WO2018052730A1 - Procédés et dispositifs d'imagerie d'objets sur une puce microfluidique - Google Patents

Procédés et dispositifs d'imagerie d'objets sur une puce microfluidique Download PDF

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Publication number
WO2018052730A1
WO2018052730A1 PCT/US2017/049731 US2017049731W WO2018052730A1 WO 2018052730 A1 WO2018052730 A1 WO 2018052730A1 US 2017049731 W US2017049731 W US 2017049731W WO 2018052730 A1 WO2018052730 A1 WO 2018052730A1
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cells
interest
cell
filter
image
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PCT/US2017/049731
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English (en)
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Fanqing Chen
Tania Chakrabarty
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Unimed Biotech (Shanghai) Co., Ltd.
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Publication of WO2018052730A1 publication Critical patent/WO2018052730A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G01N15/0227—Investigating particle size or size distribution by optical means using imaging; using holography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/1452—Adjustment of focus; Alignment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493—Particle size

Definitions

  • the embodiments disclosed herein relate to methods and devices for cell isolation, detection, imaging, and extraction using microfluidics devices, such as microfluidic chips.
  • Isolation of cells of interest from cell samples containing both cells of interest and cells not of interest for non-invasive diagnosis presents various challenges.
  • isolating circulating fetal cells (CFC) from maternal blood containing other maternal and fetal cells not of interest for non-invasive prenatal diagnosis presents challenges due to the rarity of fetal red cells in the maternal blood.
  • CTC rare circulating tumor cells
  • various approaches have been attempted to extract and analyze cells of interest for downstream genomic analysis and diagnostic assays, but the success and purity of extraction has been very poor. Additionally, throughput of such detection and extraction systems remains low, presenting another challenge in the field of non-invasive testing.
  • Fluorescence activated cell sorting remains the most commonly used method for cell separation, collection and analysis but imaging cytometry-based FACS offers much more information by offering visualization of the sorted entities.
  • the Amnis® system is an example of ID imaging cytometry, in which each cell passing in a single file through a capillary is imaged with a high speed TDI camera.
  • ID imaging cytometry In addition to ID imaging cytometry, 2D imaging cytometry can image thousands of cells in one snap shot image onto a CCD/CMOS image sensor with better resolution and a less expensive camera, as opposed to cells flowed and imaged in a file as in the case of the Amnis® system.
  • some methods of imaging cytometry utilize cell samples plated or spread on a slide or plate for analyzing, isolating, and extracting cells for further analysis.
  • the spreading methods employed present challenges because cells often clump together in more than one layer and overlap with each other, making it very difficult to identify boundaries of each cell to determine if the cell is a cell of interest.
  • Imaging cytometry to date has mostly relied on high resolution imaging.
  • High resolution imaging is memory intensive and uses expensive equipment in a slow, labor intensive process, and further fails to account for precisely identifying individual cells in an efficient and highly accurate manner.
  • Imaging of cells spread onto microscope slides or imaged in wells also require memory intensive image stitching as backend processing.
  • Other imaging cytometry systems such as the Amnis® system can currently only accept cells in a tube and do not offer a flexible platform. It also lacks the ability to allow the user to collect the cells of interest.
  • the Amnis® system is further limited by the speed of the camera, so the processing speed is usually limited to less than 2000 cells/second.
  • methods and devices described herein simplify the search and identification of rare cells by partitioning the imaging area and yet delivering the multi-color high resolution microscopic images that are required for the purposes of rare cell analysis.
  • methods and devices described herein can also capture, identify, and collect other objects of interest, such as microbeads.
  • high resolution imaging as described herein is less memory intensive because it does not require stitching as it matches the imaging area to the field of view of the low magnification of the scanning objective lens. This makes the described process fast and yet precise to identify individual cells in an efficient and highly accurate manner. Using grid based, partitioned imaging areas and having a sense of the localization of an object, the scanning process no longer requires high resolution cameras which reduces the cost for the instrument and scanning and imaging system.
  • detecting can include imaging cells captured in a filter or a plurality of filters on the microfluidic chip.
  • Imaging cells captured in the filters of the microfluidic chip can include an initial low resolution imaging procedure to determine which of the captured cells are potentially or likely to be cells of interest (using fluorescent labels or physical parameters as the initial screening criteria), and a second higher resolution imaging procedure to confirm the potential cells of interest are in fact cells of interest (using more detailed high resolution fluorescent image and morphology information as the determining criteria).
  • Embodiments of the initial imaging procedure can include imaging cells captured on a filter of the microfluidic chip using a low magnification objective of a microscope platform (also referred to herein as a microscope system), for example, a 5x or lOx objective, and determining which of the captured cells are likely cells of interest using user-defined criteria.
  • the user-defined criteria include the size and shape of the captured cell.
  • the user-defined criteria includes fluorescently-tagged characteristics of the captured cell.
  • Embodiments of the second imaging procedure can include imaging the likely cells of interest using a high magnification objective (e.g., 20x, 40x, 60x, or lOOx objective) of a microscope platform, and determining which of the likely cells of interest are confirmed cells of interest.
  • the initial imaging scan is performed by an imaging device with a field-of-view ("FOV") that matches the size and shape of one filter of a plurality of filters in the microfluidic chip.
  • FOV field-of-view
  • One innovation includes a method of controlling a microscope platform for determining cells of interest in a sample containing a plurality of cells, each cell being disposed in a through hole on one of a plurality of filters on a microfluidic chip, each of the plurality of filters having an active area including a plurality of through holes arranged in a pattern.
  • Various embodiments of such a method may include one or more of the features, or other features, disclosed herein.
  • the method includes generating, using an imaging device, one or more first images of the plurality of filters on the microfluidic chip, each of the first images having a first resolution and each of the one or more first images depicting at least a portion of a filter of the microfluidic chip.
  • the method may also include determining using the one or more first images, with an electronic hardware processor, location information for candidate cells of interest that are disposed in one of the through holes, the determined location information including information indicating a particular filter of the plurality of filters and information indicating the location of the through hole of the particular filter where each candidate cell of interest is located.
  • the method may also include storing the location information in a memory device.
  • the method may further include aligning the microscope platform to capture an image of each candidate cell of interest, wherein aligning the microscope platform includes using the stored location information to align the microscope platform to capture an image at the location of the through hole where the candidate cell of interest is disposed.
  • the method may also include generating, with the imaging device of the microscope platform, a second image depicting each candidate cell of interest, the second image having a resolution higher than the first resolution.
  • Some methods further include determining cells of interest in the second images of the candidate cells of interest.
  • generating the one or more first images comprises setting a field-of-view (FOV) of the imaging device to substantially match the shape and size of the active area of one of the plurality of filters.
  • dimensions of an active area of one of the plurality of filters is approximately 0.9 mm x 0.9 mm.
  • the location information comprises an X, Y coordinate indicting the location of the through hole on the particular filter where each candidate cell of interest is located.
  • storing the location information comprises storing the location information in a look-up table.
  • Such methods may also include, prior to generating the plurality of first images, the methods includes applying a stain to the plurality of cells, the stain including a biomarker that indicates fetal cells; and illuminating the plurality of cells with a light source that excites a stained cell, and where determining location information for candidate cells of interest in the one or more first images includes determining one or more candidate cells of interest in one of the one or more first images that are fluorescing at a particular wavelength corresponding to the stain. Some methods further include aligning the plurality of filters on the microfluidic chip with an imaging grid.
  • Such methods may include staining the plurality of cells with one or more stains, illuminating the plurality of cells with at least one illumination source that excites each of the one or more stains; and generating a first image of each of the plurality of filters at the first resolution while said illuminating the plurality of cells.
  • Some methods further comprise for each of the one or more first images, cross-referencing the determined cells of interest with at least one of the other first images of the same filter to determine candidate cells of interest.
  • illuminating the plurality of cells includes using a fast filter wheel configured to pass a portion of wavelengths of light to illuminate the plurality of cells.
  • Some methods further include using user-defined criteria to determine candidate cells of interest and/or cells of interest.
  • user-defined criteria may include one or more of a size of a cell, a florescence intensity level, and/or the absence or presence of nuclear stains, biomarkers, and/or fluorescent dyes.
  • the criteria of cell size includes cells that are less than 30 ⁇ in size.
  • the criteria of cell size includes cells that are from 5 ⁇ to 12 ⁇ in size..
  • the criteria of cell size includes cells that are less than 5 ⁇ in size.
  • the user-defined criteria includes cells that have greater than 100 counts in the channel for FITC fluorescence and the channel for DAPI fluorescence. In some embodiments of such methods, the user-defined criteria further includes cells that have less than 10 counts in the channel for PE fluorescence. Some methods further comprise auto- focusing the microscope imaging system while generating the first images of the plurality of filters.
  • Another innovation is a microscope system for determining cells of interest in a sample containing a plurality of cells, each cell being disposed in a hole of one of a plurality of filters arranged in an order on a microfluidic chip, each of the plurality of filters having an active area including a plurality of holes arranged in a pattern.
  • the microscope system can include an optical system configured for generating one or more first images at a first resolution and one or more second image at a second resolution, the second resolution being higher than the first resolution and the one or more second images each including a portion of one of the one or more first image, at least one electronic hardware processor coupled to an electronic memory component.
