some embodiments, optional valve 350 is connected to a sensor for sensing the amount of fluid in fluid container 312, and valve 350 functions to close when the fluid amount falls below a predetermined value or amount. This can prevent the fluid system from trying to operate without sufficient available sheath fluid. In some embodiments, valve 350 is connected to a sensor for sensing the level of fluid in tank 314, and valve 350 functions to close when that fluid level rises above a predetermined value or amount. This can prevent tank 314 from overfilling and potentially causing fluid spillage. In some embodiments, valve 350 is connected to both a fluid sensor in container 312 and a fluid level sensor in tank 314, and functions to close whenever readings by either sensor reach respective predetermined values as described above. Actuation of valves 350-359 is effected through any number of means known in the art, including, without limitation, manual actuation of individual stop valves, electronic actuation of individual valves (such as, e.g., rocker valves or solenoid valves), pilot actuation of individual valves (e.g., using pneumatically-controlled pinch valves in combination with solenoid valves), and joint actuation of multiple valves (such as, e.g., by a multi-position selector switch simultaneously controlling all nine valves). Different types of valves may be more suitable for different applications, according, e.g., without limitation, to their size, pressure rating, dead volume, wetted materials, reliability, and / or (for electrically operated valves) power consumption and switching speeds.

[0078] FIG. 4 schematically illustrates a configuration of a sheath pressure control module 400 for a flow nanocytometry device in accordance with some embodiments of the present disclosure. An air intake 410 (which intake is optionally filtered to remove or reduce particulates, dust, debris, and / or water vapor) conveys ambient air to the sheath pressure control module 400. A pump 420 (or optionally a set of two or more pumps, or a source of pressurized air) pressurizes air from intake 410 and conveys air to regulator 430, which in turn conveys the pressurized, regulated air to regulator 440. Regulators 430, 440 connected in series provide successive layers of isolation to the downstream part of the circuit from possible pulsatile pressure fluctuations due to operation of the pump 420 or to system variations in the air source. The output of the second regulator 440 is displayed by an optional pressure gauge 450, optionally for monitoring proper functioning of the module. Sealed sheath fluid tank 460 provides yet another layer of isolation between upstream fluctuations and downstream flow. Sheath fluid tank 460 includes a removable top or side (such as a securable sealing lid), a sealed connector 455 for introduction of the pressurized, regulated air from second regulator 440, and a flexible, sealed, gas-impermeable container 465 (including, without limitation, a sealed plastic container with flexible walls, a sealed plastic bag, and a vessel with a sealed flexible diaphragm; in each case further including a sealing connector 473, including, without limitation, a self-sealing fitment, a sealing quick-disconnect connector, and a septum) that allows the pressurization of its sheath fluid contents through pressurization of the sheath fluid tank 460 with a reduced or negligible gas exchange taking place across the wall materials of container 465. Sealing connector 473 allows pressurized sheath fluid 470 to flow out of container 465 through internal conduit 475, through external sealing connector 477, and through conduit 480. Pressurized sheath fluid 470 optionally flows through sheath fluid filter 485 (including, without limitation, a filter with 0.01 pm, 0.02 pm, 0.03 pm, 0.04 pm, 0.05 pm, 0.1 pm, 0.2 pm, 0.22 pm, 0.3 pm, 0.4 pm, 0.45 pm, 0.5 pm, 0.65 pm, 0.8 pm, or 1 pm typical pore size. In some embodiments, sheath fluid filter 485 includes a filter with a 0.05 pm pore size, or in some embodiments a filter with other pore sizes, to remove certain particulates suspended in the sheath fluid. Pressurized and optionally filtered, pressurized sheath fluid 470 flows through conduit 490 to the flow cell for hydrodynamic focusing. Pressurized sheath fluid 470, as provided to the rest of the device through conduit 490, connectors, tees, and other conduits are as known in the art, can also be used for filling and backflushing the flow cell and / or to wash the sample probe (more details on washing the sample probe are described elsewhere herein). The first sheath pressure regulator 430 can be set (e.g., during manufacture) at a pressure level that prevents potential damage to the system (e.g., without limitation, a pressure between about 10 psi and about 20 psi, or in some embodiments a pressure lower than about 10 psi or greater than about 20 psi), while the second regulator 440 can be adjustable (e.g., manually or automatically) to achieve the desired sheath flow rate in the flow cell, and thereby achieve the desired average particle transit time (e.g., for a laser beam waist dimension in the flow direction of about 10 pm, without limitation, a transit time between about 1 ps and about 20 ps, and, in some embodiments, a transit time shorter than about 1 ps or longer than about 20 ps or even longer than about 50 ps; and correspondingly for laser beam waist dimensions smaller than or greater than 10 pm). Sheath pressure control module 400 may include several submodules, or it may be provided as an integral module; for example, without limitation, sheath fluid tank 460 and its contents, along with connectors 455 and 477, conduits 480 and 490, and optionally filter 485, may be provided as separate from pump 420, regulators 430, 440, and optional pressure gauge 450, e.g., to allow easier and more convenient replacement of a depleted sheath fluid container 465 with a fresh and full such container. Pump 420, regulator(s) 430, 440, and optional pressure gauge 450 may be provided as integrated into the main body of the flow nanocytometry device of this disclosure.

[0079] FIG. 5 schematically illustrates a configuration of a sample pressure control module 500 of a flow nanocytometry device in accordance with some embodiments of the present disclosure. A pump 520 (or optionally a set of two or more pumps, or a source of pressurized air) with an air intake 510 (which intake is optionally filtered to remove or reduce particulates, dust, debris, and / or water vapor) conveys pressurized air to regulator 530, which in turn conveys the pressurized, regulated air to regulator 540. Regulators 530, 540 connected in series provide successive layers of isolation to the downstream part of the circuit from possible pulsatile pressure fluctuations due to operation of the pump 520 or to system variations in the air source. The output of the second regulator 540 is optionally displayed by a pressure gauge 550, optionally for monitoring proper functioning of the sample pressure control module 500. This output pressurizes the sample pressure vessel 560, providing yet another layer of isolation between upstream fluctuations and downstream flow. The output 570 of the sample pressure vessel 560 pressurizes the sample tube in “Run” mode, impelling the sample suspension to traverse the sample probe and enter the flow cell to be hydrodynamically focused. The first sample pressure regulator 530 can be set (e.g., during manufacture) at a pressure level that prevents potential damage to the system (e.g., without limitation, a pressure between about 10 psi and about 20 psi, or in some embodiments a pressure lower than about 10 psi or greater than about 20 psi), while the second sample pressure regulator 540 can be adjustable (e.g., manually or automatically) to achieve the desired sample flow rate in the flow cell (e.g., without limitation, a sample flow rate of between about 10 pL / min and about 100 pL / min, between about 1 pL / min and about 10 pL / min, between about 0.1 pL / min and about 1 pL / min, between about 0.1 pL / min and about 100 pL / min, lower than about 0.1 pL / min, or greater than about 100 pL / min), and thereby achieve the desired dimensions of the sample core stream and the rate at which sample particles in suspension are detected.

[0080] In some embodiments, the sheath module pump (e.g., 420 in FIG. 4) is separate from the sample module pump (e.g., 520 in FIG. 5). In another embodiment, the sheath pressure control module 400 and sample pressure control module 500 in FIGs. 4-5, respectively, share the same pump. In some embodiments, each module is served by two or more pumps connected in series. In some embodiments, each module is served by two or more pumps connected in parallel. In some embodiments, a module is served by one pump while another module is served by two or more pumps (in series or in parallel). The choice among these embodiments is driven by considerations such as, without limitation, system pressure requirements and pump flow rate capacity, size, noise level, and / or cost.

[0081] FIG. 6 schematically illustrates several modes of operation of a flow nanocytometry device in accordance with some embodiments of the present disclosure. Shown in FIG. 6 are a sample boost control switch 670, a sheath fluid conduit 610, a valve 640, a flow restrictor 645, a sample pressure conduit 665, a check valve 660, a flow cell module 650, a probe 655, a flow cell 620, a waste line 695, a sample tube 680, a sample 690, and a flip arm 645a first position and flip arm 645b second position. For operation of the system in some embodiments, the sample pressure and the sheath pressure are in such a relationship that pressurized air or other gas causes the sample to be injected into the sheath fluid flowing in the flow cell 620. Due to differences in hydrodynamic resistance in the paths that the sheath fluid and the sample take, the difference in pressure values as optionally monitored by the gauges of the apparatuses described herein (e.g., shown in FIGs. 3, 4, and 5) does not necessarily correspond to the difference in sheath and sample pressures at the point of sample injection in the flow cell 620. However, under particular operating conditions, for a given set sheath pressure (or sheath pressure at the point of sample injection in the flow cell 620), a minimum sample pressure can be found that results in sample injection into the flow cell 620. Increasing the sample pressure above this minimum threshold results in larger sample flow rates, and therefore comparatively larger sample core stream dimensions. This minimum threshold depends on the sheath pressure; the higher the set sheath pressure, the higher the minimum sample pressure needed to achieve sample injection.

[0082] In some cases, it is desirable to operate flow cytometers with the smallest achievable sample core stream dimensions. That can reduce the variability in measurement of the various quantities of interest (e.g., without limitation, light scattering, absorption, extinction, fluorescence). It also reduces the fraction of events subject to “coincidence,” i.e., those instances where more than one particle simultaneously occupy the interrogation region. A sample core stream as small as practical can generally be achieved in two ways — by increasing the sheath pressure (e.g., depending on the hydrodynamic characteristics of the system, up to about 5 psi, up to about 10 psi, up to about 15 psi, or in some cases even greater than about 15 psi) or by decreasing the sample pressure (e.g., depending on the hydrodynamic characteristics of the system, down to about 5 psi, down to about 2 psi, down to about 1.5 psi, or in some cases even below about 1.5 psi). All other things being equal, changing the sheath pressure changes the flow rate. However, changing the flow rate also changes the average particle transit time, which can be undesirable. Decreasing the sample pressure beyond a minimum threshold described above stops the sample flow altogether, and in some cases, can result in sheath fluid reversing flow through the sample probe 655 and backfdling the sample tube 680, thereby diluting and possibly contaminating the sample 690 itself. Therefore, it is often desirable to operate just above that minimum threshold.

[0083] In some embodiments, in the “Run” mode (as described above with respect to FIG. 3), the sample pressure conduit 665 is opened (e.g., by actuation of an electrically controlled valve) to drive the sample 690 through probe 655 and into flow cell 620. The pressurized air (conveyed from output 570 in FIG. 5, through, e.g., other conduits, connectors, tees, valves, and other components as are known in the art), is brought through conduit 665 to check valve 660. Check valve 660 is oriented to allow flow of pressurized air from conduit 665 to flow cell module 650, but to prevent the flow of fluid (such as, e.g., sample 690, or sheath fluid from conduit 610) through flow cell module 650 and back into conduit 665. The pressure starts building up in sample tube 680 once it is put in position around the sample probe 655, the seal is formed, and flip arm 645a, 645b is moved in position “a” under sample tube 680, triggering the sample pressure line valve to open. The sample pressure in sample tube 680 then builds up to the desired, set level, causing sample 690 to be injected and the sample core stream to take on the desired dimensions. Under certain circumstances, however, and particularly when operating near the minimum threshold described above, reaching that steady state may take several seconds, and in some cases several tens of seconds. This is undesirable as it delays acquisition of measurements, and until sample 690 starts flowing, back flowing sheath could dilute and contaminate sample 690. The sample boost module illustrated in FIG. 6 provides a technical solution to this technical problem. In “Sample Boost” mode, using switch 670, a valve 640 on conduit 610 carrying sheath fluid to the flow cell is closed, preventing the sheath fluid from flowing unimpeded through valve 640 and forcing the sheath fluid to take an alternate path through a flow restrictor 645. Switch 670 may be, for example, without limitation, a two-position non-latching switch that is engaged after insertion of the sample tube 680 in the sampling port; or can be automatically engaged, for, e.g., a preset time, by a pressure sensor pneumatically connected to the conduit 665 (e.g., sample pressure line) and positioned optionally relatively close to the sample probe assembly, responding to the increase of pressure in the sample tube 680 following insertion. The alternate sheath fluid path through flow restrictor 645 causes a drop in the sheath fluid pressure at flow cell 620, which causes a drop in the minimum pressure threshold required for sample 690 to be injected. By appropriately (e.g., empirically, analytically, or with a combination of the two) choosing the properties (e.g., the size of the restricting orifice, the length of the restriction, or the number of restrictor units including the restrictor placed in series, in parallel, or in a combination of in series and in parallel) of flow restrictor 645, the drop obtained in the minimum sample pressure can be such that, even in the early stages of pressure build-up in sample tube 680, the sample pressure exceeds the new, temporarily lower minimum threshold. This causes sample 690 to be injected into flow cell 620 faster than the sample would be injected without the boost module (i.e., the sample injection receives a “boost”), instead of being subject to a delay while the sample pressure builds up. Once sample injection is stably established, switch 670 can be released, returning the sheath pressure to its original higher value, returning the minimum sample pressure threshold to its original higher value, and causing the sample stream to reduce in size to the desired, steady-state, relatively small value desirable for minimum measurement variability.

[0084] In some embodiments, the characteristics of the sheath line flow restrictor (valve 640) are chosen so as to cause a relatively larger drop (such as, without limitation, an about 20% drop, about 30% drop, about 50% drop, or between about 20% and about 50%, or greater than about 50%) in the sheath fluid pressure at the flow cell 620, and an attendant relatively larger drop in the minimum sample pressure threshold (e.g., without limitation, a drop of up to about 1 psi, a drop of up to about 5 psi, or between about 1 psi and about 5 psi, or in some cases a drop greater than about 5 psi). In such cases, the difference between the set sample pressure and the (new) minimum threshold therefore becomes relatively larger, resulting in relatively larger dimensions of the sample core stream (e.g., without limitation, up to about 100 pm, up to about 200 pm, up to about 250 pm, or even greater than about 250 pm in one linear cross-sectional dimension). With suitable choice of flow restrictor 645 and operating parameters, the change in sample core stream dimensions can be large, reaching even a substantial fraction of flow cell 620 cross-sectional dimensions, such as about 30%, about 50%, about 70%, or higher. This increase in the sample stream dimensions can be desirable in certain circumstances. In some applications, when looking for rare (e.g., 1 in up to 100,000, or 1 event in up to 100 pL), very rare (e.g., 1 in up to 10,000,000, or 1 event in up to 10 mL), or extremely rare (e.g., 1 in up to 1,000,000,000 or more, or 1 event in up to 1 L or more) events, it may be advantageous to increase the sample flow rate as much as possible, in order to shorten the time it takes for the entire sample volume to be assayed. While normally increasing the sample stream cross-sectional dimensions to a substantial fraction of the flow cell cross-sectional dimensions is not desirable due to the greater variability of measurement in such instances, when performing assays to detect rare, very rare, or extremely rare events, such variability may be acceptable as a trade-off to a much shortened assay time. Additionally, in such cases, the variability in measurement can be mitigated by adopting optical interrogation architectures, e.g., those with substantially flat transversal beam profdes (called “flat-top” or “top-hat” profiles), thereby reducing or eliminating altogether the negative consequences of a larger sample stream with low abundance of events of interest.

[0085] In some embodiments, in “Sample Boost” mode, sample boost switch 670 is engaged whenever it is desirable to obtain, momentarily or for extended periods of time, a larger sample core stream than would normally be desirable, such as, e.g., without limitation, during manufacture or service, when optical alignment of the flow nanocytometry device may benefit from the presence of a relatively large sample core stream. In such cases, for example, so-called alignment microspheres at high concentration in a sample suspension to generate bright scattering, absorption, and / or fluorescence signals to aid in the alignment process of the optical module of the flow cytometry device may be used. In such cases, the use of sample boost switch 670 may further increase the brightness of such signals, further aiding alignment of optical components in the optical module. Sample boost switch 670 may optionally be configured to be latching (e.g., with a latching toggle, pushbutton, rocker, or other mechanisms as are known in the art) instead of nonlatching to permit the operator unencumbered use of both hands for extended periods of time after engaging the switch.

[0086] FIG. 7 illustrates a sample probe wash module of a flow nanocytometry device in accordance with some embodiments of the present disclosure. A sheath fluid line 730 carrying sheath fluid from the sheath tank (e.g., from conduit 490 in FIG. 4) is routed to a valve 740 controlled (dashed line) by a probe wash control switch 770. Sheath fluid line 730 in this example is separate from the sheath fluid line 710 used during the “Run” mode and can, therefore, be operated independently from it. Switch 770 can be, e.g., without limitation, a two-position nonlatching switch that may be engaged after removal of the sample tube from the sampling port. In other cases, switch 770 can be automatically engaged, for, e.g., a preset time, by a pressure sensor pneumatically connected to the sample pressure line (e.g., conduit 665 in FIG. 6), where the pressure sensor is optionally positioned relatively close to the sample probe assembly 750, and the pressure sensor responds to the decrease of pressure in the sample pressure line following removal. Actuation of switch 770 opens valve 740, causing sheath fluid in line 730 to flow through a path to an opening in the sample probe assembly 750 and adjacent, near, above, or surrounding the sample probe 755. The sheath fluid flowing through line 730 removes residue from the prior sample, carrying it in solution or suspension to the bottom end of the sample probe 755, where it forms a probe a wash runoff stream or series of drops 760 that fall into a probe wash receptacle 780 (e g., a funnel-shaped receptacle). The bottom of the receptacle 780 is connected by conduit 785 to a drain pump 790. Drain pump 790 drains the probe wash receptacle, aspirating the runoff through conduit 785 and conveying it through conduit 795 to a waste tank (not shown). In some embodiments, drain pump 790 operates in optional conjunction (dash-dotted line) with probe wash control switch 770, whereby the pump operates continuously whenever the probe wash control switch is activated. In some embodiments, drain pump 790 operates independently of probe wash control switch 770, operating instead on a preset timer circuit (e.g., without limitation, with a duty cycle of 5%, 10%, 15%, or other percentage; and a cycle time of 10, 20, 30 seconds, or other duration). In some embodiments, drain pump 790 operates on a circuit (implemented in hardwarebased logic, software-based logic, or a combination of the two) that disables it whenever a sample tube is being pressurized for analysis by sample probe assembly 750 and the system is in “Run” mode, and enables it for timed operation otherwise.

[0087] FIG. 8 illustrates a sheath boost module of a flow nanocytometry device in accordance with some embodiments of the present disclosure. Some elements in the apparatus in FIG. 8 are the same as those described with respect to the apparatus shown in FIG. 6. The sheath boost module includes a sheath boost control switch 870, a waste conduit 895, a valve 840, a flow restrictor 845, and a downstream waste conduit 898. Under normal operation, valve 840 is closed, forcing the waste stream from flow cell 820 in conduit 895 (which waste stream may include sheath fluid conveyed to flow cell 820 by line 810, and sample 890 forced into flow cell 820 from sample tube 880 through probe 855 and probe assembly 850 by sample pressure conveyed in line 865) to flow through restrictor 845 and into conduit 898, which then conveys the waste stream (optionally through other conduits, connectors, tees, or valves, as is known in the art) to a waste tank. Using switch 870, valve 840 is opened, allowing the waste fluid from the flow cell to flow into conduit 898, through restrictor 845 and also substantially unimpeded through the open valve 840. This additional, less restrictive flow path reduces the hydrodynamic resistance of the waste flow, allowing a greater flow rate through the device, all other things being equal. Such increase in flow rate may be desirable under several circumstances, such as, e.g., without limitation: during priming of the device; when it is desirable to clear more rapidly from the flowcell possible residue from a prior sample; and when it is desirable to use the higher flow to dislodge bubbles or other undesirable particles from the interrogation area of the flow cell or from fluid lines connected with it. The characteristics (e.g., the size of the restricting orifice, the length of the restriction, or the number of restrictor units including the restrictor placed in series, in parallel, or in a combination of in series and in parallel) of restrictor 845 are chosen (e.g., empirically, analytically, or with a combination of the two) to allow the device to achieve a desired value or range of overall sheath flow rates when operated in the specified normal sheath pressure range. For example, restrictor 845 may be chosen to allow the regulated pressurized sheath flow to achieve, without limitation, a sheath flow rate of between about 5 mL / min and about 10 mL / min, between about 1 mL / min and about 5 mL / min, between about 1 mL / min and about 10 mL / min, lower than about 1 mL / min, or greater than about 10 mL / min). In some embodiments, the sheath boost switch 870 may be nonlatching (e.g., with a non-latching toggle, pushbutton, rocker, or other mechanisms as are known in the art) to allow the momentary operation of the sheath boost without requiring the switch to be returned manually to its default position. In some embodiments, the sheath boost switch 870 may be latching (e.g., with a latching toggle, pushbutton, rocker, or other mechanisms as are known in the art) instead of non-latching to permit the operator unencumbered use of both hands for extended periods of time after engaging the switch.

[0088] FIGs. 9A-9C schematically illustrate the optical layout (also referred to as optical bench, optical module, or optical subassembly) architecture of a flow nanocytometry device in accordance with some embodiments of the present disclosure. The optical layout module 900 includes laser excitation sources 910a, 910b, 910c, beam shaping elements 915, beam combiners 920, lens 930, flow cell 940, lens 942, obscuration mask 944, optional spectral filter 946, detector 948, collection lens 941, mirror 950, lens 955, aperture 960, lens 965, one or more side scatter detection modules 970a, 970b, one or more fluorescence detection modules 971a, 971b, 971c, 971d, 971e, and microscope module 995. Several components are present in multiples; for example, each of the beams from light sources 910a, 910b, 910c pass through a similar set of beam shaping elements 915; both side scatter detection modules 970a, 970b include a similar set of optical elements (see scattering detection module 970x in FIG. 9B: i.e., a beamsplitter 972x; one or more optional spectral filters 974x; a lens 976x; a pinhole 977x; one or more lenses 978x; and a detector 980x; where, for each side scatter detection modules 970a and 970b, “x” stands for either “a” or “b”); and each of the fluorescence detection modules 971a, 971b, 971c, 971d, 971e include a similar set of optical elements (see fluorescence detection module 971y in FIG. 9C: i.e., a beamsplitter 972y or, as in FIG. 9A, mirror 973; one or more optional spectral filters 974y; a lens 979y; and a detector 980y; where for each channel 971a, 971b, 971c, 971d, 971e, “y” stands for either “a” or “b”); however, only some of these elements are labeled for simplicity.

[0089] FIG. 9A illustrates an embodiment including three laser sources 910a, 910b, 910c and eight detection channels (FSC, detected by detector 948; side scatter detection module 970a [“SSC1”]; side scatter detection module 970b [“SSC2”]; and five fluorescence detection modules 971a, 971b, 971c, 971d, 971e [FL2-FL6]). It will be appreciated by those skilled in the art that the present disclosure is not limited to such choices of number of laser sources and detection channels. The disclosed architecture is capable of supporting fewer light sources (e.g., without limitation, as few as one) as well as a greater number of them (e.g., without limitation, up to five, up to 10, or more than 10), and is also capable of supporting fewer detection channels (e.g., without limitation, as few as one) as well as a greater number of them (e.g., without limitation, up to 20, up to 30, up to 40, up to 50, or more than 50). The optical architecture of this disclosure makes it possible to instantiate these alternative embodiments without major design changes by the use of substantially collimated beams in both the excitation and the detection arms. Collimated beams include substantially parallel light rays; therefore, optical components placed along their paths can, to a high degree, be translated longitudinally along the propagation path without substantially affecting performance. In addition, substantially zero-power optical components (such as, e.g., without limitation, mirrors, dichroic beamsplitters, and fdters) can be inserted or removed from a collimated-beam optical path without substantially altering the downstream focusing behavior. Relatively thin substantially zero-power transmissive components inserted or removed at nonzero incidence (such as, e.g., dichroic beamsplitters used at a 45-degree angle of incidence) may cause a slight transversal shift in the beam centerline (also called chief ray), which can be easily accommodated in the disclosed architecture; and otherwise have substantially negligible effects on the transmitted beams. The ability to accommodate different optical layouts based on the architecture described herein is an advantage of the flow cytometry devices and method described herein over conventional flow cytometry devices.

[0090] In this illustration, and as further described herein in reference to FIGs. 10A-B, the beams from multiple laser excitation sources 910a, 910b, 910c are combined, shaped, and focused into the flow cell 940, where the hydrodynamically focused sample stream carries particles to be analyzed. In some embodiments, the flow cell 940 may optionally be continuously monitored by a dedicated microscope module 995, as described in International Patent Application PCT / US22 / 16670, filed February 16, 2022, the contents of which are herein incorporated by reference in their entirety. A microscope module may be used to improve assembly, alignment, operation, troubleshooting, and maintenance of the flow nanocytometer. In some embodiments, a microscope module may include a housing for reversibly attaching to a portion of any of the flow nanocytometers described herein (e.g., any of FIGs. 1, 3-8, 9A-9C, 10A-10B, 11-15, 17-18), such that an optical system supported by or attached to the housing is aligned with a monitoring region within a flow cell of the flow nanocytometer. The optical system of the microscope module may continuously capture and monitor at least a portion of the optical energy emanating from the monitoring region of the flow cell (e.g., at least a portion of the optical energy may emanate from the interaction between at least one light beam from at least one excitation light source and a core stream as the core stream flows through the flow cell). For example, in some embodiments, the optical system of the microscope module may include at least one sensor for detecting the optical energy captured by the optical system and generating, based on the detected optical energy, an image (or a series of images) of the monitoring region; and at least one processor for continuously providing the image to an electronic device. In some embodiments, the microscope module may detect the occurrence of one or more anomalies in the optical energy emanating from the monitoring region; and cause the at least one processor to generate indications about the one or more anomalies.

[0091] Interaction of the articles with the laser beam(s) produces light emitted from the flow cell, e g., without limitation, scattered and fluorescent light from the sample within the flow cell. As further described herein with reference to FIG. 12, lens 942 captures light scattered by a particle in a sample within a narrow cone (e.g., without limitation, a cone with about a 10-degree half-angle) aligned with the beam propagation, and relays it, after passing through obscuration mask 944 and one or more optional spectral filters 946, to a forward- scatter detector 948. Obscuration mask 944 blocks a substantial portion of undesired laser and statically scattered light (“background”), while passing through to optional filter(s) 946 and detector 948 a substantial portion of the desired particle-scattered light collected by lens 942. Side scattering and fluorescence emissions are captured by a separate, high-NA (Numerical Aperture) collection lens (or compound collection lens, collection objective, or collection subassembly) 941 centered around 90 degrees from the forward direction. An optical processing module, as further described herein in reference to FIG. 13, removes undesired background, stray light, or stray scattered light from the collected beam, and relays it, after recollimation, to several detectors 980x and 980y. For each channel of detection except the last one (fluorescence detection module 971e), a dichroic beamsplitter 972x / y separates out a spectral band from the forward-propagating beam, and relays it toward the respective detector 980x / y; in the last fluorescence detection module 971e, a mirror 973 directs to detector 980y of detection module 971e the light transmitted by beamsplitter 972y of detection module 971d).

[0092] As schematically illustrated by FIG. 9B, for each of the scattering detection modules 970x (representing, e.g., side scatter detection modules 970a and 970b in FIG. 9A), a spectral band of the substantially collimated input light (from the right in this figure) is selectively reflected by beamsplitter 972x, optionally filtered by one or more spectral filters 974x, and focused by a lens 976x onto a pinhole 977x, after which it re-expands and is then relayed by lens(es) 978x (such as, e.g., without limitation, a piano convex lens, a biconvex lens, a best-form lens, an aspherical lens, a piano convex lens pair, or an aspherical lens pair) onto the active surface of a detector 980x (such as, e.g., without limitation, a photomultiplier tube [PMT], a silicon photomultiplier [SiPM], an avalanche photodiode [APD], or a photodiode [PD]), where it is converted to an electrical signal and conveyed to downstream electronics (such as, e.g., one or more electronic amplifiers, one or more noise filters, an analog-to-digital converter, digital filters, and other electronics known in the art as desirable for the collection, processing, and storage of optical particle interrogation signals). The dichroic beamsplitters 972x may separate the light emitted from the sample in the flow cell into different wavelength bands for detection in the different detection channels (or modules). For example, the wavelength bands detected by different detection channels can be centered, without limitation, around 266 nm, around 280 nm, around 320 nm, around 355 nm, around 375 nm, around 405 nm, around 488 nm, around 525 nm, around 532 nm, around 561 nm, around 574 nm, around 594 nm, around 615 nm, around 635 nm, around 667 nm, around 785 nm, or around other wavelengths corresponding to the wavelength(s) of the one or more excitation light sources 910a, 910b, 910c

[0093] In some embodiments, light from more than one excitation light source is scattered by a particle in the sample and directed by a beamsplitter 972x into a single detector. For example, without limitation, in some embodiments, light from a 375-nm excitation source 910a and light from a 405-nm excitation source 910b, both scattered by a particle in the sample into collection lens 941, are relayed together (e.g., by long-pass beamsplitter 972x of detection module 970a) into detector 980x of side scatter detection module 970a, while light from a 488-nm excitation source 910c also scattered by the same particle in the sample into collection lens 941 is relayed (e.g., by long-pass beamsplitter 972x of detection module 970b) into detector 980x of side scatter detection modules 970b. In some embodiments, light from a 375-nm excitation source 910a scattered by a particle in the sample into collection lens 941 is relayed (e.g., by long-pass beamsplitter 972x of detection module 970a) into detector 980x of side scatter detection module 970a, while light from a 405-nm excitation source 910b and light from a 488-nm excitation source 910c, both also scattered by the same particle in the sample into collection lens 941, are relayed (e.g., by long-pass beamsplitter 972x of detection module 970b) into detector 980x of side scatter detection module 970b. In some embodiments, light from a 375-nm excitation source 910a and light from a 488-nm excitation source 910c, both scattered by a particle in the sample into collection lens 945, are relayed together (e.g., by dual-band beamsplitter 972x of detection module 970a) into detector 980x of side scatter detection module 970a, while light from a 405-nm excitation source 910b also scattered by the same particle in the sample into collection lens 941 is relayed (e.g., by long-pass beamsplitter 972x of detection module 970b) into detector 980x of side scatter detection module 970b

[0094] As schematically illustrated by FIG. 9C, for each of the fluorescence detection modules 971y (representing, e.g., channels 971a, 971b, 971c, 971d, 971e in FIG. 9A), a spectral band of the substantially collimated input light (from the right in this figure) is selectively reflected by beamsplitter 972y, optionally spectrally filtered by one or more spectral filters 974y, and focused by a lens 979y onto the active surface of a detector 980y (such as, e.g., without limitation, a photomultiplier tube [PMT], a silicon photomultiplier [SiPM], an avalanche photodiode [APD], or a photodiode [PD]), where it is converted to an electrical signal and conveyed to downstream electronics (such as, e.g., one or more electronic amplifiers, one or more noise filters, an analog- to-digital converter, digital filters, and other electronics known in the art as desirable for the collection, processing, and storage of optical particle interrogation signals). The dichroic beamsplitters 972y can separate the light emitted from the sample in the flow cell into different wavelength bands for detection in the different detection channels (or modules). For example, the wavelength bands detected by different detection channels can be centered, without limitation, around 440 nm, around 525 nm, around 580 nm, around 615 nm, around 697 nm, around 755 nm, or around other wavelengths corresponding to one or more of the Raman emissions of the sample, one or more of the autofluorescence emissions of the sample, or one or more of the fluorescence emissions of the one or more exogenous labels used. In some embodiments, the wavelength band detected by a detection channel is selected (by the use of an appropriate dichroic beamsplitter 972y and of one or more spectral fdters 974y) to maximize collection of the emission spectrum of one or more fluorophores while minimizing collection of unwanted Raman scattering light generated in the sheath fluid, the sample suspension fluid, or the sample itself by the one or more excitation light sources 910a, 910b, 910c. In some embodiments, the one or more spectral fdters 974y include a longpass fdter, a notch fdter, a rejection fdter, a multi-notch fdter, a multiband fdter, or other similar spectral element to reduce the amount of light from one or more of the excitation light sources 910a, 910b, 910c scattered by particles in the sample, by the suspension medium, or by sheath fluid (collectively, Mie scattering and Rayleigh scattering) reaching the respective detectors.