  • the at least one electronic hardware processor may be configured to determine candidate cells of interest in the one or more first images, store location information for each determined candidate cell of interest, the location information identifying one of the plurality of filters on which each candidate cell of interest is located and identifying the location of a corresponding hole, of the identified filter, in which each candidate cell of interest is within, for each candidate cell of interest: control the movement of at least a portion of the microscope system to align the optical system, for generating the one or more second images, at a position identified by the stored location information corresponding to a candidate cell of interest, generate the one or more second images, and determine cells of interest in the second images.
  • the at least one electronic hardware processor may also be configured to determine cells of interest in the second images of the candidate cells of interest.
  • the at least one electronic hardware processor may be further configured to set a field-of-view (FOV) of the imaging device to substantially match the shape and size of the active area of one of the plurality of filters.
  • the location information can include information representative of a coordinate (e.g., an X,Y coordinate) indicting the location of the through hole on the particular filter where each candidate cell of interest is located.
  • the system may further include an illumination system for illuminating the plurality of cells with at least one illumination source that excites a stain applied to the plurality of cells, wherein the one or more first images are generated while illuminating the plurality of cells.
  • the illumination system includes a fast filter wheel configured to pass a portion of wavelengths of light to illuminate the plurality of cells.
  • Another innovation includes a method of controlling a microscope system for determining cells of interest in a sample containing a plurality of cells, each cell being disposed in a through hole on one of a plurality of filters on a microfluidic chip, each of the plurality of filters having an active area including a plurality of through holes arranged in a pattern.
  • the method may include generating, using an imaging device, a first image of the plurality of filters on the microfluidic chip, determining from the first image, with one or more electronic hardware processors, location information for candidate cells of interest that are disposed in one of the through holes in the first image, the determined location information indicating the location of a through hole where a candidate cell of interest is located, aligning the microscope system to capture a second image of determined candidate cells of interest using the stored location information, generating, with the imaging device, a second image depicting each candidate cell of interest, the second image having a resolution higher than the first resolution, and determining cells of interest in the second image.
  • Such methods may include other aspects.
  • such methods further include prior to generating the of first image, applying a stain to the plurality of cells, the stain including a biomarker that indicates a fetal cell, and illuminating the plurality of cells with a light source that excites a stained fetal cell, where determining location information for candidate cells of interest comprises determining cells that are fluorescing at a particular wavelength corresponding to the stain.
  • the method includes using by the one or more electronic hardware processors, criteria to determine candidate cells of interest or cells of interest.
  • the criteria may be, for example, cell size, a florescence intensity level, and/or the absence or presence of nuclear stains, biomarkers, and/or fluorescent dyes.
  • the criteria may indicate a threshold of the size of the cells, for example a cell size less than 30 ⁇ in size, a cell size from 5 ⁇ to 12 ⁇ in size, or a cell size less than 5 ⁇ in size.
  • Another innovation includes a non-transitory computer-readable medium storing instructions that, when executed by one or more computing devices, perform a method for controlling a microscope system to determine cells of interest in a sample containing a plurality of cells, each cell being disposed in a through hole on one of a plurality of filters on a microfluidic chip, each of the plurality of filters having an active area including a plurality of through holes arranged in a pattern, where the method includes generating, using an imaging device, one or more first images of the plurality of filters on the microfluidic chip, each of the first images having a first resolution and each of the one or more first images depicting at least a portion of a filter of the microfluidic chip, determining using the one or more first images, with an electronic hardware processor, location information for candidate cells of interest that are disposed in one of the through holes, the determined location information including information indicating a particular filter of the plurality of filters and information indicating the location of the through hole of the particular filter where each candidate cell of interest
  • Another innovation includes a microscope system for determining cells of interest in a sample containing a plurality of cells, each cell being disposed in a hole of one of a plurality of filters arranged in an order on a microfluidic chip, each of the plurality of filters having an active area including a plurality of holes arranged in a pattern, the microscope system including means for generating one or more first images of the plurality of filters on the microfluidic chip, each of the first images having a first resolution and each of the one or more first images depicting at least a portion of a filter of the microfluidic chip, determining, using the one or more first images, location information for candidate cells of interest that are disposed in one of the through holes, the determined location information including information indicating a particular filter of the plurality of filters and information indicating the location of the through hole of the particular filter where each candidate cell of interest is located, means for storing the location information, means for aligning the microscope system to capture an image of each candidate cell of interest using the stored location information, and means
  • Methods and devices described herein are broadly applicable to the capture and study of objects of interest from a sample, such as cells, beads, microbeads, and other particles that are subject to filtration.
  • Figure 1 illustrates one embodiment of a filter for capturing and positioning objects of interest, such as cells of interest, according to the present disclosure.
  • Figure 2 illustrates an embodiment of a microfluidic chip including a plurality of filters such as that illustrated in Figure 1.
  • Figure 3 is an example flow diagram illustrating one method for obtaining cells of interest from a sample according to the present disclosure.
  • Figure 4 is an example flow diagram illustrating one method for analyzing cells of interest according to the present disclosure.
  • Figure 5 illustrates an example imaging grid and an example microfluidic chip according to the present disclosure.
  • Figure 6 illustrates an example scan pattern for imaging a microfluidic chip according to the present disclosure.
  • Figure 7 illustrates an example look up table generated in response to or during an initial imaging procedure according to the present disclosure.
  • Figures 8A - 8C illustrate images taken during an example method for obtaining and analyzing cells of interest according to the present disclosure.
  • Figures 9A and 9B illustrate images taken during another example method for obtaining and analyzing cells of interest according to the present disclosure.
  • Figure 10 illustrates an embodiment of a system 1000 for analyzing cells of interest captured in a microfluidic device according to the present disclosure.
  • Figure 11 is an example flow diagram illustrating a method of controlling a microscope platform for determining cells of interest in a sample containing a plurality of cells, each cell being disposed in a through hole on one of a plurality of filters on a microfluidic chip, each of the plurality of filters having an active area including a plurality of through holes arranged in a pattern.
  • Embodiments of methods and devices disclosed herein can obtain cells of interest using morphology-based isolation combined with affinity and/or biomarker-based detection and identification. By combining these processes on an integrated microfluidic device, such as a microfluidic chip, the methods and devices disclosed herein resolve challenges associated with isolating specific cells of interest from a sample of cells. Unlike fluorescence-activated cell sorting ("FACS") utilized in flow cytometry, embodiments disclosed herein are visualization-based methods similar to imaging cytometry that are performed on a microscope platform (or microscope system), but advantageously address drawbacks associated with prior imaging cytometry-based systems and methods. Methods described herein can be partially or fully automated which adds another benefit to embodiments described in the current disclosure.
  • FACS fluorescence-activated cell sorting
  • Cytometry is the measurement and/or identification of cell characteristics. Cytometry methodologies are configured to measure any of a number of parameters, including for example cell size, cell count, cell shape and structure, cell cycle phase, DNA content, and the existence or absence of specific proteins on the cell surface or within the cell. There are many applications in which the different cytometry methods can be used. For example, cytometry can be used in characterizing and counting blood cells in a sample of blood, cell biology research, and medical diagnostics to characterize cells in diseases and disorders (e.g., cancer and AIDS). Imaging cytometry is a type of cytometry that operates by imaging single cells or a large number of cells using optical microscopy.
  • imaging cytometry provides information on localization of fluorescence markers within an analyzed cell.
  • cells Prior to analysis, cells can be stained to enhance contrast or detect specific molecules by labeling these with nuclear stains, cytosolic or membrane based biomarkers, and/or fluorescent dyes.
  • Methods and devices disclosed herein advantageously use a filter device integrated on a microfluidic device, such as a microfluidic chip.
  • the filter device can be a "lattice,” a “filter platform,” or a “filter grid,” but the feature will be referred to as "a filter” throughout this disclosure.
  • the filter is used at an early step of the process described herein to isolate cells of interest from a sample containing cells of interest. Isolating cells of interest can include capturing a cell in a filter while simultaneously positioning the cell in a distinct, precisely-defined location of the filter that is spatially separated from other distinct, precisely-defined locations of the filter.
  • the sample may contain non-cellular matter and/or cells that are not of interest, in addition to cells of interest.
  • Embodiments of the filters described herein allow for enrichment of the cells of interest, such that the cells of interest may be isolated from samples containing numerous cells, at least some of which may be cells not of interest.
  • the filter is also used at later stages of the process to detect the precise location of cells that have been captured in the filter, assess the characteristics of captured cells to determine if they are cells of interest, and harvest or pluck cells that have been determined to be of interest for downstream analysis, such as genetic and/or diagnostic analysis.
  • the filter may comprise multiple through holes specifically shaped and dimensioned to capture cells of interest while permitting cells not of interest to pass through the through holes in the filter and thereby remain uncaptured.
  • the size and shape of the holes on the filter can be adjusted for the particular size and shape of the cell of interest to be captured, for example the holes can have a cross-section shape that is square, circular, oval, rectangular, or another suitable shape.
  • these features on the filter that capture some cells while allowing other cells to pass through the filter can be referred to as pores, wells, recesses, filter holes, through holes, or other terms, these features will be referred to as "through holes" throughout this disclosure.
  • the filter does not include through holes, but includes features that capture and position cells on the filter in a precisely-defined, repeating pattern of shallow wells.
  • Methods and devices disclosed herein may be used for fetal cell isolation from maternal blood samples for non-invasive prenatal diagnosis.
  • methods and devices disclosed herein isolate and analyze such cells for downstream genetic analysis and diagnostic assays.
  • the filter is a morphology-based selection filter that separates cells of interest (such as fetal nucleated red blood cells (“fnRBCs”)) based on two criteria: morphology and biomarkers specific to the cells of interest.
  • fnRBCs fetal nucleated red blood cells
  • Embodiments of filters described herein can separate, or filter, fetal nucleated red blood cells (fnRBCs) from a maternal blood sample containing mature (non-nucleated) maternal RBCs and fetal nucleated RBCs.
  • Fetal nucleated RBCs circulating in the maternal blood are extremely rare, with some estimates as low as 1-10 in a billion.
  • Mature human RBCs are oval biconcave disks and generally lack a cell nucleus.
  • fetal nucleated RBCs are slightly larger than mature maternal RBCs and generally spherical rather than disk-shaped.
  • Embodiments of the morphology-based selection filters described herein include through holes with a specific shape, size, and arrangement such that most or all of the mature red blood cells (RBCs) in a sample pass through the through holes in the filter while some, most, or all of the fetal nucleated RBCs are retained or "captured" in the through holes.
  • RBCs mature red blood cells
  • Embodiments of filters described herein can advantageously be used to distinguish captured cells of interest from captured cells that are not of interest based on a second criterion: biomarkers specific to the cells of interest (in this non-limiting example, fetal nucleated RBCs).
  • the cells can be stained and/or labeled with nuclear stain, specific biomarkers, and/or fluorescent dyes for positive or negative selection of a subset of the captured cells (for example, positive selection of captured fetal nucleated RBCs and negative selection of captured cells that are not fetal nucleated RBCs).
  • embodiments of microfluidic chips including filters described herein represent a capture platform having a specifically arranged, predetermined, and repeating grid-like pattern of through holes.
  • the capture platform such as a microfluidic chip
  • the location of each filter in the capture platform can be precisely determined based on registration marks on the filter or on the microfluidic chip. Additionally, the precise location of each through hole in each filter can be very accurately determined, down to the submicron range in some aspects.
  • methods and devices disclosed herein simultaneously (1) filter cells of interest from a population of cells in a sample, (2) retain or "capture” the filtered cells of interest in a filter, and (3) position the captured cells of interest in distinct, precisely-defined, and easily-mapped locations in the capture platform.
  • One location can correspond to one through hole in a plurality of through holes regularly arranged in a gird-like pattern of the capture platform.
  • Embodiments of the methods and devices described herein can then advantageously develop a representation (for example, obtain an image or take a picture) of all of the captured cells in a specific area of interest in a single image.
  • the specific area of interest is one filter of a plurality of filters arranged in the capture platform.
  • the specific area of interest is one filter arranged in a capture platform including one single filter. Due to the precisely-defined and repeating grid pattern of through holes in the filters, the exact position of each captured cell can be identified using the unique position of its corresponding through hole in the filter.
  • Capturing and simultaneously positioning of cells of interest in this way allows the cells of interest to be analyzed in two steps: a first quick imaging scan of all of the captured cells on the filter using, for example, a low magnification objective, followed by higher resolution imaging analysis of a subset of cells that were identified during the quick scan as likely "cells of interest” using, for example, a high magnification objective.
  • This two-step imaging analysis allows for very high throughput because high resolution imaging resources are expended on cells which are likely to be "cells of interest," that is, cells that have been initially captured in a through hole as a result of their morphology and subsequently identified as having one or more biomarkers of interest during a quick imaging scan at low resolution.
  • captured cells with those characteristics may be readily identified and their position can be easily returned to for subsequent, more detailed analysis of a captured cell or for manipulation or extraction of a captured cell.
  • embodiments of filters described herein capture and position cells in a way that increases the speed at which the captured cells can be analyzed and harvested.
  • information about the position or location of a cell in the filter (for example, a precise x, y coordinate on the filter) that is obtained during the quick scan at low resolution is later used again to (1) direct high resolution imaging resources to that specific x, y coordinate to confirm the captured cell is actually a cell of interest, and (2) direct cell harvesting or plucking devices to that specific x, y coordinate to harvest the cell after it has been confirmed as a cell of interest.
  • filters for capturing and positioning cells described herein can increase the speed at which cells of interest are removed from the filter, or "harvested,” for downstream testing.
  • One non-limiting advantage of methods and devices disclosed herein includes, but is not limited to, the ability to automatically create a monolayer of cells as a stained sample flows over a microfluidic chip, which is not possible in imaging cytometry methods that spread or plate the sample on a slide.
  • Embodiments of microfluidic chips described herein may be configured to create a monolayer of cells by preventing one potential cell of interest of the sample from sitting or lying on top of another potential cell of interest, which can cause imaging and resolution complications due to the imaging procedure needing to distinguish the two closely-spaced cells from one another.
  • Implementations of the microfluidic chips disclosed herein can contain a single layer of through holes, where each through hole is spatially separated from other through holes and configured to capture only a single cell.
  • each through hole that captures a cell contains a single, isolated cell whose characteristics (such as size, morphology, biomarkers) can be readily distinguished because the cell is spatially separated from other cells, and is at an distinct, precisely-defined location for further analysis. Additionally, embodiments of methods and devices herein can advantageously maximize the density of cells of interest on a single microfluidic chip.
  • Methods and devices disclosed herein can be configured for automated scanning and imaging of stained samples to improve efficiency and decrease time associated with imaging cytometry methods. Methods and devices disclosed herein can quickly and efficiently scan captured cells at a low magnification objective; generate a look-up table containing information about captured cells based on user-defined criteria, including a precise position for each captured cell in a filter of a microfluidic chip; return to the precise location to perform verification that the captured cell is a cell of interest; and manipulate the verified cells of interest for downstream genetic or diagnostic analysis.
  • Methods and devices disclosed herein may also permit: 1) cell filtration, staining, enrichment (if needed) on one integrated platform minimizing manual labor and intervention; and 2) use of automation to streamline processing of cell samples.
  • Methods and devices disclosed herein may be described in reference to an exemplary, non-limiting application of cell-based non-invasive prenatal testing (cbNIPT) and the isolation of fetal nucleated RBCs from maternal RBCs and isolation of fetal trophoblasts.
  • cbNIPT cell-based non-invasive prenatal testing
  • CTC circulating tumor cells
  • the skilled artisan will understand, however, that the principles and concepts of the methods and devices are broadly applicable to the capture and study of objects of interest from a sample, such as cells, beads, microbeads, and other particles that are subject to filtration with or without fluorescence- based staining.
  • methods and devices disclosed herein can be configured for the isolation of microbeads, tumor cells for oncology, or any other pathological condition where cells of one kind can be differentiated from cells of another kind based on size, morphology, nuclear staining, and/or biomarker identification.
  • Methods and devices disclosed herein allow cell capture and analysis on a single platform, thereby minimizing manual labor and intervention, and use of automation to streamline processing of cell samples. Methods and devices described herein are also compatible with multi-spectral image analysis.
  • a filter includes one or more filters.
  • microfluidic device or “microfluidic chip” generally refers to a device through which materials, particularly fluid borne materials, such as liquids, can be transported, in some embodiments on a micro-scale.
  • microfluidic chips described herein can include microscale features, nanoscale features, and combinations thereof.
  • the samples delivered on such a device may be fluids alone or fluids with suspended components such as cells and particles.
  • An exemplary microfluidic chip can include structural or functional features dimensioned on the order of a millimeter-scale or less, which are capable of manipulating a fluid at a flow rate on the order of 5 mL/min or less.
  • Examples of microfluidic chips described herein can be 5 millimeters x 5 millimeters in size.
  • a microfluidic chip includes a plurality of filters arranged in a grid-like pattern. Examples of filters described herein can be approximately 0.9 millimeters by 0.9 millimeters in size. Other sizes are possible.
  • a microfluidic chip includes 25 filters arranged in a grid-like pattern.
  • a microfluidic chip includes additional features such as, but not limited to channels, fluid reservoirs, reaction chambers, mixing chambers, and separation regions.
  • a channel includes at least one cross-sectional dimension that is in a range of from about 0.1 ⁇ to about 10 millimeters.
  • a microfluidic chip can exist alone or can be a part of a microfluidic system which, for example and without limitation, can include: pumps and valves for introducing fluids, e.g., samples, reagents, buffers and the like, into the system and/or through the system; detection equipment or systems; data storage systems; and control systems for controlling fluid transport and/or direction within the device, monitoring and controlling environmental conditions to which fluids in the device are subjected, e.g., temperature, current, and the like using sensors where applicable.
  • the valves in such systems may be pressure or vacuum driven.
  • a sample is subjected to one or more steps that enrich cells of interest relative to the total components of the sample and/or enrich the cells of interest relative to the total cells in the sample.
  • the cell of interest is a fetal nucleated RBC
  • the maternal sample can be used as is, pre-diluted in desirable buffer or diluted on chip if required prior to applying it to a filter.