[0095] In some embodiments, one or more detection channels may be configured to operate as scattering detection channels or as a fluorescence detection channel. Selection of the beamsplitter reflecting light into the detection channel path, and of the optional spectral filter(s) in that path, determine whether a given detection channel operates as a scattering or fluorescence detection channel. For example, side scatter detection module 970b in FIG. 9A may be a scattering detection channel “SSC2” (e.g., without limitation, by selecting beamsplitter 972x of detection module 970b to reflect light around 488 nm and to transmit light longer than, e.g., 495 nm) or a fluorescence channel “FL1” (e.g., without limitation, by selecting beamsplitter 972x of detection module 970b to reflect light emitted by fluorescence in a sample in the wavelength band around 440 nm and to transmit light longer than, e.g., 475 nm). A scattering channel may additionally be reconfigured to detect scattered light from a different excitation source, also by appropriate selection of the respective beamsplitter and optional spectral filters. Likewise, a fluorescence channel may be reconfigured to detect a different wavelength band, again by appropriate selection of the respective beamsplitter and optional spectral filters. Such reconfigurations, as also described herein with reference to FIG. 18, may be executed repeatedly to perform different sample analyses, or a given configuration may be selected during manufacturing and offered as a default.

[0096] In some embodiments, different types of photodetectors can be used to detect the light in different wavelength bands. Collection of side scattering and fluorescence emissions can, in some embodiments, be arranged to be centered around an angle of less than about 90 degrees from the forward direction and, in other embodiments, around an angle of greater than about 90 degrees from the forward direction, up to about 180 degrees (in which case the resulting collected scattering signal is also referred to as backscattering). Tn some embodiments, a spatial mask is employed (e.g., just downstream of collection lens 945) to select light scattered into certain angular ranges and not others. Such spatial mask may define one or more rectangular areas of collection, one or more polygonal shaped areas of collection, one or more round areas of collection, one or more areas of collection with shapes other than a circle or a rectangle (e.g., without limitation, one or more annuli, or one or more annular segments), and such areas of collection may be symmetrically disposed around the scattering axis (e.g., 90 degrees from the direction of excitation light propagation), or they may be disposed in asymmetric geometries.

[0097] FIGs. 10A-10B illustrate an embodiment where the substantially collimated, substantially Gaussian light beam 1011 emitted by one of the excitation light sources 1010 passes through beam shaping elements 1015 prior to being focused into flow cell 1040 by global focusing lens 1030. The geometry of the system is indicated by the Cartesian axes. For example, in this embodiment, y (out of the page in 10B) represents an axis transverse to the beam and aligned with the vertical direction and with the prevailing direction of fluid flow in the flow cell, x (into the page in 10A) represents an axis transverse to the beam and aligned with a horizontal direction transverse to the prevailing direction of fluid flow in the flow cell, and z represents an axis longitudinal with respect to the beam and aligned with a second horizontal direction also transverse to the prevailing direction of fluid flow in the flow cell. In this embodiment, beam shaping elements 1015 include cylindrical lenses 1016, 1017. Cylindrical lens 1016 is a negative-power lens (e.g., without limitation, a plano-concave spherical lens or a negative aspherical lens) while cylindrical lens 1017 is a positive-power lens (e.g., without limitation, a plano-convex spherical lens or a positive aspherical lens). The curvatures, or powers, of both cylindrical lenses 1016, 1017 are in the y direction (FIG. 10A); in the x direction (FIG. 10B) both cylindrical lenses 1016, 1017 are powerless. In such an arrangement, the y characteristics of the beam transmitted through cylindrical lenses 1016, 1017 are altered, while the x characteristics are substantially unaltered. The y profile of beam 1011 (schematically indicated by its centerline, or optical axis, the dash-dotted line 1012; and by the two dashed lines 1013, corresponding, e.g., to the points in the beam where the intensity drops to a value approximately 1 / e2of the peak intensity obtained along the dash-dotted line 1012) diverges after passing through cylindrical lens 1016, and is re-collimated after passing through cylindrical lens 1017. The x profile of beam 1011 (schematically indicated by its centerline, or optical axis, the dash-dotted line 1012; and by the two dashed lines 1014, corresponding, e g., to the points in the beam where the intensity drops to a value approximately 1 / e2of the peak intensity obtained along the dash-dotted line 1012) remains substantially unchanged by its propagation through both cylindrical lenses 1016, 1017. This kind of arrangement, known as cylindrical beam expansion, is achieved through selection and orientation of the focal lengths of cylindrical lenses 1016, 1017. For example, a cylindrical lens 1016 with an effective focal length of -50 mm, combined with a cylindrical lens 1017 with a focal length of + 100 mm, positioned approximately 50 mm apart, achieve an approximately “2x” beam expansion in they direction, where the resulting beamy profile is twice as wide as in the input beam; while the x beam profile is unchanged. It will be apparent to those skilled in the art that many variations in this arrangement are possible that are entirely encompassed by this disclosure; e.g., beam expansion of factors other than 2 (e.g., without limitation, factors of 1.5, 2.5, 3, 4, 5, 6, 8, 10, more than 10, or intermediate values between 1 and 10).

[0098] It is common for excitation light sources (e.g., without limitation, lasers) to produce approximately circular beams (e.g., without limitation, 0.7 mm, 1 mm, 3 mm, or other values, of 1 / e2beam dimensions in both transversal directions), such beams having substantially or predominantly Gaussian intensity profdes in both transversal directions (but also optionally in the case of flat-top beams and other beam profiles). The arrangement illustrated in FIGs. 10A-10B achieves an asymmetric expansion of the excitation light beam, with (in the case of an approximately circular input beam 1011) the y dimension becoming larger than the x dimension after passage through beam shaping elements 1015. Due to the properties of light propagation, a larger beam dimension in the collimated portion results, after focusing by lens 1030 (which may be, e.g., without limitation, a spherical piano convex lens, an aspherical positive lens, an achromatic positive lens, a positive achromatic asphere, or other two-dimensional focusing element) in a tighter beam waist on the focal plane (in this case, in the flow channel within flow cell 1040). For sheath-flow-based analysis of particles, it is desirable for excitation beams to be focused asymmetrically into the flow cell: the horizontal beam waist in the plane perpendicular to the sample core stream flow direction (x) being larger than the vertical beam waist along the stream flow direction (y), considerably larger than the core stream transversal dimension (which may be, e.g., without limitation, 5 pm, 10 pm, 20 pm, smaller than 5 pm, larger than 20 pm, or having intermediate values between 5 pm and 20 pm), and smaller than the relevant cross-sectional dimension of the flow cell internal channel (which may be, e.g., without limitation, 50 pm, 100 pm, 150 gm, 200 gm, 250 gm, and in some embodiments smaller than 50 gm, larger than 250 gm, or having intermediate values between 50 gm and 250 gm). This results in a relatively broad horizontal waist that presents an approximately uniform light intensity to particles flowing in the relatively tight sample core stream, while minimizing light scattering from the flow cell internal channel walls, and while vertically focusing the excitation beam into a relatively tight waist to achieve greater instantaneous scattering and fluorescence signals. The specific values of the beam waists can be chosen from a range of dimensions, according to the specific application of interest, the nature of the particles to be interrogated, the wavelength of laser light being used, the internal cross-sectional dimensions of the flow cell, and other considerations. For a core stream of approximately 10 pm in transversal cross-sectional dimension and a flow cell of approximately 200 gm in transversal cross-sectional dimension, a suitable horizontal (x) beam waist may be in the range of about 80 gm to about 160 gm (1 / e2full waist, or uniform portion of a flat-top beam), although in certain embodiments it may be desirable to obtain horizontal beam waists larger than about 160 gm or smaller than about 80 gm. For analysis of most particles, a suitable vertical (y) beam waist may be in the range of about 20 gm to about 40 gm (1 / e2full waist), although in certain embodiments it may be desirable to obtain vertical beam waists larger than about 40 gm (such as, e.g., without limitation, for the analysis of tumor spheroids, other cell aggregates, or pollen) or smaller than about 20 gm (such as, e.g., without limitation, for the analysis of exosomes or other nanoparticles).

[0099] Not illustrated in FIGs. 10A-10B are alternative embodiments of the disclosed architecture that achieve the same purpose using other devices known in the art. For example, instead of two cylindrical lenses, the collimated laser beams can be subjected to asymmetric expansion through an anamorphic prism pair. Alternatively, the laser beam y profile may be expanded using two positive cylindrical lenses, wherein the second lens (in the order of propagation) has a larger focal length than the first lens, and the two lenses are placed a distance apart approximately equal to the sum of their focal lengths. Yet alternatively, the laser beam x profile may be reduced (instead of the y profile being expanded). For example, cylindrical lenses 1016, 1017 may be reversed, with the positive lens being upstream of the negative lens, and oriented along the x axis instead of the y axis; or two positive cylindrical lenses, with their curvatures along the x axis, may be employed with the second one (in the order of propagation) having a smaller focal length than the first one, the two lenses being placed a distance apart approximately equal to the sum of their focal length. In each case, the resulting asymmetrical beams (with the vertical resulting; / beam dimension larger than the horizontal x beam dimension, in the collimated portion between the beam shaping elements 1015 and the global focusing lens 1030) can produce asymmetrically focused beam waists substantially similar to the waists described above. In some embodiments, the output beams from one or more of the excitation light sources may be specified to have a desired degree of ellipticity (e.g., without limitation, a 4: 1 ratio of vertical to horizontal), obviating the need for the asymmetric beam shaping elements 1015 in order to produce the asymmetric focusing on the center plane of the sample core stream as described above. In yet another embodiment, the substantially circular excitation laser beam(s) are asymmetrically focused by a combination of two crossed cylindrical lenses rather than by a single global focusing lens. It will be appreciated by those skilled in the art that other ways exist to achieve substantially similar results that do not depart from the spirit and principle of the present disclosure. Detailed optical propagation calculations can inform the precise selection and relative placement of lenses 1016, 1017, and 1030, and other optical components in the systems described herein in order to account for second-order effects, aberrations (such as, without limitation, chromatic, spherical, coma, and astigmatic aberrations), beam offsets, manufacturing variations, and other possible departures from an approximate model.

[0100] For embodiments including more than one excitation wavelength, the beam shaping elements 1015 may be placed prior to the beams having different wavelengths being combined. In one such embodiment, beam combiners (920 in FIG. 9A) are placed downstream of beam shaping sections (beam shaping elements 915 in FIG. 9A; also beam shaping elements 1015 in FIGs. 10A- B) in the respective beams from the individual light sources, and upstream of the global focusing lens (930 in FIG. 9A; also 1030 in FIGs. 10A-B). In another embodiment, a beam combiner combining two substantially overlapping beams may be placed upstream of a common beam shaping section. In yet another embodiment, light sources producing multiple wavelengths (such as, e.g., without limitation, light hubs, light engines, or multi-laser engines) produce substantially overlapping, substantially collimated beams 1011 each having distinct wavelength characteristics. In embodiments where a beam shaping section transmits beams having distinct wavelength characteristics, the elements including the beam shaping section (beam shaping elements 915 in FIG. 9A; also beam shaping elements 1015 in FIGs. 10A-B) may be approximately achromatic at the wavelengths of the respectively combined beams. In embodiments involving multiple excitation source wavelengths, the global focusing lens (global focusing lens 930 in FIG. 9A; also global focusing lens 1030 in FIGs. 10A-B) may be approximately achromatic at the wavelengths of the combined beams. The beams from each excitation light source may be conveyed in free space, through optical fibers, through optical waveguides, or through other means of beam transport. For means of beam transport involving focusing (such as, e.g., without limitation, into an optical fiber or waveguide), the generally divergent output beam is collimated using means known in the art (including, e.g., without limitation, an aspherical lens) before entering the beam shaping section as described herein (beam shaping elements 915 in FIG. 9A; also beam shaping elements 1015 in FIGs. 10A-B). In one embodiment involving multiple excitation source wavelengths, the combined and shaped excitation beams are all substantially collimated and substantially overlapping prior to entering the global focusing lens (global focusing lens 930 in FIG. 9A; also global focusing lens 1030 in FIGs. 10A-10B).

[0101] FIG. 11 illustrates an externally adjustable laser fine alignment module of a configurable or modular flow cytometry apparatus in accordance with some embodiments of the present disclosure. In the example shown in FIG. 11, the global focusing lens (global focusing lens 1130; also global focusing lens 930 in FIG. 9A and global focusing lens 1030 in FIGs. 10A-10B) is mounted using hardware 1131 known in the art (such as, without limitation, custom mounts, lens mounts, lens tubes, lens tube reducers, lens tube couplers, and / or retaining rings) to a lens translation stage 1132 (such as, without limitation, an X stage, an XY stage, an XYZ stage, a 4- axis stage, a 5-axis stage, a flexure mount, a dovetail stage, a crossed-bearings stage, a threaded mount, or other mechanical positional motion adjuster as are known in the art). The translation stage 1132, which is mounted to supporting elements (such as, e.g., without limitation, custom holders, clamps, brackets, posts, post holders, bases, pedestals, holding forks, bolts, or other elements as are known in the art) stationary relative to the rest of the optical module, may be a stage capable of adjusting the horizontal position, or it may be capable of adjusting the horizontal and vertical positions, or it may be capable of additional adjustments beside the horizontal and vertical positions. The externally adjustable laser fine alignment module can adjust the propagation direction of an input light beam 1112a to an adjusted propagation direction of light beam 1112b.

[0102] The horizontal adjustment element 1133 of the translation stage 1132, e.g., a threaded bolt that translates linearly the spring-loaded movable member of the stage 1132 when turned, is actuatable using a tool such as a hexagonal key (also known as, e.g., Allen key, Allen wrench, or hexagonal ball driver), a screwdriver (such as, e.g., a Phillips head, a flat head, star, or other type), or other suitable tool comprising a shaft and a shaped point and capable of conveying torque. A guide tube 1136 is mounted (e.g., without limitation, to a base, breadboard, plate, or horizontal panel) using hardware 1135 known in the art (such as, without limitation, custom holders, clamps, brackets, posts, post holders, bases, pedestals, holding forks, and / or bolts) so as to align such torquing tool with and onto the knob of the adjustment element. The guide tube 1136 is capped with an inner funnel 1134 at the distal end (closest to the knob) that reduces the variability in transversal position of the torquing tool to aid with insertion into the mating recess of the knob. The guide tube 1136 is also provided with a light baffle 1137 including an outer flare and optional optical gasket at the proximal end (e.g., closest to an apparatus cover) that reduces or substantially eliminates the amount of stray light permitted to either exit or enter the optical bench. The light baffle 1137 is coupled to the guide tube 1136 so as to make contact with the inner surface of a front panel of the apparatus, into which panel a hole is provided that aligns with the guide tube and permits the insertion of the torquing tool through the panel, along the tube, past the funneled cap, and onto the adjustment knob. In other embodiments, adjustment elements that rely on different actuation mechanisms (such as ratchets, pulleys, springs, and other mechanisms known in the art) are actuated in similar ways using a tool capable of reaching and actuating the adjustment element from outside the apparatus.

[0103] The module described herein with respect to FIG. 11 therefore permits the adjustment of the horizontal cylindrical lens externally or by external means, i.e., without lifting the apparatus cover, remove the front or side panels, or otherwise alter the intended configuration of the instrument in ways that could impair its performance. Horizontal adjustment of the horizontal cylindrical lens in the optical layout described herein causes an angular shift, on a plane perpendicular to the sample core stream flow direction, in the propagation direction of the beam(s) 1112b refracted through the lens. Such controlled shift aids in fine-tuning the alignment of the beam(s) with the sample core stream in the flow cell (which is flowing vertically in this embodiment), allowing substantial maximization of excitation intensity and substantial maximization of the optical signals generated in the interactions with particles of interest in the sample stream. Therefore, the performance of the apparatus may be rapidly optimized using inexpensive means and tools without unduly burdening the measurement workflow.