  • a microfluidic chip can be made from any suitable materials, such as PDMS (Polydimethylsiloxane), glass, silicon, metal, ceramics, paper, PMMA (polymethylmethacrylate), PET (polyethylene terephthalate), PC (Polycarbonate), etc., or a combination thereof.
  • the filter integrated in the chip may be made from similar materials or different materials.
  • filters described herein are made from silicon oxynitride, such as but not limited to SiON or Si0 2 .
  • the term "filter” refers to a material that separates objects of interest from other objects that are not of interest.
  • a filter separates objects of interest by retaining objects of interest in through holes in the filter, while objects that are not of interest pass through the through holes in the filter.
  • the objects of interest can be, but are not limited to, cells, beads, or microbeads.
  • Embodiments of filters described herein can include a single-layer of material, or include multiple layers, such as two, three, or more layers.
  • the term "through hole” refers to an opening or recess extending through a structure, such as a filter.
  • the structure included a first side and a second side, and a through hole extends entirely through the structure between the first and the second side.
  • Through holes allow objects to translocate through the substrate.
  • through holes can allow an object initially present on one side of the structure to translocate through the structure to a region on the opposing side of the structure.
  • through holes do not allow an object to pass through the structure, and retain the object on one side of the structure.
  • Objects retained in a through hole can be disposed partially or entirely within a through hole.
  • Through holes described herein can be specifically shaped and dimensioned to separate objects of interest from other objects that are not of interest.
  • Through holes may also be referred to as pores, wells, filter holes, or other terms representing an opening in the filter, however, these features will be referred to as "through holes" throughout this disclosure.
  • the through holes facilitate the separation of objects from objects not of interest.
  • the through holes can be designed to have specific dimensions corresponding to the shape and size of the objects of interest. In this way, single instances of objects of interest (e.g., a single cell) can be captured in a through hole, while permitting objects not of interest to either be passed entirely through the through hole or inhibited from entering (or being retained within) the through hole.
  • objects of interest can be, but are not limited to, cells, beads, or microbeads.
  • Through holes may be designed in any shape or size, for example circular, rectangular, oval, etc. The shape and size of each through hole may be determined based on the objects of interest being captured by the filter.
  • Cells captured in through holes may be referred to as "potential cells of interest” or “candidate cells of interest” which may indicate cells for further examination. For example, in methods of scanning and imaging filters containing cells using a microscope platform, a first image may be generated where candidate cells of interest are identified and location information of the candidate cells of interest are stored. Subsequently, a second image of each identified candidate cell of interest is generated at a higher resolution.
  • the stored location information can be used as control information to guide the microscope platform (or the imaging system) to a location to generate a second image of the candidate cell of interest, and the second (higher resolution) image can be used for further image evaluation of the candidate cells of interest to determine cells of interest.
  • microscope platform refers to a system and/or device configured to perform scanning and/or imaging of cells.
  • a microscope platform includes an epifluorescence microscope.
  • a microscope platform includes other types of microscopes that can perform scanning and/or imaging of cells.
  • the microscope platform may include an imaging device configured with an adjustable or multiple magnification objective (e.g., l Ox, 40x, 60x, l OOx, etc.), and an image sensor configured to obtain an image based on the light received through an imaging device lens.
  • the imaging device includes a field-of-view (“FOV”) that is configured to match the size and shape of at least one filter of the microfluidic chip.
  • FOV field-of-view
  • the microscope platform can be configured to scan across multiple pre-defined imaging locations and obtain at least one image at each pre-defined imaging location.
  • the microscope platform may be configured to scan along a microfluidic chip including a plurality of filters and obtain at least one image of each filter, where the dimension of each filter corresponds to the FOV of the imaging device.
  • cell samples are labeled or stained with fluorophores, fluorescent chemical compounds that can re-emit light upon light excitation.
  • Cells samples can be labeled or stained with multiple kinds of fluorophores, each kind designed to emit a specific color of light upon light excitation.
  • Embodiments of the microscope platform include an illumination source configured to illuminate fluorescently-stained cells in a filter with light of a specific wavelength which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e, of a different color than the absorbed light).
  • the specific wavelength can be selected based on the nuclear staining and/or biomarker identification used to fluorescently stain the cell sample.
  • the microscope platform further includes a detector or a sensor configured to detect the spectral emission characteristics of the fluorophore used to label the fluorescently-stained cell.
  • the distribution of a single fluorophore (color) can be imaged by the microscope platform. Multi-color images of several kinds of fluorophores can be developed using several single- color images.
  • the microscope platform is configured to have multiple illumination sources or modify the illumination of the captured cells to cause fluorescence of multiple different dyes.
  • Methods and devices disclosed herein can be configured for automated scanning and imaging of stained samples to improve efficiency and decrease time associated with some other imaging cytometry methods.
  • Embodiments of microscope platforms described herein can quickly and efficiently scan captured cells at a low magnification objective; generate a look-up table containing information about captured cells based on user-defined criteria, including a precise position for each captured cell in the microfluidic chip; return to the precise location to perform verification that the captured cell is a cell of interest; and manipulate the cells of interest for downstream genetic and/or diagnostic analysis.
  • Methods and devices described herein can use the highly specific binding of an antibody to its antigen in order to label specific proteins or other molecules within a cell of interest.
  • a cell sample is treated with a primary antibody specific for the molecule of interest.
  • a fluorophore is directly conjugated to the primary antibody.
  • a secondary antibody, conjugated to a fluorophore, which binds specifically to the first antibody is used.
  • an "antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule, and can be an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE.
  • IgY which is the major antibody type in avian species such as chicken, is also included within the definition.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
  • the term "specifically binds" refers to the binding specificity of a specific binding pair. Recognition by an antibody of a particular target in the presence of other potential targets is one characteristic of such binding. Specific binding involves two different molecules wherein one of the molecules specifically binds with the second molecule via chemical or physical means. The two are related in the sense that their binding with each other is such that they are capable of distinguishing their binding partner from other assay constituents having similar characteristics.
  • the members of the binding component pair are referred to as ligand and receptor (anti-ligand), specific binding pair (SBP) member and SBP partner, antibody-antigen and the like.
  • a molecule may also be an SBP member for an aggregation of molecules; for example an antibody raised against an immune complex of a second antibody and its corresponding antigen may be considered to be an SBP member for the immune complex.
  • the examples may be described as a process, which is depicted as a flowchart, a flow diagram, a finite state diagram, a structure diagram, or a block diagram. Although a flowchart may describe the operations as a sequential process, many of the operations can be performed in parallel, or concurrently, and the process can be repeated. In addition, the order of the operations may be re-arranged.
  • a process is terminated when its operations are completed.
  • a process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc. When a process corresponds to a software function, its termination corresponds to a return of the function to the calling function or the main function.
  • Integrated microfluidic chips for non-invasive isolation of cells are described herein.
  • the integrated microfluidic chips can include a single filter or a plurality of filters.
  • the filter can include a sheet or layer of filter material supported by a substrate.
  • the microfluidic chip can include a plurality of filters arranged in a grid-like structure.
  • Microfluidic chips described herein can also include a binding moiety or affinity molecule.
  • the system can include a binding moiety or affinity molecule that specifically binds to a cell-specific antigen or a non-fetal cell-specific antigen for positive selection of fetal cells or negative selection of unwanted cells.
  • Systems described herein can include a microscope platform configured to image one or more filters on the microfluidic chip.
  • the system includes software including instructions to direct the microscope platform to image the one or more filters in a specific pattern and at a specific magnification objective.
  • the integrated microfluidic chip may be configured to be visualized on a microscope platform.
  • the integrated microfluidic chip may comprise at least one "filter" that is transparent and visualizable under a microscope.
  • the filter comprises multiple through holes that are arranged in a repeating grid pattern and are configured to capture and simultaneously position cells of interest in precisely-defined, clearly-distinguishable locations on the filter.
  • the through holes are specifically arranged in a regular and repeating grid pattern where each through hole can be precisely located based on a unique, predetermined X, Y coordinate on the filter.
  • each filter may include several thousand through holes (e.g., 8,000 or more), thus enabling the capture and imaging of several thousand cells.
  • FIG. 1 illustrates one exemplary microfluidic chip with a filter 100 having a regular, repeating pattern of through holes 110 according to the present disclosure.
  • a microfluidic chip according to embodiments described herein may include a plurality of filters 100, as will be described with reference to Figure 2 below.
  • the size, shape, and relative spacing of each through hole 110 can be specifically designed and configured to capture any desired cell, microbead, or other object based on known characteristics (such as, but not limited to, the size and morphology) of the sought-after cell, microbead, or other object, such that a single object of interest is captured in each through hole 110.
  • By changing the shape, size, and/or relative spacing of the through holes multiple filters can be designed and manufactured for the isolation of specifically sought after cells or objects.
  • the filter 100 is designed to include circular through holes 110 that are shaped and sized to capture specifically identified bacterial cells of interest.
  • the filter 100 is designed to include circular through holes 110 are designed to capture a single RBC in the through hole 110 based on the disk-like shape of RBCs.
  • a circular through hole 110 may be dimensioned to allow mature disk-shaped RBCs (such as maternal RBCs) to pass through the through hole 110, while a single fetal nucleated RBC is captured in a single through hole 110 based on the spherical shape and slightly larger size of fetal nucleated RBCs.
  • each through hole 110 may be specifically selected based on the shape and size of the objects of interest to be captured in each through hole 110. Further, the density of through holes 110 on a single filter 100, and the relative position or arrangement of the through holes 110 relative to each other, can be selected to optimize the number of objects of interest that are retained or "captured" in the filter 100.
  • filters disclosed herein are the ability to automatically create a monolayer of cells as the sample flows over the filter, which is not possible in imaging cytometry methods using a plating of the sample on a slide. Due to the specifically designed size and shape of the through holes, the filter can be configured to prevent one potential cell of interest in the sample from obscuring, overlapping with, or lying on top of another potential cell of interest.
  • methods and systems described herein do not need expensive imaging resources, such as high resolution imaging resources, to determine where specific cell boundaries lie, to trace cell outlines to distinguish two closely- spaced cells from one another, or to ascertain if an object is actually two or more cells clumped together - activities that are typically required in conventional cell plating before a potential cell of interest is actually studied and confirmed to be a cell of interest.
  • expensive imaging resources such as high resolution imaging resources
  • FIG. 2 illustrates an exemplary microfluidic chip 200 according to one embodiment, includes a plurality of filters 210.
  • the microfluidic chip 200 includes 25 filters 210.
  • the microfluidic chip 200 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 50, 100, 125, 150, 200, 250, 1000, or some other number of filters 210.
  • the filters 210 in microfluidic chip 200 are similar to filter 100 described above with reference to Figure 1. It will be understood, however, that any filter, not only that illustrated in Figure 1 , may be employed based on the objects of interest to be captured, imaged, and analyzed.
  • the area of a filter 210 containing through holes may be referred to as the active area of the filter.
  • the sum of the active area of each filter 210 in the microfluidic chip 200 can be referred to as the active area of the microfluidic chip.
  • the number of filters 210 included in the microfluidic chip 200 can be increased or decreased to obtain an optimal active area for the microfluidic chip 200.
  • the plurality of filters 210 are arranged in an NxN grid arrangement.
  • the microfluidic chip 200 includes 25 filters 210, arranged in 5 columns and 5 rows. Filters 210 can be arranged in microfluidic chip 200 in other grid arrangements.
  • the following description provides for methods for isolation, identification, and harvesting of fetal nucleated RBCs for non-invasive prenatal diagnosis. While example the exemplary embodiment disclosed herein may describe isolation of fetal nucleated RBCs from a maternal blood sample for non-invasive prenatal diagnosis, the skilled artisan will understand that the principles and concepts of the methods and devices described herein are applicable beyond NIPT. For example, methods and devices disclosed herein may be configured for the isolation of microbeads, tumor cells for oncology, or any other pathological condition where cells of one kind can be differentiated from cells of another kind based on size, morphology, nuclear staining, and/or biomarker identification.
  • the fetal cells will be isolated from a biological sample, for example, a maternal blood sample.
  • the methods may comprise applying a maternal blood sample to a filter integrated on a microfluidic chip to thereby separate the fetal nucleated RBCs from the maternal blood sample.
  • the methods may comprise labeling the isolated fetal nucleated RBCs within the microfluidic chip, for example, with a fluorescent binding moiety or affinity molecule that specifically binds to a fetal cell-specific antigen or a non-fetal cell-specific antigen for positive selection or negative selection.
  • Figure 3 is an example flow diagram illustrating one method 300 for obtaining cells of interest (such as fetal nucleated RBCs) from a sample according to the present disclosure. As illustrated in Figure 3, the method 300 can include one or more functions, operations or actions as illustrated by one or more operations 310-370.
  • cells of interest such as fetal nucleated RBCs
  • Method 300 can begin at operation 310, "Providing a sample.” Operation 310 can be followed by operation 320, "Applying the sample to a filter integrated on a microfluidic chip.” Operation 320 can be followed by operation 330, “Labeling cells in the sample.” Operation 330 can be followed by operation 340, "Isolating cells of interest.” In some cases, operation 320 and operation 340 are performed simultaneously.
  • Operation 340 can be followed by operation 350, "Scanning isolated cells to determine potential cells of interest.” Operation 350 can optionally be followed by operation 360, "Imaging potential cells of interest to identify confirmed cells of interest.” Operation 360 can be followed by operation 370, “Harvesting confirmed cells of interest.” Operation 330 can be followed by optional operation 340, “Removing cells of no interest.” Operation 330 or operation 340 can be followed by operation 350, "Isolating the cells of interest.”
  • a sample containing cells of interest may be provided.
  • maternal samples containing one or more fetal nucleated cells such a red blood cells
  • the maternal sample can be taken during the first trimester (about the first three months of pregnancy), the 2nd trimester (about months 4-6 of pregnancy), or the third trimester (about months 7-9 of pregnancy).
  • the sample obtained is a blood sample.
  • the amount of sample can vary depending upon size, gestation period, and the condition being screened. In one embodiment, up to 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 mL of a sample is obtained. In one embodiment, 5-200, 10-100, or 30-50 mL of sample is obtained. In one embodiment, more than 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 150 mL of a sample is obtained. In one embodiment between about 10-100 or SO- SO ml of a peripheral blood sample is obtained from a pregnant female.
  • a blood sample is obtained from a pregnant human mother within 36, 24, 22, 20, 18, 16, 14, 12, 10, 8 weeks of conception, or a range between any of the above values.
  • a blood sample is obtained from a pregnant human mother as early as 8 weeks of conception.
  • a blood sample is obtained from a pregnant human mother even after a pregnancy has terminated.
  • the sample can be subjected to one or more steps that enrich the fetal nucleated cells relative to the total components of the sample and/or enrich the fetal nucleated cells relative to the total cells in the sample.
  • the maternal sample can be used as is, pre-diluted in desirable buffer or diluted on chip if required prior to applying it to a filter.
  • a filter may comprise through holes that have a size and/or shape which allows mature RBCs to pass through, but retains the fetal nucleated RBCs.
  • the through holes may be a size that is, is about, or is less than, 4.0 ⁇ , 4.1 ⁇ , 4.2 ⁇ , 4.3 ⁇ , 4.4 ⁇ , 4.5 ⁇ , 4.6 ⁇ , 4.7 ⁇ , 4.8 ⁇ , 4.9 ⁇ , 5.0 ⁇ , 6.0 ⁇ , 7.0 ⁇ , 8.0 ⁇ , 9.0 ⁇ , 10.0 ⁇ , 11.0 ⁇ , 12.0 ⁇ , 13.0 ⁇ , 14.0 ⁇ , 15.0 ⁇ , 16.0 ⁇ , 17.0 ⁇ , 18.0 ⁇ , 19.0 ⁇ , 20.0 ⁇ , 21.0 ⁇ , 22.0 ⁇ , 23.0 ⁇ , 24.0 ⁇ , 25.0 ⁇ , 26.0 ⁇ , 27.0 ⁇ , 28.0 ⁇ , 29.0 ⁇ , 30.0 ⁇ , or a range between any two of the above values, for example, between 2.0 ⁇ to 2.5 ⁇ , between 1.8 ⁇ to 3.0 ⁇ , etc.
  • the shape of the through holes may be rectangular, circular, oval, triangular, etc., or an irregular shape.
  • the "size" of the through hole refers to the smallest effective dimension of the through hole.
  • the microfluidic chip and filter used in this non-limiting example are substantially similar to the microfluidic chip 200 of Figure 2 and the filters 100a of Figure 1A or filters 100b of Figure IB. Accordingly, in some embodiments, fetal nucleated RBCs may be captured when mature RBCs pass through holes having a size and/or shape that allow mature RBCs to pass through, but not fetal nucleated RBCs.
  • a filter may be coated with a binding moiety or affinity molecule that selectively binds fetal nucleated cells, such as fetal nucleated RBCs.
  • a binding moiety or affinity molecule that selectively binds fetal nucleated cells, such as fetal nucleated RBCs.
  • an antibody that specifically binds to fetal nucleated RBCs may be used to coat the filter, so that fetal nucleated RBCs are retained while the mature RBCs pass through the filter.
  • an enriched product such as a sample applied to a filter at operation 320
  • can be dominated (>50%) by cells not of interest e.g., nucleated maternal red blood cells.
  • the nucleated fetal cells of an enriched sample make up at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% of all cells in the enriched sample.
  • a maternal blood sample of 10-20 mL from a pregnant human can be enriched for one or more nucleated fetal cells, such as nucleated red blood cells, such that the enriched sample has a total of about 1 thousand to about 10 million cells, 2% of which are nucleated fetal cells and the rest of the cells are maternal.
  • nucleated fetal cells such as nucleated red blood cells
  • the enrichment steps performed have removed at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8 or 99.9% of all unwanted analytes (e.g., maternal cells such as platelets and leukocytes, mature RBCs) from a sample.
  • unwanted analytes e.g., maternal cells such as platelets and leukocytes, mature RBCs
  • cells may be labeled, directly or indirectly, with a dye in a staining process. Any fluorescent dye that is used in fluorescence microscopy can be used.
  • the nucleated fetal RBCs may be labeled, directly or indirectly, with a dye indicative of certain characteristics of the cell.
  • the labeling procedure of operation 330 may be performed prior to, during, or after operation 320.
  • a dye that stains DNA such as Acridine orange (AO), ethidium bromide, hematoxylin, Nile blue, Hoechst, Safranin, or DAPI, may be used.