[0104] It will be apparent to those skilled in the art that other configurations of the components and elements described herein are encompassed by this disclosure. In another embodiment, for example, the main direction of sample stream flow in the flow cell is horizontal, and the direction of the interrogating light beam(s) transversal to the flow is also horizontal. Cylindrical lenses 1016, 1017 of FIGs. 10A-10B are in such embodiment horizontally oriented (i.e., along the x axis); and the module of FIG. 11 is modified so as to provide vertical adjustment to the position of global focusing lens 1130. The light baffle in such embodiment contacts the internal surface of the cover of the apparatus, and fine adjustment of alignment of laser beam(s) with the sample core stream by external means is achieved by insertion of the torquing tool through a hole provided in such top cover. Other embodiments, e.g., where the direction of sample stream flow is horizontal, the direction of beam propagation is vertical, and the orientation of cylindrical lenses 1016, 1017 of FIGs. 10A-10B is horizontal perpendicular to the flow direction; or where the direction of sample stream flow is neither horizontal nor vertical and where the orientation of cylindrical lenses 1016 and 1017 of FIGs. 10A-10B is neither vertical nor horizontal; are also possible and encompassed by this disclosure.

[0105] In another embodiment, the lens translation stage 1132 of FIG. 11 is provided with motorized adjustment(s), capable of remote actuation through, e.g., electrical or wireless control. In such embodiment, insertion of a mechanical torquing tool from the exterior is unnecessary, as are the guide tube 1136, the inner funnel 1134, the light baffle 1137, and the guide tube mounting hardware 1135. In such cases, the positional adjustment(s) of the movable member of the lens translation stage 1132 can be controlled through direct electrical actuation (e.g., without limitation, by application of a voltage impulse to a DC motor, by application of series of impulses to a stepper motor, or by application of voltage signals to a galvo driver and motor or to a piezoelectric actuator) or by wireless signals received by suitable wireless communication modules as are known in the art, translated into actuation signals, and conveyed to an electrical actuator such as those described above. External adjustment of laser alignment with the sample stream is therefore achieved in this embodiment using electrical or wireless signals conveyed to actuator(s) for the translation of a cylindrical lens in the path of the laser beam(s).

[0106] As described above with reference to FIGs. 10A-10B, the cylindrical beam shaping section illustrated in FIGs. 10A-10B may be replaced in other embodiments with other means of obtaining asymmetric focusing of the beam(s), such as, e.g., without limitation, using anamorphic prism pair(s), positive cylindrical lens pair(s), cylindrical beam reducer(s), natively elliptical laser beams, internally elliptically shaped laser beams, and other devices known in the art, and one or more spherical or aspherical lenses for focusing the beam(s) onto the sample core stream in the flow cell. In such embodiments, the element subject to translation using the configuration illustrated in FIG. 11 may be one of the one or more spherical or aspherical lenses, another optical element with nonzero optical power in the direction of translation, the laser(s) themselves, or the flow cell itself. And also as described above, the present disclosure is not limited to horizontal translation of such element(s) for effecting external adjustment of the laser alignment to the sample stream, but encompasses any direction at all.

[0107] The same principle illustrated above in reference to FIG. 11 for transversal adjustment of laser alignment to the sample core stream also applies to adjustment of laser alignment along the sample stream flow direction. In some embodiments, the elements illustrated and described in reference to FIG. 11 refer to vertical adjustments of global focusing lens 1130. The adjustment obtained using the module described above as applied in the vertical (y) direction achieves changes in propagation direction of the laser beam(s) on a plane that includes both such propagation direction and the direction of fluid flow. Such adjustment can be desirable to obtain maximum coupling of the emitted (e.g., without limitation, scattered and fluorescent) light into the one or more photodetectors shown in FIGs. 9A-9C and therefore to obtain maximum signal from the interaction of sample particles with the laser beam(s). As above, it is not necessary for sample flow to be in the vertical direction; sample flow may be oriented in any direction at all, with the translation module described herein providing translation in substantially the same direction as the flow. In another embodiment, adjustment by external means of laser alignment along the direction of flow is based on at least partial overlap of the direction of translation with the direction of flow, with greater overlap producing the desired effects with greater efficiency.

[0108] FIG. 12 illustrates a stray -light blocking module 1200 of a flow nanocytometer in accordance with some embodiments of the present disclosure. One or more sources of excitation light (as in, e.g., without limitation, laser(s)) are focused onto the sample stream in the flow cell 1240 (also flow cell 940 in FIG. 9A; and flow cell 1040 in FIGs. 10A-10B) as described herein. The excitation light beam(s) interact with particles in the sample stream and generate optical signals of interest, such as, without limitation, forward scatter, small-angle scatter, intermediate-angle scatter, side scatter, backscatter, absorption, extinction, fluorescence, Mie, Rayleigh, Raman, coherent anti-Stokes Raman scattering, and other signals. At least a portion of such signals is collected and selected using optics such as, without limitation, lenses, mirrors, beamsplitters, filters, gratings, prisms, optical fibers, and / or diffractive optical elements, and conveyed to photodetector(s), as described herein. FIG. 12 shows a portion of an FSC detection module of an apparatus in accordance with an embodiment of the disclosure, together with an element designed to reduce unwanted optical background. A relay element 1242 (such as, without limitation, a bestform lens, a biconvex lens, a piano convex lens, an aspherical lens, an achromat, a compound lens, or an off-axis parabolic mirror) collects light (aligned along beam propagation direction along optical axis 1212) emerging from the flow cell 1240 in a cone approximately centered on the forward direction (i.e., the direction of propagation of the excitation light beam(s)). The relay element 1242 is housed in a translation mount or stage 1243 that comprises transversal adjustments (such as an XY stage). Light relayed by the lens is optionally passed through one or more spectral filters 1246 (such as, without limitation, neutral-density, bandpass, shortpass, longpass, multiband, and / or color filters) before reaching the active surface of a photodetector 1248 (such as, without limitation, aPMT, an SiPM, an APD, or aPD), where it is converted to an electrical signal.

[0109] Light emerging from the flow cell 1240 in the forward direction includes desired optical signals, such as forward-scattering signals from particle(s) in the flow cell; but generally also includes undesired (static) contributions to the optical signal, such as scattering from flow cell surfaces, imperfections, or deposits, as well as diffraction, refractions, and / or reflections of the excitation beam(s), which can propagate in the forward direction in substantial overlap with the desired (transient) optical signals. Such undesired contributions can cause elevated background, elevated optical and electrical noise, and can act to degrade or hide the desired signals altogether. To reduce the impact of such undesired contributions, in some embodiments, an FSC module of the disclosure includes an obscuration mask 1244 fastened onto the element housing the relay element 1242 (such as, without limitation, the lens mount, the lens translation mount, or the lens translation stage 1243). The obscuration mask 1244 includes one or more elements designed to block unwanted optical contributions from reaching the FSC photodetector 1248. In some embodiments, the obscuration mask 1244 is substantially in the shape of a cross whose arms are aligned, respectively, with the longitudinal axis (the y direction, as shown in FIG. 12) of the flow cell 1240 (i.e., the axis substantially along which fluid flows) and in the x direction (i.e., at a right angle to both the longitudinal axis (in the y direction) and the light propagation (z) direction). The two arms of the obscuration mask, in this embodiment, are dimensioned to block as much of the unwanted light as possible (e.g., without limitation, up to about 50%, up to about 80%, up to about 90%, or greater than about 90%), while simultaneously allowing as much of the desired optical signals to pass through (e.g., without limitation, up to about 30%, up to about 50%, or greater than about 50%).

[0110] In some embodiments, the relay element 1242 is placed in such a position so as to relay desired scattered light from a particle in the sample core stream onto the active surface of the photodetector 1248, according to principles of fundamental optics well known in the art. In some cases, the position of the relay element 1242 is also such to subtend a cone of desired scattered light having the desired angular extent (e.g., without limitation, an FSC cone with a half-angle of less than about 2 degrees, between about 2 degrees and about 5 degrees, between about 5 degrees and about 10 degrees, or greater than about 10 degrees). In some cases, the lens translation stage

[0111] 1243 onto which the obscuration mask 1244 is affixed allows translation of the obscuration mask

[0112] 1244 into an alignment relationship with the propagation direction of the laser beam(s) (centered on optical axis 1212) to permit maximal rejection of unwanted light while also permitting maximal transmission of desired light. In some cases, the arms of the obscuration mask 1244 are formed out of non-reflective material (such as, without limitation, black-anodized aluminum, other mattefinish metal, matte-finish hardened resin, or other matte-finish material able to withstand absorption of a substantial portion of the optical power in each of the beam(s) as well as in all the beam(s) combined). In some cases, each of the arms of the obscuration mask 1244 is wide enough (such as, e.g., without limitation, more than about 0.5 mm wide, more than about 1 mm wide, more than about 2 mm wide, or more than about 4 mm wide) to block the laser beam(s) and a substantial portion of the light diverted from the beam(s) in the absence of particles of interest in the sample core stream. In some cases, each of the obscuration mask 1244 arms is narrow enough to allow a substantial portion of the light scattered by particle(s) of interest in the sample core stream to pass through (such as, e.g., without limitation, less than about 1 mm wide, less than about 2 mm wide, less than about 3 mm wide, or less than about 6 mm wide). In some cases, each of the arms of obscuration mask 1244 stretches across the entire diameter of the relay element 1242; and each of the arms of obscuration mask 1244 is placed as close to the relay element 1242 as practical.

[0113] Other embodiments that also fall under the scope of the present disclosure include, without limitation: placement of the obscuration mask at other positions than just before the relay lens (e.g., downstream of, including downstream of and substantially adjacent to, the relay lens, as illustrated by lens 942 and obscuration mask 944 in FIG. 9A; and an obscuration mask with shapes other than a symmetric cross with equal straight arms, such as a mask with a cross with flared arms (arms becoming wider away from the center), a cross with tapered arms (arms becoming thinner away from the center), a cross with one arm wider than the other, a cross with arms in mixed combinations (e.g., one arm tapered and one arm flared), a cross where one or more of the mask arms is arranged to stretch across only portion of the relay lens (such as, e.g., without limitation, through the central 30%, 50%, or 70% of the diameter of the relay lens), a “tee” (i.e., a cross with one full arm and half of the other), a single arm (e.g., a single bar, whether straight, tapered, flared, or otherwise), a single arm with a central bulge (e.g., a circular, elliptical, rectangular, or othershaped element substantially aligned so as to effectively block light from the excitation beams), a cross with a central bulge, a tee with a central bulge, and other mask shapes and configurations that perform a similar function to that described. In some embodiments, an FSC module may not have a relay lens, where the mask is placed as close as practical to the active area of the photodetector, and the photodetector is placed at such a distance from the flow cell so that its active area subtends a cone of desired scattered light having the desired angular extent (e.g., without limitation, a FSC cone with a half-angle of less than about 2 degrees, between about 2 degrees and about 5 degrees, between about 5 degrees and about 10 degrees, or greater than about 10 degrees). In some embodiments, stray-light blocking module 1200 includes, in addition or as an alternative to obscuration mask 1244, a spatial filter, including a light-blocking element such as, e.g., an aperture, slit, window, mask, iris, or pinhole placed substantially on the image plane of lens 1242 between lens 1242 and detector 1248.

[0114] FIG. 13 illustrates a spatial filter or stray-light blocking module of a flow nanocytometry device in accordance with some embodiments of the present disclosure. Light generated by the interaction of the laser beam(s) with particle(s) in the sample stream flowing in the flow cell 1340 (such as, without limitation, SSC and fluorescence) is collected by an optical element or collection lens subassembly 1341. The collection lens element or subassembly 1341, also called a collection lens, a condenser lens, or a high-numerical aperture (high-NA) lens, can be a single lens, a compound lens, a set of multiple lenses, a microscope objective, a parabolic mirror, an off-axis paraboloid, and / or an assembly of optical elements as are known in the art. The collection lens subassembly 1341 gathers as much of the desired optical light signals as practical. High-NA lenses, high-NA compound lenses, and high-NA microscope objectives (where the NA can be 0.3 or less, 0.5 or less, 1.0 or less, 1.25 or less, and / or less than or greater than 1.3, and where for an NA of approximately 1.0 or greater the first element in the collection subassembly 1341 can be an immersion optical element such as, without limitation, an oil immersion lens or an index-matching- gel-coupled lens) collect more of the desired light signals, generally, than elements or subassemblies with lower NA. In some embodiments, the collection subassembly is designed to be substantially achromatic over a certain wavelength range of interest, or over one or more wavelength bands of interest. It is not necessary for the collection lens subassembly 1341 to be perfectly achromatic, or even substantially achromatic, in all embodiments. In some embodiments, illustrated schematically in FIG. 13, the emitted light collected by the collection lens subassembly 1341 exits the collection lens subassembly as a substantially collimated beam or substantially collimated bundle of beams 1351. In some embodiments, such emitted light beam 1351 exiting the collection lens subassembly 1341 is substantially converging, and in other embodiments, it is substantially diverging. In yet other embodiments, beams 1351 with certain wavelengths or range of wavelengths exit the collection lens subassembly 1341 substantially collimated, while beams 1351 with other wavelengths or range of wavelengths exit such collection lens subassembly 1341 substantially converging, and beams 1351 with yet other wavelengths or range of wavelengths exit such collection lens subassembly 1341 substantially diverging.

[0115] The collected light beam 1351 is relayed by an optional turning mirror 1350 toward a focusing lens 1355. This focusing lens 1355 focuses the collected emission light 1351 (indicated in FIG. 13 by three representative rays, including the center or chief ray and two side rays) toward an aperture 1360 (or alternatively a pinhole, window, slit, iris, or mask). By arranging the relative position of the flow cell 1340 and the positions and focal lengths of the collection lens subassembly 1341 and the focusing lens 1355, a real image of the sample core stream can be substantially formed onto the plane of aperture 1360, according to principles of fundamental optics well known in the art. The dimensions of aperture 1360 are chosen so as to pass a substantial portion of the desired collected emission light (e.g., without limitation, up to about 80%, up to about 90%, or more than about 90%) while blocking a substantial portion of undesired stray or scattered light (e.g., without limitation, up to about 80%, up to about 90%, or more than about 90%). For example, for a sample core approximately 30 pm in transversal extent (as viewed from the side of the collection lens subassembly 1341), a flow cell 1340 cross-sectional dimension of approximately 400 pm (again as viewed from the collection lens) and a transverse optical magnification ratio of lOx, the real images of the core stream and flow cell 1340 have approximate dimensions of, respectively, 300 pm and 4 mm. An aperture 1360 with a transversal dimension greater than about 300 pm and less than about 4 mm, centered on the core stream image, can transmit a substantial portion of the desired light (e.g., scattered and fluorescent light emitted by particles interrogated in the sample stream) while rejecting a substantial portion of the undesired light (e.g., without limitation, stray light from the flow cell walls, fluorescent light from unbound dye, and / or Raman scattering from the sheath fluid). In some embodiments corresponding to the approximate dimensions indicated above for the core stream and flow cell 1340, an aperture 1360 can be provided with a diameter of about 2 mm, which performs the desired rejection while maintaining a substantial tolerance margin against variations in the sample core stream dimensions and position. In embodiments with, e.g., different transverse optical magnification ratios, different flow cell cross-sectional dimensions, and / or different typical core stream dimensions, an aperture may be provided with a different transversal dimension, according to optimization principles known in the art.

[0116] After the aperture 1360, a collimating lens 1365 (e.g., without limitation, the same collimating lens identified in and further described below, e.g., in reference to FIG. 14) substantially collimates or recollimates the emission light beams 1351 and relays them in the direction 1366 toward the detection arm (also identified in and further described below, e.g., in reference to FIG. 14). The focal length and position of the collimating lens 1365 can be chosen, in some embodiments, to provide a collimated relayed beam size such that substantially no clipping occurs throughout the rest of the downstream optical paths.