  • a cell type-specific dye for example, a dye that specifically labels a fetal cell or a non-fetal cell
  • the cell type-specific dye may be used to label the cells directly or indirectly, for example, through a cell type-specific antibody.
  • the labeling strategy involved may be sequentially carried out or simultaneously carried out.
  • stains as used herein may refer to dyes, stains, and/or biomarkers throughout this disclosure, unless the context of the usage indicates otherwise.
  • any of a variety of fluorescent molecules or dyes can be used to label cells in methods provided herein, including, but not limited to, Alexa Fluor 350, AMCA, Alexa Fluor 488, Fluorescein isothiocyanate (FITC), GFP, RFP, YFP, BFP, CFSE, CFDA- SE, DyLight 288, SpectrumGreen, Alexa Fluor 532, Rhodamine, Rhodamine 6G, Alexa Fluor 546, Cy3 dye, tetramethylrhodamine (TRITC), SpectrumOrange, Alexa Fluor 555, Alexa Fluor 568, Lissamine rhodamine B dye, Alexa Fluor 594, Texas Red dye, SpectrumRed, Alexa Fluor 647, Cy5 dye, Alexa Fluor 660, Cy5.5 dye, Alexa Fluor 680, Phycoerythrin (PE), Propidium iodide (PI), Peridinin chlorophyll protein (PerCP), PE-Alexa Fluor 700
  • Such fluorescent molecules or dyes may produce a corresponding light signature or spectrum upon being illuminated or excited by a light source having a particularly corresponding wavelength of light.
  • using specific fluorescent molecules or dyes may produce an indicator of a particular nucleic acid, antibody or antibody-based fragment probe present on or within a fetal cell.
  • fetal biomarkers can be used to label one or more fetal cells at operation 330 of Figure 3. For example, this can be performed by distinguishing between fetal and maternal cells based on relative expression of a gene (e.g., DYS1, DYZ, CD-71, MMP14) that is differentially expressed during fetal development.
  • a gene e.g., DYS1, DYZ, CD-71, MMP14
  • detection of transcript or protein expression of one or more genes including, MMP14, CD71, GPA, HLA-G, EGFR, CD36, CD34, HbF, HAE 9, FB3-2, H3-3, erythropoietin receptor, HBE, AFP, APOC3, SERPINC1, AMBP, CPB2, ITIHl, APOH, HPX, beta-hCG, AHSG, APOB, J42-4-d, 2,3-biophosphoglycerate (BPG), Carbonic anhydrase (CA), or Thymidine kinase (TK), is used to enrich, purify, enumerate, identify, detect, or distinguish a fetal cell.
  • genes including, MMP14, CD71, GPA, HLA-G, EGFR, CD36, CD34, HbF, HAE 9, FB3-2, H3-3, erythropoietin receptor, HBE, AFP, APOC3, SERPINC1, AMBP, C
  • the expression can include a transcript expressed from these genes or a protein.
  • expression of one or more genes including MMP14, CD71, GPA, HLA-G, EGFR, CD36, CD34, HbF, HAE 9, FB3-2, H3-3, erythropoietin receptor, HBE, AFP, AHSG, J42-4-d, BPG, CA, or TK is used to identify, purify, enrich, or enumerate a fetal nucleated cell such as a fetal nucleated RBC.
  • fetal cells known as trophoblasts are a cell of interest that is isolated using filters described herein.
  • Biomarkers specific to trophoblasts can be labeled and used to distinguish the fetal trophoblast cells (that are captured in the filter and are objects of interest) from maternal cells (that are also captured in the filter but are not objects of interest).
  • Biomarkers that can be used to label, identify, detect, or distinguish a fetal trophoblast cell include (but are not limited to) cytokeratin 5, 6, 7, 8, 10, 13, 14, 18, 19; CD147, CD47, CD105, CD141, CD9, HAI-1, CD133, HLA-G, human placental lactogen, PAI-1, and IL-35.
  • biomarkers that are not specific to fetal trophoblast cells but that can be used to label, identify, detect, or distinguish fetal cells of interest from maternal cells that are not of interest, include (but are not limited to) CD68, CD105, placental alkaline phosphatase (PLAP), NDOG, GB25, ⁇ -hCG, and 3b- hydroxy-5-ene steroid dehydrogenase.
  • biomarkers provides examples of suitable biomarkers for labeling, identifying, detecting, or distinguishing a fetal cell from a maternal cell and is not intended to limit methods and devices described herein, which can capture and identify any cell of interest that is subject to filtration, whether or not the cell of interest has biomarkers that are used to distinguish the cell of interest captured in the filter from another object that is also captured in the filter but is not a cell of interest.
  • Isolating cells of interest cells of interest such as fetal cells may be isolated using embodiments of filters described herein. Isolating cells of interest can include positioning a single cell of interest at a distinct, precisely-defined location, such as a single through hole, in a filter. In one example embodiment, fetal nucleated RBCs are isolated by capturing and positioning the cells in a filter such as filter 100A or filter 100b when the sample is applied to the filter at operation 320.
  • each fetal nucleated RBC may isolated from other cells in the sample (other fetal nucleated RBCs, non-nucleated fetal cells, maternal cells, etc.) when the fetal nucleated RBC is retained in a single through hole of the filter while other cells that are not of interest (such as mature maternal RBCs) pass through the through holes of the filter and are not retained in the filter. Accordingly, the isolation operation 340 may be performed at the same as operation 320.
  • analysis 350 “Scanning isolated cells to determine potential cells of interest,” the cells captured in the filter at operation 320 and/or operation 340 are analyzed to determine which cells are likely to be cells of interest. Such analysis can include imaging the cells captured in the filter at low resolution. As will be described in detail below, analysis at operation 350 can also include imaging each filter of a plurality of filters using a microscope platform with a field of view (FOV) that matches the dimensions of a single filter.
  • FOV field of view
  • Information obtained during operation 350 can be used at operation 360, "Imaging potential cells of interest to identify confirmed cells of interest.”
  • Operation 360 can include imaging the potential cells of interest (determined at operation 350) at high resolution to confirm the cell in question is a cell of interest (such as, for example, a fetal nucleated RBC).
  • a cell of interest such as, for example, a fetal nucleated RBC.
  • “Harvesting confirmed cells of interest” the confirmed cells of interest that were identified at operation 360 are removed from the filter for genetic and/or diagnostic analysis.
  • operation 370 can optionally include removing cells not of interest from the filter, rather than removing cells of interest from the filter.
  • operation 360 is not performed or is optionally performed.
  • cells identified as potential cells of interest at operation 350 are next harvested at operation 370, without obtaining a second, higher resolution image of the likely cells of interest.
  • a second, higher resolution image of the particular cell may not be needed for confirmation at operation 360.
  • the method 300 can vary from cell to cell.
  • a first cell that only meets 2 or 3 user-defined criteria e.g., fluoresces in 2 or 3 different fluorescent channels
  • a second, different cell that meets 4 or 5 user-defined criteria e.g., fluoresces in 4 or 5 different fluorescent channels
  • operation 360 may be performed on the first cell but not on the second cell.
  • cells not of interest may be isolated in operation 340 and/or identified as a cell not of interest in step 360.
  • operation 370 may be also be performed to destroy, fragment, and/or remove the captured cell from its respective through hole, thereby permitting capture of cells of interest in the now-cleared through hole that was previously occupied by the cell not of interest.
  • operations 310-340 can be repeated after cells that are not of interest are removed during operation 370. Repeating method 300, or certain operations in method 300, in this manner can result in a microfluidic chip having a large number of cells of interest captured in filters of the microfluidic chip.
  • Microfluidic chips with a maximum density of cells of interest can thus be obtained by repeating method 300, or certain operations of method 300, on the same microfluidic chip after cells not of interest are removed at each iteration of operation 370.
  • harvesting of confirmed cells of interest in operation 370 is only performed after a significant number of through holes have captured confirmed cells of interest.
  • the distinct, precisely-defined position of each through hole within the microfluidic device enables the extraction and/or manipulation of captured cells that are of interest, as well as cells that are not of interest.
  • a non-limiting example of a method 400 for analyzing captured or "isolated" cells to determine which cells are cells of interest (such as fetal nucleated RBCs) is illustrated in the flow diagram shown in Figure 4.
  • the method 400 can include one or more functions, operations or actions as illustrated by one or more operations 410-460.
  • the method of 400 may be performed during operations 350, 360, and 370 of Figure 3.
  • the method 400 may be performed by a microscope platform having captured cells disposed in the FOV of the imaging device of the microscope platform.
  • the method 400 can be implemented as a software solution by one or more processors coupled to a memory component ("memory") that are included either in the microscope platform or a computer that may be in data communication with the microscope platform, or has access to data generated and/or stored by the microscope platform, for example, by an optical system included in the microscope platform.
  • the data may include a first image of a particular filter on a microfluidic chip at a first resolution, the first image having a first resolution.
  • the data may further include a second image at a second resolution that includes at least a portion of the particular filter in the first image, the second resolution being at a higher resolution than the first image. That is, the second image can be captured at a higher magnification than the first image.
  • candidate cells of interest may be identified in the first image, and then each candidate cell of interest can be captured in a second image and further evaluated to determine if it is in fact a cell of interest, as defined by certain criteria /thresholds.