[0117] In some embodiments, bundle of beams 1351 in a certain desired wavelength range exits collection lens subassembly 1341 substantially converging, focusing lens 1355 is not present, and aperture 1360 is placed on the conjugate focal plane of collection lens subassembly 1341, on which plane a real image is formed of the sample core stream, according to principles of fundamental optics well known in the art. In some embodiments, collection lens subassembly 1341 functions also as a substantially achromatic or partially achromatic focusing lens, outputting substantially converging bundle of beams 1351 directly toward aperture 1360, which aperture is placed on the conjugate focal plane of collection lens subassembly 1341, on which plane a real image is formed of the sample core stream, according to principles of fundamental optics well known in the art. As described herein, the dimension of the aperture can be chosen to transmit a substantial portion of the desired light while rejecting a substantial portion of the undesired light, whether focusing lens 1355 is present, absent, or incorporated into collection lens subassembly 1341. In FIGs. 14-15, the relative orientation of fluid flow, light propagation, and transverse directions is shown, respectively, as the set of axes , z, and x. The process steps involved in the performance of some embodiments of the present disclosure are described here with reference to FIGs. 14-15, and are also further summarized in flow-chart fashion in FIG. 16.

[0118] FIG. 14 illustrates a cross-section, perpendicular to the direction of fluid flow, of an embodiment of a light collection configuration of a flow nanocytometry device in accordance with some embodiments of the present disclosure. A flow cell 1440 (as also described elsewhere herein), of which the inner part is schematically indicated in the figure, provides a channel for fluid flow. Sheath fluid 1481 is provided to confine the sample core stream 1482 carrying the particles 1483 to be analyzed, as described, e.g., herein in reference to FIGs. 3-6. The sheath fluid 1481 and the sample core stream 1482 are focused into the flow cell 1440 lumen, optionally by hydrodynamic means; such focusing produces a sample core stream 1482 bounded by the sheath fluid 1481. One or more interrogating light beams 1418 are provided to interact with the particles in the sample core stream, as described, e.g., herein with reference to FIG. 1. The interrogating beam or beams 1418, usually having a Gaussian intensity profile in both transversal directions, but also optionally having, e g., flat-top or other profiles in one or both transversal directions, are generally focused into a relatively tight spot in the plane of the sample core stream, as described, e.g., herein with reference to FIGs. 10A-10B. Particles 1483 to be analyzed in the sample core stream 1482 interact with light in the interrogating beam or beams 1418 to generate optical signals 1484 by optical processes including, for instance, scattering, absorption, or fluorescence, as described, e.g., herein with reference to FIG. 1. The optical signals 1484 are collected by collection optics 1441, as described, e.g., above with reference to FIG. 13. The collected optical signals 1451 are then conveyed (relayed) to a spatial filter (as described, e.g., herein with reference to FIG. 13) and then to spectral filtering optics 1472 to select appropriate spectral bands of the optical signals for detection, as described, e.g., herein with reference to FIGs. 9A-9C. The spectral filtering optics 1472 may include, without limitation, reflective, transmissive, absorptive, diffractive, or holographic means, or means based on interference, or a combination thereof. The resulting spectrally filtered optical signals 1485 are then conveyed (relayed) as light signals 1486 by focusing optics 1479 to detector(s) 1480, as described, e.g., herein with reference to FIGs. 9A-9C. The detector converts the light signals 1486 into electrical signals 1487, which are then conveyed to a processing unit 1490 for further analysis, processing, and optionally storage, as described below. Together, the collection optics 1441, the spatial filter optics, and the focusing optics 1479 may be referred to as relay optics.

[0119] In some embodiments, more than one spectral band output may be generated. For instance, FIG. 15 illustrates a cross-section, perpendicular to the direction of fluid flow, of an embodiment of a light collection configuration of a flow nanocytometry device in accordance with some embodiments of the present disclosure. It is similar in concept to the configuration illustrated in FIG. 14 except that the spectral filtering optics 1572 produce more than one spectral band output 1585a-1585z, separated according to spectral characteristics. Each spectral band is then conveyed (relayed) to a separate set of focusing optics 1579a-1579z and separate detectors 1580a-1580z, resulting in respectively separate electrically converted signals 1587a-1587z. The resulting electrical signals are then routed to signal processing unit 1590 for further elaboration. FIG. 15 depicts, for the sake of clarity, two sets “a” and “z” of spectral bands, focusing optics, and detectors. It will be apparent to those skilled in the art that an arbitrary number of such sets is encompassed by the scope of the present disclosure, as described, e.g., herein with reference to FIGs. 1, 9A-9C, 12, and 18.

[0120] FIG. 16 is a flowchart that describes a method of particle analysis that can be performed using a flow nanocytometry device in accordance with some embodiments of the present disclosure. For example, the method of FIG. 16 can be performed using any of the flow cytometry devices illustrated in FIGs. 1, 3-8, 9A-9C, 10A-10B, and / or 11-15. Step 1695a includes generating one or more beams of light for optical interrogation of particles in a sample, as described, e.g., herein with reference to FIGs. 10A-10B. Step 1695b including forming, by means, e.g., without limitation, of hydrodynamic focusing of the sample by sheath fluid, a core stream of particles flowing substantially one at a time through the laser beam(s) in the microchannel portion of the flow cell for optical interrogation. An optional step 1695c includes adjusting, including by automatic means such as through an algorithm and a feedback circuit, sheath pressure to achieve a desired average particle transit time across the light beam(s), as described, e.g., herein with reference to FIG. 3 and / or FIG. 4. An optional step 1695d includes adjusting, including by automatic means such as through an algorithm and a feedback circuit, sample pressure to achieve desired cross-sectional dimensions of the sample core stream, as described, e.g., herein with reference to FIG. 3 and / or FIG. 5. An optional step 1695e includes adjusting, including by automatic means such as through an algorithm and a feedback circuit, the position of the light beam(s) (e.g., transversally to the direction of fluid flow) by external actuation of lens positioning stage(s), as described, e.g., above with reference to FIG. 11. Step 1695f includes interrogating, by optical interaction, a substantially single particle in the sample core stream by the one or more light beams, resulting in the generation of optical interaction signals, as described, e.g., herein with reference to FIGs. 12, 14, and 15. Step 1695g includes collecting the optical interaction signals, the optical filtering of the collected optical signals, and the spectral isolation of the filtered optical signals, as described, e.g., herein with reference to FIGs. 9A-9C and 12-15. Step 1695h includes detecting the spectrally isolated optical signals, transducting the signals into analog electrical signals, digitizing the analog electrical signals into digital signals, and processing the digital signals. Step 1695i includes determining, based on the detected and processed signal parameters, including, without limitation, fluorescence intensity in one or more spectral bands, phase shift, scattering intensity from one or more excitation sources, and absorption. Step 1695j includes triggering a call for action, which may be automated or displayed by the system through a processing unit, on whether to analyze additional particles; if the choice is positive, the method workflow returns to the step 1695f; if the choice is negative, the method workflow continues to optional step 1695k or step 1695m. Optional step 1695k includes classifying a portion or a totality of the events detected and analyzed according to certain criteria (which may include, without limitation, entities commonly referred to in the art as “triggers,” “thresholds,” and “gates,” in conjunction with logic operators known in the art, such as, without limitation, OR, AND, NOT, and combinations thereof), which may be predetermined and preloaded into the apparatus or may be selected or modified or created by the operator. Step 1695m includes displaying the processed data (which may include, without limitation, the raw detected time-varying signals, a list of detected particle-interrogation events, and / or graphs or plots of detected events displayed according to characteristics such as, e.g., fluorescence intensity and scattering intensity) by means of a user interface such as, e.g., a screen, a computer monitor, a printout, or other such means.

[0121] In some embodiments, the method of FIG. 16 may further include introducing a sample to the apparatus (e.g., by a user or operator or automatically). The method of FIG. 16 may further include mixing, reacting, and incubating the sample with one or more reagents (e.g., fluorescently labeled markers, for example antibodies, that couple to PKD associated proteins or control proteins), such that the reagents may be preloaded onboard the apparatus or may be introduced to the apparatus. FIG. 17 shows a block diagram of an exemplary embodiment of a data processing system 1700 to provide a particle analysis system as described herein. In some embodiments, data processing system 1700 is a part of the control system to perform a method that includes providing a light beam; exposing a sample to the light beam; detecting optical emissions from the exposed sample; and receiving electrical analogues of the emissions, as described herein. In some embodiments, data processing system 1700 is represented by any one of signal processing units 190, 290, 1490, and 1590 depicted in FIGs. 1, 14, and 15, respectively, and further optionally incorporates data storage unit 192 depicted in FIG. 1.

[0122] Data processing system 1700 includes a processing unit 1701 that may include a microprocessor or microcontroller, such as Intel microprocessor (e.g., Core i7, Core 2 Duo, Core 2 Quad, Atom), Sun Microsystems microprocessor (e.g., SPARC), IBM microprocessor (e g., IBM 750), Motorola microprocessor (e g., Motorola 68000), Advanced Micro Devices (“AMD”) microprocessor, Texas Instrument microcontroller, and any other microprocessor or microcontroller.

[0123] Processing unit 1701 may include, e.g., without limitation, a personal computer (PC), such as a Macintosh® (from Apple Inc. of Cupertino, California), Windows®-based PC (from Microsoft Corporation of Redmond, Washington), Android®-based PC (from Alphabet of Mountain View, California), or one of a wide variety of hardware platforms that run the UNIX operating system (e g., Linux®), or other operating systems. For at least some embodiments, processing unit 1701 includes a general purpose or specific purpose data processing system based on Intel, AMD, Motorola, IBM, Sun Microsystems, IBM processor families, or any other processor families. As shown in FIG. 17, a memory 1703 is coupled to the processing unit 1701 by a bus 1702. Memory 1703 has instructions and data 1704 stored thereon which when accessed by processing unit 1701 cause the processing unit 1701 to perform methods to provide, for example, particle analysis, as described herein.

[0124] Memory 1703 can be dynamic random access memory (“DRAM”) and can also include static random access memory (“SRAM”). A bus 1702 couples processing unit 1701 to memory 1703 and also to a non-volatile storage 1707 and to a display controller 1705 (if a display is used) and to input / output (I / O) controller(s) 1708. Display controller 1705 controls in the conventional manner a display on a display device 1706 which can be a cathode ray tube (CRT), a liquid crystal display (LCD), a light-emitting diode (LED) monitor, a plasma monitor, or any other display device. Input / output devices 1722 can include a keyboard, disk drives, printers, a scanner, a camera, and other input and output devices, including a mouse or other pointing device. I / O controller 1708 may also be coupled to one or more audio input devices 1709 such as, for example, one or more microphones. Additionally, or alternatively, I / O controller 1708 may be coupled to one or more digital image input devices 1721 (e.g., computing device, image sensor, etc.).

[0125] Display controller 1705 and I / O controller 1708 can be implemented with conventional well-known technology. An audio output 1723 such as, for example, one or more speakers, may be coupled to I / O controller 1708. Non-volatile storage 1707 can be a magnetic hard disk, an optical disk, a solid-state drive, a flash drive, or another form of storage for large amounts of data. Some of this data is often written, by a direct memory access process, into memory 1703 during execution of software in data processing system 1700 to perform methods described herein.

[0126] One skilled in the art will immediately recognize that the terms “computer-readable medium” and “machine-readable medium” include any type of storage device that is accessible by processing unit 1701. Data processing system 1700 can interface to external systems through a modem or network interface 1724. It will be appreciated that, in some cases, modem or network interface 1724 can be considered to be part of data processing system 1700. This interface 1724 can be an analog modem, ISDN modem, cable modem, token ring interface, satellite transmission interface, Wi-Fi, Bluetooth, cellular network communication interface, or other interfaces for coupling a data processing system to other data processing systems.

[0127] It will be appreciated that data processing system 1700 is one example of many possible data processing systems which have different architectures. For example, personal computers based on an Intel microprocessor often have multiple buses, one of which can be an input / output (I / O) bus for the peripherals and one that directly connects processing unit 1701 and memory 1703 (often referred to as a memory bus). The buses are connected together through bridge components that perform any appropriate translation due to differing bus protocols.

[0128] Network computers are another type of data processing system that can be used with the embodiments as described herein. Network computers do not usually include a hard disk or other mass storage, and the executable programs are loaded from a network connection into memory 1703 for execution by processing unit 1701. A typical data processing system may usually include at least a processor, memory, and a bus coupling the memory to the processor. It will also be appreciated that data processing system 1700 can be controlled by operating system software which includes a file management system, such as a disk operating system, which is part of the operating system software. Operating system software can be, e.g., without limitation, the family of operating systems known as Macintosh® Operating System (Mac OS®) or Mac OS X® from Apple Inc. of Cupertino, California, the family of operating systems known as Windows® from Microsoft Corporation of Redmond, Washington, the family of operating systems known as Linux®, or the family of operating systems known as Android® from Alphabet of Mountain View, California, and their associated file management systems. The file management system is typically stored in non-volatile storage 1707 and causes processing unit 1701 to execute the various acts used by the operating system to input and output data and to store data in memory, including storing files on non-volatile storage 1707.

[0129] In various embodiments, hardwired circuitry may be used in combination with software instructions to implement methods described herein. A non-transitory machine-readable medium can be used to store software and data which when executed by a data processing system causes the system to perform various methods described herein. This executable software and data may be stored in various places including for example ROM, volatile RAM, non-volatile memory, and / or cache. Portions of this software and / or data may be stored in any one of these storage devices.

[0130] Thus, a machine-readable medium includes any mechanism that provides (i.e., stores and / or transmits) information in a form accessible by a machine (e.g., a computer, network device, or any device with a set of one or more processors, etc.). For example, a machine-readable medium includes recordable / non-recordable media (e.g., read only memory (ROM); random access memory (RAM); magnetic disk storage media; optical storage media; flash memory devices; and the like).

[0131] It will be further appreciated that data processing system 1700 may be functionally implemented by allocating several of its functions to distributed units or modules separate from a central system. In some embodiments, some or all of the signal processing functions as depicted, e.g., in FIG. 1 and illustrated in FIGs. 14-15, may be performed by signal processing units or modules physically separate from data processing system 1700, yet connected with it for performance of other functions, such as, e.g., input / output, display, data storage, memory usage, bus usage, additional signal processing functions, and both specific-purpose and general -purpose data processing functions. In some embodiments, some or all of the data storage functions as depicted, e.g., in FIG. 1 and illustrated in FIGs. 14-15, may be performed by data storage units or modules physically separate from data processing system 1700, yet connected with it as described above. In some embodiments, some or all of the signal processing functions mentioned may be performed by processing unit 1701 internal to data processing system 1700, and in some embodiments, some or all of the data storage functions mentioned may be performed by nonvolatile storage 1707 and / or memory 1703 internal to data processing system 1700.

[0132] The methods as described herein can be implemented using dedicated hardware (e.g., using Field Programmable Gate Arrays, Digital Signal Processing chips, Application Specific Integrated Circuits, or Graphic Processing Units) or shared circuitry (e.g., microprocessors, microcontrollers, single-board computers, standalone computers, or cloud-based processors on remote servers) under control of program instructions stored in a machine-readable medium. The methods as described herein can also be implemented as computer instructions for execution on a data processing system, such as data processing system 1700 of FIG. 17.