  • Method 400 can begin at operation 410, "Quick scan of stained clinical sample using low magnification objective.” Operation 410 can be followed by operation 420, "Analyze scanned images based on user defined criteria.” Operation 420 can be followed by operation 430, “Generate look up table for fluorescent tagged cells.” Operation 430 can be followed by operation 440, "Look up table records intensity for all fluorescent channels (FITC, DAPI, PE) and the X, Y coordinates.” Operation 440 can be followed by operation 450, "Confirm staining pattern of top hits from look up table with higher magnification objective.” Operation 450 can be followed by operation 460, "Move micromanipulator/cell selecting device to the confirmed cell of interest for cell plucking for downstream genetic analysis.”
  • a quick scan of the stained enriched cells may be performed by the microscope platform.
  • a microfluidic chip may be provided in accordance with method 300 described with reference to Figure 3.
  • the microfluidic chip may be, for example, substantially similar to the microfluidic chip 200 described with reference to Figure 2, having cells captured or disposed in through holes.
  • the captured cells may also have been labeled in accordance with operation 330 of method 300, including but not limited to, being stained and/or labeled with nuclear stain, specific biomarkers, and/or fluorescent dyes used to identify user-defined characteristics of the cells of interest, such as fetal nucleated RBCs.
  • the maximum number of fluorescent dyes and/or biomarkers considered to identify fetal nucleated RBCs is four, including but not limited to, DAPI, FITC, PE, and APC plus bright field.
  • a user may select any number of nuclear stains, biomarkers, and/or fluorescent dyes to label cells (before or after applying the sample to a filter to isolate cells of interest).
  • the selection of the number and type of nuclear stains, biomarkers, and/or fluorescent dyes may be based on user-defined criteria for identifying cells of interest from the plurality of cells that were initially captured in the filter as a result of a cell's shape and size.
  • a user may select any number of nuclear stains, biomarkers, and/or fluorescent dyes to be analyzed during a scan of the labeled cells.
  • cells in a sample may be labeled with 5 fluorescent dyes, but specifically analyzed for fewer than 5 (such as 4, or 3, 2, or 1) of the applied dyes during the quick scanning procedure performed at operation 410.
  • the microscope platform may be configured to obtain a single image of each filter.
  • the microscope platform may include an illumination source configured to excite the nuclear stains, biomarkers, and/or fluorescent dyes used to label the captured cells which may cause captured cells containing any of the nuclear stains, biomarkers, and/or fluorescent dyes to fluoresce based on the presence of the nuclear stains, biomarkers, and/or fluorescent dyes in the captured cell.
  • the microscope platform may be configured to obtain an image of fluorescence, and evaluate the fluorescence (e.g., by intensity, presence, or other known indicators of light) to determine whether the captured cell is a likely cell of interest or a confirmed cell of interest.
  • the microfluidic chip enables the simultaneous capture and positioning of cells into distinct, precisely-defined locations on a repeating gridlike pattern of through holes on a filter.
  • the position of each captured cell within the microfluidic device may be precisely identified and recorded.
  • a microfluidic chip can be configured to provide a readily-determinable, precisely-defined position for each captured cell based on the specific location of the through hole in which the captured cell is disposed.
  • the precise location of a captured cell can be based on which filter of the microfluidic chip its corresponding through hole is located and the coordinates of the through hole in that filter.
  • the number of through holes and size of each through hole may be stored in memory, and used by the image evaluation software to help identify the location of cells of interest on the filter in images generated of the filters.
  • Figure 5 illustrates an example imaging grid and an example microfluidic chip according to the present disclosure.
  • the plurality of filters on the microfluidic chip may be aligned with a reference system, or "an imaging grid," such that each of the plurality of through holes can be individually referenced by information indicating a particular filter on the microfluidic chip and a location within each particular filter.
  • Figure 5 depicts a portion of a microfluidic chip 500 including multiple filters 510 arranged in a 5 x 5 grid arrangement on the microfluidic chip 500.
  • the microfluidic chip 500 is substantially similar to microfluidic chip 200 described in reference to Figure 2.
  • Figure 5 also depicts multiple imaging regions 560 which collectively make up grid 550.
  • grid 550 includes x-axis coordinates 555 labeled 1-5 and y-axis coordinates 557 labeled A-E.
  • each imaging region 560 based on the location of each imaging region 560 within the grid 550, each imaging region 560 has a uniquely identifiable position (e.g., Al, Bl, CI , Dl, El , A2, B2, etc.) and may correspond to a particular filter positioned at that location.
  • the microfluidic chip 500 may be aligned onto grid 550.
  • each filter may be individually aligned with an imaging region 560, and each filter 510 may be associated with a unique identifiable position (e.g., Al, A2, etc.).
  • filter 510 may be positioned on grid 550 at imaging region 560 having an identifiable location of Al .
  • the microfluidic chip 500 may include position markers or fiducials (not shown) on the exterior portion (for example, exterior portion 220 of Figure 2) of the microfluidic chip 500 to enable alignment with similarly-positioned markers on the grid 550 (not shown).
  • magnétique platform can be configured to have autofocus capabilities, thereby enabling the alignment and calibration of each filter relative to the image plane of the image sensor (e.g., X, Y, and Z directions) as the quick scan is performed.
  • each through hole of each filter such as filter 510
  • the unique location of each through hole of each filter is precisely defined by known X and Y coordinates of the filter, as described above with reference to Figures 1 and 2.
  • captured cells can be identified by a unique identification scheme for downstream genetic analysis and manipulation.
  • Window 515 illustrates a zoom-in view of a portion of the filter 510, such that the multiple through holes are shown in greater detail having regular, repeating, and known positions on the filter 510.
  • the microscope platform may be further configured to include multiple magnification objectives or an adaptable magnification objective, such that the microscope platform can re-image captured cells at a higher magnification objection for verification and confirmation that the captured cell is a cell of interest.
  • through hole 516 may represent a through hole that either does not contain a captured cell or the captured cell does not satisfy the user-defined criteria, and thus is not identified as a potential or likely "cell of interest" during the quick scan of operation 410.
  • hole 517 represents a through hole containing a captured cell satisfying the user-defined criteria, and will thus be identified as a potential or likely "cell of interest" during the quick scan of operation 410.
  • the user-defined criteria that is used to identify a captured cells as a likely cell of interest during the quick scan operation 410 may depend on which nuclear stains and/or fluorescent dyes have been used to label the captured cells.
  • the quick scan operation 410 may determine that the captured cell in through hole 517 is fluorescing at a particular wavelength corresponding to a particular nuclear stain and/or fluorescent dye.
  • a verification and confirmation operation performed after the quick scan can identify whether the potential cells of interest identified during the quick scan are confirmed cells of interest.
  • Figure 6 illustrates an example scan pattern for imaging a microfluidic chip according to the present disclosure.
  • a microfluidic chip is aligned with grid 550.
  • a scan pattern 610 can be performed by the microscope platform during operation 410.
  • the scan pattern 610 is configured to provide minimal time to scan the entire microfluidic chip and obtain an image at each imaging region (such as imaging region 560 shown in Figure 5).
  • the microscope platform can be configured to obtain an image for each imaging region, and obtain a single image for each filter that is positioned in each imaging region.
  • the specific FOV of a given magnification objective of an image sensor of the microscope platform can be selected to match the shape and size of each filter located at an imaging region 560 in the grid 550.
  • the microfluidic chip may be an 8 millimeter x 8 millimeter microfluidic chip comprising 25 filters making up the 5 mm x 5 mm interior portion, each filter having an active area that is 0.9 mm x 0.9 mm.
  • the FOV of the microscope platform is selected to obtain a representation of one entire filter (e.g., one 0.9 mm x 0.9 mm area) in a single image using a low magnification objective (for example, a lOx magnification objective).
  • the adjustment/setting of the magnification such that the FOV represents one filter may be performed using physical optical elements, in other embodiments the adjustment/setting of the magnification may be performed using a combination of physical optical elements and sensor magnification adjustments, while in some embodiments the adjustment/setting of the magnification may be done using sensor magnification control. While specific examples and sizes are disclosed herein, it will be understood that the shape and size of the FOV of a microscope platform and/or imaging device can be selected to match the specific size and shape of a single filter in the microfluidic chip.
  • One non-limiting advantage of matching the FOV of the microscope platform to the size and shape of a single filter is that the approach avoids stitching together multiple images to obtain a composite image of a single filter.
  • Embodiments of the quick scan operation 410 that match the filter size and shape to the FOV of the microscope platform in this way save time in the overall scanning and imaging of captured cells, enable more cells to be quickly isolated and confirmed as cells of interest for further downstream genetic analysis, and thereby increase throughput.
  • higher resolution imaging resources can also be conserved for cells that are identified as potential cells of interest during the quick scan operation 410, rather than initially taking high resolution imaging of all cells that are plated or spread on a slide.
  • imaging cytometry methods typically require a high magnification objective for all images so that the resolution is high enough to analyze (for example, differentiate) the cells in the image from one another (a process that is required even before the differentiated cells are analyzed to determine if they are cells of interest).
  • the high resolution results in a smaller area of the overall sample being captured in a single image, and requires more time to obtain the image and more storage space to record the image.