[0133] FIG. 18 is a mechanical drawing of an optical module 1800 (also referred to as optical bench, optical subsystem, or optical layout) of a flow nanocytometry device in accordance with some embodiments of the present disclosure. This Computer-Aided Design (CAD) drawing illustrates many of the elements, modules, and subsystems described herein; some of the components, such as, e.g., the optical fibers delivering laser light to the rest of the module, and certain mechanical fasteners, have been omitted for clarity. In some embodiments, the optical subsystem of a flow cytometry apparatus described herein comprises mechanical and optomechanical supports such as, optionally and without limitation, rods, rod collars, rod adapters, cage plates, removable cage plates, cage mirror mounts, cage lens mounts, cage translation stages, cage rotation stages, cage brackets, removable cage brackets, cage reducers, cage adapters, cage lens tube adapters, lens tubes, lens tube couplers, lens tube reducers, lens tube caps, cage filter holders, kinematic cage filter holders, magnetic kinematic filter holders, kinematic cage beamsplitter cubes, magnetic kinematic cage beamsplitter cubes, longitudinal-translation rodmounted adjustments, micrometer actuators, fasteners, pillars, pillar posts, pedestals, posts, post holders, holding forks, bases, brackets, blocks, plates, clamps, custom mounts, custom plates, custom brackets, custom holders, custom housings, custom inserts, custom stages, custom blocks, custom spacers, custom clamps, custom fasteners, and other optomechanical components as are known in the art. In one embodiment, a cage plate or other rod-mounted cage element is allowed to translate along one or more cage rods in order to flexibly achieve longitudinal positioning of one or more optical elements attached to such a cage element. The use of, e.g., without limitation, a rod-and-cage architecture as illustrated in FIG. 18 (see, e.g., submodule 1850a and submodule 1850b) permits ease of adjustments during manufacturing to optimize performance of the device.

[0134] In the example illustrated in FIG. 18, five excitation light sources are provided. Light engine 1810a provides (e.g., by optical fiber, not shown) collinear light beams at two wavelengths (such as, e.g., without limitation, any two of approximately 266, 320, 355, 360, 375, 405, 460, 488, 514, 532, 561, 594, 615, 632, 638, 640, or 785 nm, or other wavelengths as are available in the art) to beam shaping assembly 1815a (as also described herein in reference to beam shaping elements 915 in FIG. 9A and beam shaping elements 1015 in FIGs. 10A-B); light engine 1810b provides (e.g., by optical fiber, not shown) collinear light beams at two other wavelengths (such as, e.g., without limitation, two different wavelengths from the list above for light engine 1810a) to beam shaping assembly 1815b (as also described herein in reference to beam shaping elements 915 in FIG. 9A and elements 1015 in FIGs. 10A-B); and laser module 1810c provides (e.g., in free space) light at yet another wavelength (such as such as, e.g., without limitation, yet a different wavelength from the list above for light engine 1810a) to beam shaping assembly 1815c (as also described herein in reference to beam shaping elements 915 in FIG. 9A and elements 1015 in FIGs. 10A-B). The three separate beams are combined by beam combiners (such as 920, as also described herein in reference to FIG. 9A) into a single, substantially collinear and collimated beam comprising the five separate wavelengths, and delivered to global focusing lens 1817, which may be translated with externally adjustable beam fine alignment module 1818 (both global focusing lens 1817 and externally adjustable beam fine alignment module 1818 as also described herein in reference to FIG. 11).

[0135] In the example illustrated in FIG. 18, a microscope module 1820 (as also described herein in reference to microscope module 995 in FIG. 9A) allows monitoring of the sample core stream (or absence thereof) in the flow cell (housed in flow cell assembly 1830). In some embodiments, microscope module 1820 includes an externally controllable focusing element (such as, without limitation, a liquid-lens element, a motorized stage, a motorized lens, an electrically controllable zoom, and other devices known in the art). By providing the ability to adjust the focus of the microscope module 1820 externally without needing to physically access the microscope module 1820 itself or the optional translation stage it may be mounted on, the externally controllable focusing element provides flexibility and ease of operation in fine-tuning, adjusting, modifying, or adapting the focus of the microscope to a sample core stream that may vary in size and / or position within the flow cell assembly 1830.

[0136] In the example illustrated in FIG. 18, one or more brackets or holders 1822 are provided to each hold an optical element (such as, without limitation, a polarizer or a filter) between the microscope module’s digital image sensor 1825 and the flow cell assembly 1830. Such optical elements may be used to reduce the amount of light of certain wavelengths or of certain polarizations that reach the microscope, resulting in an altered balance of the detected light. For example, a longpass filter may be used to reduce the amount of scattered laser light that reaches the microscope, while accepting a substantial portion or even most of fluorescent emissions from particles in a sample stream. The brackets or holders 1822 may be removable so that the respective optical elements may be placed in a position in the optical path between the flow cell assembly 1830 and the digital image sensor 1825, or in a position outside of such optical path.

[0137] In another embodiment, a longpass beamsplitter is provided between the flow cell assembly 1830 and the digital image sensor 1825 such that scattered light from particles in the sample stream is reflected by the beamsplitter to a side-scatter photodetector (such as, without limitation, a PMT, an SiPM, an APD, or a PD), optionally after passing through additional elements such as filters, lenses, and / or mirrors, and such that fluorescent light emitted from particles in the sample stream is transmitted through the beamsplitter to the microscope. This embodiment allows the collection of, e.g., without limitation, scattered light (at wavelengths such as, without limitation, 375 nm, 405 nm, 488 nm, or other wavelengths) that may otherwise be undesirably attenuated or reduced by passage through a collection lens assembly.

[0138] In the example illustrated in FIG. 18, a forward-scattering detection channel is provided in detection assembly 1840 (as also described herein in reference to FIG. 9A and FIG. 12). A light collection and spatial filter module is provided in subassembly 1850b (as also described herein in reference to FIGs. 9A and 13). Side scattering and fluorescence detection channels are provided in detection module 1860 (as also described herein in reference to FIGs. 9A-9C), including beamsplitters, spectral filters, lenses 1885, optional pinholes 1887, and detectors 1890. Beamsplitters incorporated in the apparatus (as also described herein in reference to FIGs. 9A-C, 14, and 15) may be housed in removable kinematic inserts 1870. This embodiment allows easy and quick removal of a beamsplitter and replacement of the beamsplitter with another, also mounted in a substantially similar insert. Likewise, spectral filters incorporated in the apparatus (as also described herein in reference to FIGs. 9A-9C, 14, and 15) may be housed in removable kinematic filter holders 1880. This embodiment allows quick removal of a filter and replacement of the filter with another, also mounted in a substantially similar filter holder. The kinematic design of such filter holders and beamsplitter inserts allows swapping of like holders and like inserts without the need to perform optical realignment, which represents an advantage of an apparatus of the present disclosure in terms of workflow, time, and convenience.

[0139] In some embodiments, it may be desirable to include means to remove a substantially unvarying background from a substantially time-varying signal. In some samples, desired scattered light signals from nanoparticles may be comparable to residual undesired scattered light background. Some components of this undesired scattered light background — including, without limitation, stray scattered light from interfaces (such as internal and external walls of the flow cell) not otherwise blocked by one or more spatial filters in the detection light path, and Rayleigh and / or Raman scattering from the fluid surrounding the particles under measurement — are present as substantially fixed or unvarying (i.e., as they are known in the art, “direct currenf’-like or DC) contributions to the detected signal, while the desired signals are transient or time-varying (i.e., as they are known in the art, “alternating currenf’-like or AC, even when they are not in the form of a regular sinusoidal fluctuation). The side scattering detection modules are particularly susceptible to this kind of background, as the background light in question (stray light and Rayleigh scattering) has the same wavelength as the desired signal light, and therefore cannot be filtered by optical spectral means. Fluorescence channels may also benefit from DC level removal since it may not be practical or possible to remove, e.g., undesired Raman scattering from the desired signal purely by optical spectral means. By substantially removing the fixed DC component of the detected signal, the signal-to-noise ratio of the resulting signal increases (i.e., improves). In addition, substantially removing the fixed DC component of the detected signal allows the remaining signal to be amplified (e.g., through detector gain, through amplifier gain, or through other stages of amplification) by larger factors, increasing (i.e., improving) detection sensitivity, resolution, and dynamic range.

[0140] In some embodiments, substantial removal of the DC component may be performed by one or more electronic components. In some embodiments, the electronic component may be incorporated into one or more detectors (e.g., detectors 170 in FIG. 1; 980x and 980y in FIGs. 9A- 9C; 1480 in FIG. 14; and 1580a-1580z in FIG. 15; and 1890 in FIG. 18): for example, the detector electronic circuit itself may provide an AC-coupled output with the DC component of the detected signal substantially removed. In some embodiments, the one or more detectors may be communicatively coupled to an electronic component that substantially removes the DC component of the detected signal (such as, e.g., an RC fdter, a high-pass filter, and other such filters as are known in the art). In some embodiments, an electronic component performing substantial removal of the DC component may be signal processing unit 190 (in FIG. 1; also 1490 in FIG. 14, 1590 in FIG. 15, and data processing system 1700 in FIG. 17). In some embodiments, the signal processing unit may include as an electronic component an amplifier with a DC offset, which offset may additionally be variable by either manual or automatic means. In some embodiments, the signal processing unit may include as an electronic component a signal digitizer (e.g., an analog- to-digital converter) with a DC offset, which offset may additionally be variable by either manual or automatic means.

[0141] FIGs. 19A-19B show two graphs of a flow nanocytometry analysis of reference nanoparticles and illustrate some embodiments of the present disclosure. The graphs in FIGs. 19A and 19B display the results of analysis of a mixture of silica nanoparticles such as may be used to characterize, benchmark, calibrate, and / or perform quality control on a flow nanocytometer according to some embodiments of the present disclosure. Each detected event from the nanoparticle sample is displayed as a larger black dot on the two-dimensional graph (also referred to as a “dot plot” in the art) of FIG. 19A. Events detected in a background run performed with the same operating parameters as the sample run are shown as smaller black dots in FIG. 19A. In the histogram of FIG. 19B, the thick black line represents gated (as further described herein) background- subtracted sample results. The three clusters 1922, 1932, and 1942 in FIG. 19A correspond, respectively, to subpopulations of silica nanospheres (Alpha Nanotech, Vancouver, BC, Canada) approximately 186 nm, 508 nm, and 1000 nm in diameter in the sample; the same three subpopulations are visible on the histogram in FIG. 19B as peaks 1925, 1935, and 1945 (again, respectively corresponding to 186 nm, 508 nm, and 1000 nm diameter nanoparticle subpopulations). The histogram in FIG. 19B plots event counts as a function of the intensity (or, as also known in the art, “peak height” or just “height”) of the detected side scattering “1” signal pulses (“SSC1 Height”). The dot plot in FIG. 19A plots individual events on a two-dimensional plane where the x axis is the same as the one used for the histogram in FIG. 19B (“SSC1 Height”). The two plots were scaled identically and aligned for ease of comparison. Because of such alignment, lines 1921, 1931, and 1941, common to both the dot plot in FIG. 19A and the histogram in FIG. 19B, illustrate as guides to the eye the correspondence, respectively, between the nanoparticle subpopulation clusters 1922, 1932, and 1942 and the subpopulation peaks 1925, 1935, and 1945. The graphs in FIGs. 19A-19B show how, in some embodiments of the present disclosure, nanoparticles spanning a wide range of sizes can be detected and characterized in a single sample, without using operational adjustments such as, without limitation, changing gain settings, changing amplifier settings, or changing detection threshold settings.

[0142] FIGs. 19A-19B also illustrate additional features of some embodiments of the present disclosure. The graph in FIG. 19B displays detected events on a two-dimensional plot of SSC1 signal pulse width (“SSC1 Width”) against SSC1 signal pulse height (“SSC1 Height”). In some embodiments of the flow nanocytometer according to the present disclosure, particle detection events occur substantially as pulses (e.g., without limitation, pulses of scattered light signals and / or pulses of fluorescence light signals), due to the transient nature of the detection process in flow cytometry and flow nanocytometry, whereby individual particles pass through the interrogation region as the fluid they are suspended in flows through the flow cell. These light pulses are then converted into electronic pulse signals (e.g., without limitation, by detectors 170 in FIG. 1; detectors 980x / y in FIGs. 9A-9C; detector 1248 in FIG. 12; detector 1480 in FIG. 14; detectors 1580a-1580z in FIG. 15; detectors 1890 in FIG. 18; and through method step 1695h in FIG. 16) and processed, e.g., by analog electronic means, by digital electronic means, or by both analog and digital electronic means (e.g., without limitation, by signal processing unit 190 in FIG. 1; 1490 in FIG. 14; 1590 in FIG. 15; data processing system 1700 in FIG. 17; and through process steps 1695h and 1695i in FIG. 16), to result in particle detection signals, also referred to in the art as “events,” that are stored (e.g., in data storage unit 192 in FIG. 1; and in one or more elements of data processing system 1700 in FIG. 17) and / or further processed and / or analyzed (e.g., without limitation, through process steps 1695i, 1695j, 1695k, and 1695m in FIG. 16 and / or using the method of FIG. 2). Signal pulses corresponding to passage of a particle under measurement through the interrogation region of the flow cell may generally be characterized in terms of their height (also known, e.g., as peak height, peak intensity, intensity, maximum), their width (e.g., the full width of the pulse at a level corresponding to half the maximum pulse height, or “Full Width Half Maximum”, or FWHM; the width corresponding to a number of standard deviations of the pulse, e.g., one, two, four, six, or another number of standard deviations; or other width metrics), and / or the area under the curve, or just “area” (defined by the area under the signal pulse over time over a defined background baseline). Conventional flow cytometric analysis is performed mainly on signal pulse heights and / or areas, as those values generally correlate substantially directly with, e.g., the size of the particle (for scattering signals) and, e.g., the level of expression of a fluorophore-labeled molecule of interest, such as, e.g., a surface protein, an antigen, a nucleic acid, or other entity (for fluorescence signals). In some embodiments of a flow nanocytometer according to the present disclosure, a width measure of particle detection event signals may be used to provide additional means of rejection of unwanted noise while preserving a substantial fraction of desired particle events. In FIG. 19A, detection events can be seen as clustering into several subpopulations, including those already described herein as corresponding to particles of size 186 nm (cluster 1922), 508 nm (cluster 1932), and 1000 nm (cluster 1942); in addition, a large cluster of events is visible (cluster 1905) that displays substantially lower SSC1 Width values than clusters 1922, 1932, and 1942. Events with such significantly lower measured signal widths may be classified as noise events, since for a given set of flow parameters, most particles traverse the interrogation region with substantially similar velocities and therefore produce signal pulses with substantially similar widths.

[0143] In some embodiments of a flow nanocytometer according to the present disclosure, detected events that satisfy certain requirements are preliminarily classified as possible particles of interest (“accepted”) and preserved for further processing, analysis, and / or storage steps; while detected events that do not satisfy such requirements are classified as noise or background (“rejected”) and are optionally discarded from further processing, analysis, and / or storage steps. In some embodiments, requirements for event acceptance are implemented in the form of thresholds: i.e., minimum values in one or more dimensions of the detection parameter space, above which events are considered valid and are accepted (or maximum values below which events are accepted. In some embodiments, requirements for event acceptance are implemented in the form of acceptance ranges, or what is known in the art as “gates:” i.e., ranges of values, in one or more dimensions of the detection parameter space, within which events are considered valid and are accepted, and outside of which events are considered noise and rejected. In FIG. 19A, one such gate 1910 is indicated as “Gate 1”; this gate defines a relatively wide range of acceptance in SSC1 Height measured values (approximately from 102to 3x10° SSC1 Height units) and a relatively narrow range of acceptance in SSC1 Width measured values (approximately from 195 to 335 SSC1 Width units). In this example, only events having both SSC1 Height values and SSC1 Width values falling within these respective ranges (including, e.g., events in clusters 1922, 1932, and 1942, corresponding to particle subpopulations of interest as described herein) are accepted for further processing, analysis, and / or storage; while events having either SSC1 Height values or SSC1 Width values falling outside these respective ranges (including, e.g., events in cluster 1905) are rejected as noise and optionally discarded.

[0144] In some embodiments of a flow nanocytometer according to the present disclosure, an acceptance gate is defined as a single range of values of a single detection parameter (e.g., without limitation, the range from 195 to 335 SSC1 Width units). In some embodiments, a gate is defined as a rejection gate including multiple ranges of values of a single detection parameter (e.g., the range below 102and above 3x10° SSC1 Height units). In some embodiments, an acceptance gate is defined as ranges of values for a plurality of detection parameters (e.g., as illustrated above in FIG. 19A for SSC1 Width and SSC1 Height). In some embodiments, an acceptance gate is defined as a parametric shape in a multidimensional detection space (e.g., values falling within a polygon, ellipse, or other arbitrary shape in a two-dimensional space comprising two measured detection parameters; values falling within a cube, sphere, ellipsoid, or other arbitrary shape in a three- dimensional space comprising three measured detection parameters; and similarly for higher dimensions). In some embodiments, multiple gates are defined in different dimensional detection spaces, and combined using logic operators (such as, without limitation, AND, OR, NOT, XOR, and other logic operators as are known in the art) to produce a set of criteria for event acceptance, rejection, or a combination thereof. It will be appreciated by those skilled in the art that a wide spectrum of criteria for acceptance, rejection, or both exists and may be implemented, e.g., without limitation, in logic, hardware, software, neural network, or other form, on any one detection parameter (such as, without limitation, signal height, signal width, signal area, signal skew, and other pulse metrics as are known in the art), or on multiple detection parameters, with criteria (gates) for each detection parameter optionally taking different forms.

[0145] In some embodiments, event acceptance and / or rejection are performed according to defined gates at one or more levels in the signal transduction path (e.g., at the detector level, amplifier level, analog signal processing level, digitizer level, digital signal processing level, FPGA level, GPU level, ASIC level, and / or CPU level). As illustrated in FIG. 19A, gate 1910 (“Gate 1”) contains 22.48% of the detected events; rejection of noise events outside of gate 1910 results therefore in the optional discarding of 77.52% of detected events. FIG. 19B shows the portion of detected events in gate 1910 — those preliminarily classified as particles of interest. In some embodiments, the discarding of rejected events may be performed automatically early on in the signal transduction path (e.g., at the digitizer level or earlier), allowing hardware and software signal processing resources to be allocated more efficiently primarily to signals likely to be dominated by events of interest. In the example of FIG. 19A, all events displayed (including those outside gate 1910) were recorded for analysis; in some embodiments, it may be desirable for events outside, e.g., gate 1910, or other gates as may be defined on SSC1 Width, SSC1 Height, other scattering detection parameters, fluorescence detection parameters, and / or combination thereof, to be discarded during the signal transduction process, so as to enable the freeing up of a substantial fraction of the events needing further processing and substantially more efficient signal processing (e.g., in the case of acceptance gate 1910 in FIG. 19A, an approximately fourfold increase in signal processing efficiency). The use of width gates (as exemplified, without limitation, by SSC1 Width in FIG. 19A; and extendable to any of the detection parameters implemented on a given apparatus) may be particularly effective in flow nanocytometry, due to the generally small size of particles of interest and the resulting relatively small scattering and fluorescence pulse height and area signals, to reduce the impact of background nanoparticles in the suspension or sheath fluid, of optical noise, of electronic noise, and / or of other sources of noise on the detected signals.

[0146] Working Examples

[0147] Working Example 1. FIGs. 21 A-21C show flow nanocytometry data from a normal sample and a PKD patient sample. Samples were diluted 1 : 105(FIG. 21 A), l : 104(FIG. 21B), and 1 : 103(FIG. 21C). As shown in FIG. 21C, at a 1 : 103dilution the PKD sample shows a higher proportion of EVs (2X more EVs) in the fluorescence (“FL2-h”) channel as compared to the normal sample. This example illustrates the importance of dilution and of fluorescence channels of the devices described herein in detecting EVs in a biological sample.

[0148] Working Example 2, FIGs. 22A-22B show flow nanocytometry data for urine samples stained with and without an R-phycoerythrin (R-PE)-labeled anti-PCl antibody (i.e., a fluorescently labeled marker coupled to PCI; “FL2 (PCI -PE)”) and a membrane marker CellBrite™ 650 (“FL3 (CellBrite 650)”). Analysis of freshly voided urine showed that the R-PE labeled anti-PCl mAb (7el2 IgG lK) could detect a population of PC1+bright PKD-EVs (event cluster in oval 2210), which EVs were additionally positive in membrane stain; and that there was also a population of PC I EVs that stained well with the membrane marker CellBrite™ 650 (CB650) but had no PCI staining (event cluster in oval 2220). This is to be expected as the PKD- EVs represent a subpopulation of all urinary EVs. Fresh early void urine was centrifuged at 2000g, 10 minutes, stained with R-PE anti-PCl (7el2) and CB650 to label all EVs. Y-axis log PCI and x- axis log membrane dye fluorescence. The anti-PCl mAb had some protein aggregates that could be removed with a fdter.

[0149] Working Example 3, FIGs. 23A-23B show flow nanocytometry data for samples stained with both a PCI stain (PCI -PE, PCI conjugated to phycoerythrin; shown in the right panels (“f!3 = h”) in each of FIGs. 23 A and 23B) and a PC2 stain (PC2-AF488, PC2 conjugated to AZ488; shown in the left panels (“f!2 - h”) in each of FIGs. 23 A and 23B). In each histogram, the raw data is represented by the thinnest line; the relevant background data is represented by a thicker black line; and the background- subtracted data is represented by a thick black line. All the events shown in FIGs. 23 A — 23B are gated (as illustrated herein, e.g., in reference to FIG. 19A) to remove a substantial fraction of background noise events, and they are further gated to show only doublepositive events-that is, events that indicate relatively elevated levels of staining in both PCI -PE and in PC2-AZ488. FIG. 23A shows the results for a healthy adult urine sample; FIG. 23B shows the results for LipolOO, a control material containing liposomes with an approximate diameter of 100 nm. These nanoparticles do not normally express either PCI or PC2, therefore they can be used as negative controls in assays involving detection of PCI and / or PC2. FIG. 23B shows substantially null signals (histogram curves 2330, 2340) for both PCI and PC2, showing that nonspecific binding was kept to a minimum in this assay. In contrast, FIG. 23A shows substantial levels of PC1+PC2+EVs (histogram curves 2310, 2320) as is normal for a healthy adult. The significance of these results is the ability of a flow nanocytometer in accordance with the present disclosure to resolve, detect, and count EVs expressing multiple PKD-associated proteins well in excess of background and with relatively high specificity.

[0150] Working Example 4, FIGs. 24A-24C show flow nanocytometry data for samples stained with both a PCI stain (PC1-PE, PCI conjugated to phycoerythrin) and a CD133 stain (CD133- AF488, CD133 conjugated to AlexaFluor 488), compensated for spectral spillover. In each histogram panel, the thin solid line (e.g., line 2410 in the top panel FIG. 24A, and corresponding lines in the other panels) represents the raw signal; the thin dashed line (e.g., line 2420) represents the relevant background for the respective sample (e.g., sheath fluid for liposomes; and stain-only control for stained urine samples); and the thin line (e.g., line 2430) represents the background- subtracted signal. In each of FIGs. 24A-24C, the histograms display events in a defined doublepositive gate for that sample: i.e., events where the detected PCI level and the detected CD133 level were both above respective background levels. In each of FIGs. 24A-24C, the top panel displays double-positive events on a histogram of PCI, and the bottom panel displays the same double-positive events on a histogram of CD133. FIG. 24A shows results for LipolOO, a control material containing liposomes with an approximate diameter of 100 nm. These nanoparticles do not normally express either PCI or CD 133, therefore they can be used as negative controls in assays involving detection of PCI and / or CD133. FIG. 24B shows results for a normal adult urine sample. In urine samples from healthy adults, EVs can normally be found expressing PCI. FIG. 24C shows results for a urine sample from an ADPKD patient (33 years old female, ThTKV of 649.7 ml / m, Mayo score of ID, indicating a relatively high severity of disease). In urine samples from ADPKD patients, the number of EVs expressing PCI, the level of PCI expression on EVs, or both, are reduced compared to corresponding levels in healthy adults. By counting only PC1+ / CD133+ double-positive EV events, this method provides a built-in control (since CD133 is a PKD- invariant protein). The total count of gated double-positive events is shown at the bottom of each of FIGs. 24A-24C. In FIG. 24A (negative control), the total double-positive count is 170, representing therefore the background level of this assay (e.g., due to nonspecific binding). In FIG. 24B (normal urine), the total double-positive count is 968, a number much higher than the background level of 170 — as is expected from a healthy donor. In FIG. 24C (PKD urine), the total double-positive count is 166, a number statistically indistinguishable from background — reflecting precisely the kind of reduction in PCI, compared to a healthy adult, expected for a patient with advanced PKD.

[0151] Prophetic Examples

[0152] Prophetic Example 5, We will obtain fresh, first void of the day urine, from 50 individuals with ADPKD of varying ages and Mayo scores age matched with 50 individuals with normal kidneys. All individuals will have an estimated glomerular filtration rate (eGFR) greater than about 60 mL / min / 1.73m2, racial composition may reflect the local population (18% black, 80% white and 2% others), APOL1 status will be noted as will age, sex, height, weight, BMI, spot BP, medications, smoking history (pack years if positive) and glycated hemoglobin. Each individual with ADPKD will be genotyped for their mutation and classified as PKD1, PKD2, or other. Mutations will be characterized as missense, in frame nonsense, frame shifting nonsense, and deletion. Individuals with ADPKD generally have a HtTKV and / or a Mayo score.

[0153] We have monoclonal antibodies that work using flow nanocytometry; e.g., without limitation, for PCI, PC2, EPCIP, and CD133 (see Table 1). These antibodies can be directly labeled with, e.g., without limitation, AlexaFluor 488, AlexaFluor 568, AlexaFluor 647, R-PE, and allophycocyanin (APC). A plurality of different antigens can be detected per PKD-EV, for example, PCI, PC2, EPCIP, CD133, and / or CEMIP2(TMEM2)). Table 1. Antibodies

[0154] We will also assess patients with different kidney diseases, 10 each of diabetic nephropathy, hypertensive kidney disease and glomerulopathy to ascertain whether these diseases generate false positives or otherwise confound the assay.

[0155] In some embodiments, a population of PCI ", PC2 , EPCIP PKD-EVs may be observed that are still CD133+, CEMIP2(TMEM2)+. Thus, this population of EVs may increase in number and percentage and correlate to increasing age, HtTKV, and Mayo score.

[0156] In some embodiments, when PCI and PC2 are involved in assembly of PKD-EVs, then a decrease in total number of PC1+, PC2+, EPCIP+, CD133+PKD-EVs per ml of urine may be observed. The number of PKD-EVs may decrease with increasing age, HtTKV, and / or Mayo score and thus may correlate with disease severity.

[0157] In some embodiments, the mean flow nanocytometry-detected intensity of PCI, PC2, EPCIP on CD133+EVs may decrease with time, HtTKV, and Mayo score, but the total number of EVs observed in the urine may remain the same. In some embodiments, the mean flow nanocytometry-detected intensity of CEMIP2(TMEM2) on CD133+EVs may increase with time. The ratio of PC 1 / CEMIP2(TMEM2) and PC2 / CEMIP2(TMEM2) may increase more than the ratio of PC1 / CD133 and PC2 / CD133 and correlate better with HtTKV and / or Mayo score.

[0158] Example Embodiments

[0159] Example 1. A flow nanocytometer configured to measure extracellular vesicles (EVs), comprising: a flow cell configured to receive a core stream therethrough, the core stream configured to carry at least one EV in a urine sample from a subject; at least one excitation source module configured to output a light beam to interrogate a portion of the core stream; a detection module configured to detect the light beam; and a processor communicatively coupled to a memory, the at least one excitation source, and the detection module, wherein the memory is configured to store instructions that when executed by the processor, cause the processor to perform a method comprising: receiving a plurality of electronic signals from the detection module; detecting an intensity of a polycystic kidney disease (PKD) associated protein expressed by the EVs based on the plurality of electronic signals; determining a quantity of the EVs in the urine sample based on the plurality of electronic signals; and determining a severity or a prognosis of polycystic kidney disease in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs in the urine sample.

[0160] Example 2. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 1, wherein the PKD associated protein is selected from the list consisting of: polycystin-1 (PCI), poly cystin-2 (PC2), fibrocystin (FC), exosomal polycystin-1 interacting protein (EPCIP), C16orf89, and CEMIP2(TMEM2).

[0161] Example 3. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 2, wherein the intensity of the PKD associated protein is one or both of: an intensity of PCI relative to an intensity of an invariant protein or an intensity of PC2 relative to an intensity of an invariant protein. Example 4. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 3, wherein the invariant protein is selected from the list consisting of: CD133, tetraspanin-4 (TSN3), tetraspanin-4 (TSN4), tetraspanin-7 (TSN7) and tetraspanin-8 (TSN8).

[0162] Example 5. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 2, wherein the determining the quantity further comprises determining one or both of: a quantity of PC1+CD133+ EVs or a quantity of PC2+CD133+ EVs.

[0163] Example 6. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 1, wherein the stored instructions further cause the processor to normalize the intensity of the PKD associated protein to an intensity of a co-expressed, invariant protein.

[0164] Example 7. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 1, wherein the EVs have a diameter of about 100 nm to 150 nm.

[0165] Example 8. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 1, further comprising a beam shaping element in a light path between the at least one excitation source module and the flow cell.

[0166] Example 9. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 1, wherein the detection module comprises at least a first side scatter detection submodule and at least a first fluorescence detection submodule.

[0167] Example 10. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 9, wherein the detection module further comprises a second side scatter detection submodule.

[0168] Example 11. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 9, wherein the detection module further comprises a second fluorescence detection submodule.

[0169] Example 12. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 9, further comprising: a light path between the flow cell and the detection module; and a light collection element in the light path between the flow cell and the detection module.

[0170] Example 13. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 12, wherein the light collection element comprises a high-numerical aperture collection lens. Example 14. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 12, further comprising a first spatial filter in the light path between the flow cell and the detection module.

[0171] Example 15. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 14, wherein the first spatial filter comprises an aperture.

[0172] Example 16. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 12, wherein the detection module further comprises a beamsplitter, a light path between the beamsplitter and the first side scatter detection submodule, and a light path between the beamsplitter and the first fluorescence detection submodule.

[0173] Example 17. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 16, wherein the detection module comprises a second spatial filter in the light path between the beamsplitter and the first side scatter detection submodule.

[0174] Example 18. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 17, wherein the second spatial filter comprises an aperture.

[0175] Example 19. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 9, further comprising an electronic component that substantially removes a direct current (DC) component from at least one of the plurality of electronic signals.

[0176] Example 20. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 9, wherein the method further comprises discarding a portion of the plurality of electronic signals from the detection module based on one or more gating criteria, one or more logic criteria, or both one or more gating criteria and one or more logic criteria.

[0177] Example 21. The flow nanocytometer of any one of the preceding embodiments, but particularly Example 20, wherein at least one of the gating criteria is a threshold pulse width of at least one of the plurality of electronic signals from the detection module.

[0178] Example 22. A computer-implemented method, performed by a processor communicatively coupled to a memory, for detecting extracellular vesicles (EVs) in a biological sample from a subject, comprising: receiving a plurality of electronic signals indicating light scattering properties of the EVs and fluorescence emissions of a marker coupled to at least one polycystic kidney disease (PKD) associated protein; detecting an intensity of the at least one PKD associated protein expressed by the EVs based on the plurality of electronic signals; determining a quantity of the EVs in the biological sample based on the plurality of electronic signals; and determining a severity or a prognosis of PKD in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs in the biological sample.

[0179] Example 23. The computer-implemented method of any one of the preceding embodiments, but particularly Example 22, wherein the at least one PKD associated protein is selected from the list consisting of: polycystin-1 (PCI), polycystin-2 (PC2), fibrocystin (FC), exosomal polycystin- 1 interacting protein (EPCIP), C16orf89, and CEMIP2(TMEM2).

[0180] Example 24. The computer-implemented method of any one of the preceding embodiments, but particularly Example 23, wherein the detecting the intensity of the at least one PKD associated protein comprises one or both of: detecting an intensity of PCI relative to an intensity of an invariant protein or detecting an intensity of PC2 relative to an intensity of an invariant protein.

[0181] Example 25. The computer-implemented method of any one of the preceding embodiments, but particularly Example 24, wherein the invariant protein is selected from the list consisting of: CD133, tetraspanin-4 (TSN3), tetraspanin-4 (TSN4), tetraspanin-7 (TSN7) and tetraspanin-8 (TSN8).

[0182] Example 26. The computer-implemented method of any one of the preceding embodiments, but particularly Example 23, wherein the determining the quantity further comprises determining one or both of: a quantity of PC1+CD133+ EVs or a quantity of PC2+CD133+ EVs.

[0183] Example 27. The computer-implemented method of any one of the preceding embodiments, but particularly Example 23, further comprising normalizing the intensity of the at least one PKD associated protein to an intensity of a co-expressed, invariant protein.

[0184] Example 28. The computer-implemented method of any one of the preceding embodiments, but particularly Example 23, wherein the EVs have a diameter of about 100 nm to 150 nm.

[0185] Example 29. The computer-implemented method of any one of the preceding embodiments, but particularly Example 23, wherein the computer-implemented method is performed on a flow nanocytometer comprising: a flow cell configured to receive a core stream therethrough, the core stream configured to carry at least one EV in the biological sample from the subject; at least one excitation source module configured to output a light beam to interrogate a portion of the core stream; a detection module configured to detect the light beam; and the processor is further communicatively coupled to the at least one excitation source, and the detection module. Example 30. The computer-implemented method of any one of the preceding embodiments, but particularly Example 29, wherein the flow nanocytometer further comprises a beam shaping element in a light path between the at least one excitation source module and the flow cell.

[0186] Example 31. The computer-implemented method of any one of the preceding embodiments, but particularly Example 29, wherein the flow nanocytometer further comprises: a light path between the flow cell and the detection module; and a light collection element in the light path between the flow cell and the detection module.

[0187] Example 32. The computer-implemented method of any one of the preceding embodiments, but particularly Example 31, where the light collection element comprises a high-numerical aperture collection lens.

[0188] Example 33. The computer-implemented method of any one of the preceding embodiments, but particularly Example 31, wherein the flow nanocytometer further comprises a first spatial filter in the light path between the flow cell and the detection module.

[0189] Example 34. The computer-implemented method of any one of the preceding embodiments, but particularly Example 33, where the first spatial filter comprises an aperture.

[0190] Example 35. The computer-implemented method of any one of the preceding embodiments, but particularly Example 29, wherein the detection module further comprises a beamsplitter, a first light path between the beamsplitter and a first side scatter detection submodule, and a second light path between the beamsplitter and a first fluorescence detection submodule.

[0191] Example 36. The computer-implemented method of any one of the preceding embodiments, but particularly Example 35, wherein the detection module comprises a second spatial filter in the first light path between the beamsplitter and the first side scatter detection submodule.

[0192] Example 37. The computer-implemented method of any one of the preceding embodiments, but particularly Example 36, where the second spatial filter comprises an aperture.

[0193] Example 38. The computer-implemented method of any one of the preceding embodiments, but particularly Example 23, wherein the plurality of electronic signals further indicates at least a first side scatter of the EVs and at least a first fluorescence emission.

[0194] Example 39. The computer-implemented method of any one of the preceding embodiments, but particularly Example 23, wherein the plurality of electronic signals further indicates a second side scatter of the EVs. Example 40. The computer-implemented method of any one of the preceding embodiments, but particularly Example 23, wherein the plurality of electronic signals further indicates a second fluorescence emission.

[0195] Example 41. The computer-implemented method of any one of the preceding embodiments, but particularly Example 29, further comprising substantially removing a direct current (DC) component from at least one of the plurality of electronic signals.

[0196] Example 42. The computer-implemented method of any one of the preceding embodiments, but particularly Example 29, further comprising discarding a portion of the plurality of electronic signals based on one or more gating criteria, one or more logic criteria, or both one or more gating criteria and one or more logic criteria.

[0197] Example 43. The computer-implemented method of any one of the preceding embodiments, but particularly Example 42, wherein at least one of the gating criteria is a threshold pulse width of at least one of the plurality of electronic signals from the detection module.

[0198] Example 44. A computer-readable medium comprising computer readable instructions that, when executed by a processor, cause the processor to execute a method comprising: receiving a plurality of electronic signals indicating light scattering properties of a plurality of extracellular vesicles (EVs) and fluorescence emissions of a marker coupled to at least one polycystic kidney disease (PKD) associated protein in a biological sample of a subject; detecting an intensity of the at least one PKD associated protein expressed by the plurality of EVs based on the plurality of electronic signals; determining a quantity of the plurality of EVs in the biological sample based on the plurality of electronic signals; and determining a severity or a prognosis of PKD in the subject, based on the intensity of the at least one PKD associated protein and the quantity of the plurality of EVs in the biological sample.

[0199] Example 45. The computer-readable medium of any one of the preceding embodiments, but particularly Example 44, wherein the at least one PKD associated protein is selected from the list consisting of: polycystin-1 (PCI), polycystin-2 (PC2), fibrocystin (FC), exosomal polycystin- 1 interacting protein (EPCIP), C16orf89, Clorf95, and CEMIP2(TMEM2).

[0200] Example 46. The computer-readable medium of any one of the preceding embodiments, but particularly Example 45, wherein the detecting the intensity of the at least one PKD associated protein comprises one or both of: detecting an intensity of PCI relative to an intensity of an invariant protein or detecting an intensity of PC2 relative to an intensity of an invariant protein. Example 47. The computer-readable medium of any one of the preceding embodiments, but particularly Example 46, wherein the invariant protein is selected from the list consisting of: CD133, tetraspanin-4 (TSN3), tetraspanin-4 (TSN4), tetraspanin-7 (TSN7) and tetraspanin-8 (TSN8).

[0201] Example 48. The computer-readable medium of any one of the preceding embodiments, but particularly Example 45, wherein the determining the quantity further comprises determining one or both of: a quantity of PC1+CD133+ EVs or a quantity of PC2+CD133+ EVs.

[0202] Example 49. The computer-readable medium of any one of the preceding embodiments, but particularly Example 46, further comprising normalizing the intensity of the at least one PKD associated protein to an intensity of a co-expressed, invariant protein.

[0203] Example 50. The computer-readable medium of any one of the preceding embodiments, but particularly Example 46, wherein the EVs have a diameter of about 100 nm to 150 nm.

[0204] Example 51. The computer-readable medium of any one of the preceding embodiments, but particularly Example 46, wherein the processor is communicatively coupled to a flow nanocytometer comprising: a flow cell configured to receive a core stream therethrough, the core stream configured to carry at least one EV in the biological sample from the subject; at least one excitation source module configured to output a light beam to interrogate a portion of the core stream; a detection module configured to detect the light beam; and the processor further communicatively coupled to a memory, the at least one excitation source, and the detection module.

[0205] Example 52. The computer-readable medium of any one of the preceding embodiments, but particularly Example 51, wherein the flow nanocytometer further comprises a beam shaping element in a light path between the at least one excitation source module and the flow cell.

[0206] Example 53. The computer-readable medium of any one of the preceding embodiments, but particularly Example 51, wherein the flow nanocytometer further comprises: a light path between the flow cell and the detection module; and a light collection element in the light path between the flow cell and the detection module.

[0207] Example 54. The computer-readable medium of any one of the preceding embodiments, but particularly Example 53, where the light collection element comprises a high-numerical aperture collection lens. Example 55. The computer-readable medium of any one of the preceding embodiments, but particularly Example 54, wherein the flow nanocytometer further comprises a first spatial filter in the light path between the flow cell and the detection module.

[0208] Example 56. The computer-readable medium of any one of the preceding embodiments, but particularly Example 55, where the first spatial filter comprises an aperture.

[0209] Example 57. The computer-readable medium of any one of the preceding embodiments, but particularly Example 51, wherein the detection module further comprises a beamsplitter, a first light path between the beamsplitter and a first side scatter detection submodule, and a second light path between the beamsplitter and a first fluorescence detection submodule.

[0210] Example 58. The computer-readable medium of any one of the preceding embodiments, but particularly Example 57, wherein the detection module comprises a second spatial filter in the first light path between the beamsplitter and the first side scatter detection submodule.

[0211] Example 59. The computer-readable medium of any one of the preceding embodiments, but particularly Example 58, where the second spatial filter comprises an aperture.

[0212] Example 60. The computer-readable medium of any one of the preceding embodiments, but particularly Example 44, wherein the plurality of electronic signals further indicates at least a first side scatter and at least a first fluorescence emission.

[0213] Example 61. The computer-readable medium of any one of the preceding embodiments, but particularly Example 44, wherein the plurality of electronic signals further indicates a second side scatter.

[0214] Example 62. The computer-readable medium of any one of the preceding embodiments, but particularly Example 44, wherein the plurality of electronic signals further indicates a second fluorescence emission.

[0215] Example 63. The computer-readable medium of any one of the preceding embodiments, but particularly Example 51, further comprising substantially removing a direct current (DC) component from at least one of the plurality of electronic signals.

[0216] Example 64. The computer-readable medium of any one of the preceding embodiments, but particularly Example 51, further comprising discarding a portion of the plurality of electronic signals based on one or more gating criteria, one or more logic criteria, or both one or more gating criteria and one or more logic criteria. Example 65. The computer-readable medium of any one of the preceding embodiments, but particularly Example 64, wherein at least one of the gating criteria is a threshold pulse width of at least one of the plurality of electronic signals from the detection module.

[0217] Example 66. A computer-implemented method, performed by a processor communicatively coupled to a memory, for detecting extracellular vesicles (EVs) in a biological sample from a subject, comprising: receiving a plurality of electronic signals indicating light scattering properties of the EVs; detecting an intensity of the at least one PKD associated protein expressed by the EVs based on the plurality of electronic signals; determining a quantity of the EVs in the biological sample based on the plurality of electronic signals; and determining a severity or a prognosis of PKD in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs in the biological sample.

[0218] Example 67. A computer-implemented method, performed by a processor communicatively coupled to a memory, for detecting extracellular vesicles (EVs) in a biological sample from a subject, comprising: receiving a plurality of electronic signals indicating fluorescence emissions of a marker coupled to at least one polycystic kidney disease (PKD) associated protein expressed by the EVs; detecting an intensity of the at least one PKD associated protein expressed by the EVs based on the plurality of electronic signals; determining a quantity of the EVs in the biological sample based on the plurality of electronic signals; and determining a severity or a prognosis of PKD in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs in the biological sample.

[0219] The systems and methods of the various embodiment and variations thereof can be embodied and / or implemented at least in part as a machine configured to receive a computer- readable medium storing computer-readable instructions. The instructions are executed by computer-executable components integrated with the system and one or more portions of the processor on the flow cytometer and / or computing device. The computer-readable medium can be stored on any suitable computer-readable media such as RAMs, ROMs, flash memory, EEPROMs, optical devices (e.g., CD or DVD), hard drives, floppy drives, or any suitable device. The computer-executable component is preferably a general or application-specific processor, but any suitable dedicated hardware or hardware / firmware combination can alternatively or additionally execute the instructions. References in the specification to “one embodiment,” “an embodiment,” “an illustrative embodiment,” “some embodiments,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0220] As used in the description and claims, the singular form “a”, “an” and “the” include both singular and plural references unless the context clearly dictates otherwise. For example, the term “treatment” may include, and is contemplated to include, a plurality of treatments. For example, the term “EV” may include, and is contemplated to include, a plurality of EVs. At times, the claims and disclosure may include terms such as “a plurality,” “one or more,” or “at least one;” however, the absence of such terms is not intended to mean, and should not be interpreted to mean, that a plurality is not conceived.

[0221] The term “about” or “approximately,” when used before a numerical designation or range (e.g., to define a length or pressure), indicates approximations which may vary by ( + ) or ( - ) 5%, 1% or 0.1%. All numerical ranges provided herein are inclusive of the stated start and end numbers. The term “substantially” indicates mostly (i.e., greater than 50%) or essentially all of a device, substance, or composition.

[0222] As used herein, the term “comprising” or “comprises” is intended to mean that the devices, systems, and methods include the recited elements, and may additionally include any other elements. “Consisting essentially of’ shall mean that the devices, systems, and methods include the recited elements and exclude other elements of essential significance to the combination for the stated purpose. Thus, a system or method consisting essentially of the elements as defined herein would not exclude other materials, features, or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. “Consisting of’ shall mean that the devices, systems, and methods include the recited elements and exclude anything more than a trivial or inconsequential element or step. Embodiments defined by each of these transitional terms are within the scope of this disclosure. The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. Other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is in fact disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMS1. A flow nanocytometer configured to measure extracellular vesicles (EVs), comprising: a flow cell configured to receive a core stream therethrough, the core stream configured to carry at least one EV in a urine sample from a subject; at least one excitation source module configured to output a light beam to interrogate a portion of the core stream; a detection module configured to detect the light beam; and a processor communicatively coupled to a memory, the at least one excitation source, and the detection module, wherein the memory is configured to store instructions that when executed by the processor, cause the processor to perform a method comprising: receiving a plurality of electronic signals from the detection module; detecting an intensity of a polycystic kidney disease (PKD) associated protein expressed by the EVs based on the plurality of electronic signals; determining a quantity of the EVs in the urine sample based on the plurality of electronic signals; and determining a severity or a prognosis of polycystic kidney disease in the subject, based on the intensity of the PKD associated protein and the quantity of the EVs in the urine sample.

2. The flow nanocytometer of claim 1 , wherein the PKD associated protein is selected from the list consisting of: polycystin-1 (PCI), polycystin-2 (PC2), fibrocystin (FC), exosomal polycystin- 1 interacting protein (EPCIP), C16orf89, and CEMIP2(TMEM2).

3. The flow nanocytometer of claim 2, wherein the intensity of the PKD associated protein is one or both of: an intensity of PCI relative to an intensity of an invariant protein or an intensity of PC2 relative to an intensity of an invariant protein.

4. The flow nanocytometer of claim 3, wherein the invariant protein is selected from the list consisting of: CD133, tetraspanin-4 (TSN3), tetraspanin-4 (TSN4), tetraspanin-7 (TSN7) and tetraspanin-8 (TSN8).

5. The flow nanocytometer of claim 2, wherein the determining the quantity further comprises determining one or both of: a quantity of PC1+CD133+ EVs or a quantity of PC2+CD133+ EVs.

6. The flow nanocytometer of claim 1 , wherein the stored instructions further cause the processor to normalize the intensity of the PKD associated protein to an intensity of a coexpressed, invariant protein.

7. The flow nanocytometer of claim 1, wherein the EVs have a diameter of about 100 nm to 150 nm.

8. The flow nanocytometer of claim 1, further comprising a beam shaping element in a light path between the at least one excitation source module and the flow cell.

9. The flow nanocytometer of claim 1, wherein the detection module comprises at least a first side scatter detection submodule and at least a first fluorescence detection submodule.

10. The flow nanocytometer of claim 9, wherein the detection module further comprises a second side scatter detection submodule.

11. The flow nanocytometer of claim 9, wherein the detection module further comprises a second fluorescence detection submodule.

12. The flow nanocytometer of claim 9, further comprising: a light path between the flow cell and the detection module; and a light collection element in the light path between the flow cell and the detection module.

13. The flow nanocytometer of claim 12, wherein the light collection element comprises a high-numerical aperture collection lens.

14. The flow nanocytometer of claim 12, further comprising a first spatial filter in the light path between the flow cell and the detection module.

15. The flow nanocytometer of claim 14, wherein the first spatial filter comprises an aperture.

16. The flow nanocytometer of claim 12, wherein the detection module further comprises a beamsplitter, a light path between the beamsplitter and the first side scatter detection submodule, and a light path between the beamsplitter and the first fluorescence detection submodule.

17. The flow nanocytometer of claim 16, wherein the detection module comprises a second spatial filter in the light path between the beamsplitter and the first side scatter detection submodule.

18. The flow nanocytometer of claim 17, wherein the second spatial filter comprises an aperture.

19. The flow nanocytometer of claim 9, further comprising an electronic component that substantially removes a direct current (DC) component from at least one of the plurality of electronic signals.

20. The flow nanocytometer of claim 9, wherein the method further comprises discarding a portion of the plurality of electronic signals from the detection module based on one or more gating criteria, one or more logic criteria, or both one or more gating criteria and one or more logic criteria.

21. The flow nanocytometer of claim 20, wherein at least one of the gating criteria is a threshold pulse width of at least one of the plurality of electronic signals from the detection module.

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