  • multiple high resolution images must be taken and stitched together to develop a complete (albeit composite) image of the sample.
  • Methods and devices disclosed herein avoid having to stitch multiple images together by capturing a complete sample (e.g., a filter) in a single image.
  • embodiments of the quick scan operation 410 can provide an X, Y coordinate for a potential cell of interest with a single low resolution image of a single filter in a microfluidic chip, thereby enabling the user to quickly and efficiently return to the known position of a captured cell for further verification that it is a cell of interest.
  • operation 410 illustrates a single image obtained for each filter of the microfluidic chip. However, operation 410 may be performed multiple times for any given microfluidic chip. The number of times operation 410 is performed can depend on the number of nuclear stains, biomarkers, and/or fluorescent dyes used to stain the cells captured on the filter. Each nuclear stain, biomarker, and/or fluorescent dye may require a separate excitation by an illuminating light source, thereby necessitating multiple iterations of operation 410.
  • operation 410 may be performed four separate times, resulting in four images (such as, for example, four low resolution images) of the filter 510 at location Al of microfluidic chip 500, four images of a filter 510 at location A2 of microfluidic chip 500, and so on.
  • images such as, for example, four low resolution images
  • the positional information of each captured cell can be retained (e.g., stored in memory) between each iteration operation 410, such that the separate images can be cross referenced and used to readily identify the captured cell for identification and downstream analysis.
  • a multicolor image may be obtained of each filter prior to the quick scan proceeding to the next filter.
  • Operation 410 may include a multicolor scan, where the microscope platform is configured to modify the illuminating light source as needed to excite the different nuclear stains, biomarkers, and/or fluorescent dyes.
  • the microscope platform may include a fast filter wheel, disposed along the emission and excitation optical paths and between the illuminating light source and the captured cells.
  • the fast filter wheel may include a filter cube set, such as a DA/FITC/PE filter cube.
  • the illuminating light source may be a white light source, where the white light comprises wavelengths of light able to excite the nuclear stains, biomarkers, and/or fluorescent dyes used to stain and/or label captured cells.
  • the white light from the white light source can be incident upon at least one of the filters in the fast filter wheel and/or the filter cube.
  • the filter wheel and/or filter cube may be configured to pass only a portion of the incident white light from the white light source, the selected portion being chosen to excite one or more nuclear stains, biomarkers, and/or fluorescent dyes. Therefore, in some embodiments of operation 410 according to the present disclosure, the microscope platform may be configured with a single modifiable illumination source to control the excitation of the nuclear stains, biomarkers, and/or fluorescent dyes.
  • the time to execute operation 410 including a multicolor scan, where the objective is 4x to lOx is less than 15 minutes.
  • One non-limiting advantage of the quick scan methods disclosed herein is that the reduction of time to image the captured cells on a microfluidic chip is inversely related to the magnification of the objective lens. For example, where the quick scan method of operation 410 utilizes a lOx objective as compared to a 60x objective of another approach, the reduction of time to obtain
  • the image (e.g., time saved) is 6 .
  • the amount of time saved by operation 410 is N where N is the ratio of objective magnification of the conventional approach to the objective used in embodiments of operation 410 described herein.
  • the scanning operation may include an autofocus capability to adjust for field flatness during each scan.
  • the scanning system may have color correction for different wavelengths and may adjust for focus drift (if any) during the entire scan period, thereby ensuring proper focus throughout the quick scan operation 410.
  • the images from operation 410 are analyzed based on user-defined criteria.
  • user-defined criteria may be based on the nuclear stains, biomarkers, and/or fluorescent dyes used to stain and/or label cells captured on filters described herein.
  • the presence or absence of user-selected nuclear stains, biomarkers, and/or fluorescent dyes on a captured cell may form a basis for rejecting and/or selecting a captured cell for further analysis or removal (for example, at operation 360 or operation 370 described with reference to Figure 3).
  • the criteria may also include the size of the captured cell and/or any criteria a user may deem necessary or desirable in determining whether a captured cell is a cell of interest or not a cell of interest.
  • the user can reject or select criteria from a plurality of criteria based on the user's objective during downstream genetic and/or diagnostic testing.
  • a captured cell that satisfies user-defined criteria during quick scan operation 410 may be referred to as a "hit.”
  • a captured cell that is identified as a "hit” can refer to a captured cell that is identified as a likely cell of interest (candidate cell of interest) during operation 350 of method 300.
  • a user can define certain criteria based on biomarkers and/or fluorescent dyes that are indicative of a fetal nucleated RBC.
  • operation 410 may be performed to illuminate the captured cells, excite any fluorescent dye indicative of a biomarker that may be present on the captured cells, and obtain images of any fluorescence that is emitted by a captured cell.
  • the images may be evaluated and analyzed to identify captured cells that emit fluorescence at an intensity that satisfies the user-determined threshold intensity, thereby indicating the captured cell satisfies the user-defined criteria and can be labeled a hit.
  • the precise location of the hit on the microfluidic chip may then be identified and recorded for further verification and analysis, such as at operation 360 described above with reference to Figure 3.
  • One non-limiting example of user-defined criteria for generating hits indicative of a fetal nucleated RBC may include counting and identifying the location of all cells that are 5 ⁇ to 12 ⁇ in size, have greater than 100 counts in the channel for FITC fluorescence and the channel for DAPI fluorescence, and less than 10 counts in the channel for PE fluorescence.
  • a look up table is created based on information obtained during the analysis performed at operation 420.
  • the look up table is generated at the same time the analysis is performed at operation 420.
  • Figure 7 illustrates one example look up table 700 generated in response to or at the same time as operation 420.
  • the microscope platform can be configured to analyze the scanned images at operation 420 and simultaneously generate a look-up table at operation 430. For example, each time operation 410 takes an image of a filter, the microscope platform can be configured to analyze the image and generate a look up table for that image, prior to the microscope platform obtaining a subsequent image or while obtaining the subsequent image.
  • Embodiments of look up table 700 described herein can be searchable by a user, and can be configured to record and display to the user the X and Y coordinates of captured cells disposed in through holes, such as through hole 517 in filter 510.
  • look up table 700 may enable identification of hits (e.g., possible cells of interest) based on user-defined criteria, for example as described with reference to operation 420 above.
  • look up table 700 may also enable the methods and devices disclosed herein to return to a hit location on the microfluidic chip, as recorded in the look up table.
  • FIG. 7 illustrates one example embodiment of look up table 700.
  • each filter such as filter 510
  • FIG. 7 illustrates the listing of the imaging region of each filter in column 750.
  • each cell captured in a through hole e.g., through hole 517) in each filter can be recorded, as illustrated in Figure 7 in the two left-most columns 710, 720. Accordingly, in some embodiments, the location of each captured cell is generated and/or recorded. Thus, the precise location of cells located in each through hole can be identified and the microscope platform can be configured to return to each location retaining a cell for further analysis.
  • multiple columns may also be included that provide further details or characteristics associated with each through hole location.
  • column 730 may indicate the size of an object in each through hole. Information on the size of an object located in a through hole, in conjunction with other information recorded in the table, may assist in determining whether the object is a hit.
  • Other embodiments may include an indication that the through hole contains a cell or does not contain a cell.
  • the intensity of the nuclear stains, biomarkers, and/or fluorescent dyes used to stain and/or label the captured cells may be listed in columns 741-74N. In one non-limiting embodiment shown in Figure 7, stains for DAPI, FITC, and PE were used to label the captured cells.
  • Operations 430 and 440 may be performed at the same time.
  • the microscope platform performed operations 410 and 420, and during operation 430 the microscope platform generated look up table 700 by recording the intensity values for each respective nuclear stain, biomarker, and/or fluorescent dye in the look up table 700.
  • operations 410-440 may be performed in the order described above, concurrently, or in any order selected by the user to enable the generating of the look up table and the analysis of the hits (e.g., possible cells of interest).
  • operation 420 "Analyze scanned images based on user defined criteria” may be performed after or during operations 430, “Generate look up table for fluorescent tagged cells,” and/or operation 440, "Look up table records intensity for all fluorescent channels and the X, Y coordinates.”
  • the analysis based on the user-defined criteria of operation 420 may be automated, based on preselected criteria, as the look up table is generated.
  • operation 450 can be performed to verify that a hit identified during operations 420-440 is in fact a cell of interest based on the user-defined criteria.
  • at least one hit is identified from the look up table and the microscope platform is configured to return to the identified location of the at least one hit.
  • the microscope platform may then return to the previously-identified X, Y coordinate of the at least one hit and re-image the at least one hit at a higher magnification objective to verify that the hit is a cell of interest.
  • the user analyzes the second, higher resolution image to confirm the staining pattern of the at least one hit is consistent with the staining pattern expected from a cell of interest, such as a fetal nucleated RBC.
  • methods and devices disclosed herein can zoom in on any one of the identified hits for further inspection based on the user-defined criteria.
  • operation 450 is not performed for a particular cell that is identified as a hit (e.g., potential or likely cell of interest), and the method 400 moves to operation 460 to remove the potential cell of interest from the filter.
  • a cell satisfies a large number (2, 3, 4, 5, or more) user-defined criteria during the initial quick scan, and the user determines further verification at operation 450 is not desirable or necessary before removing the cell from the filter.
  • look up table 700 can be configured to identify a hit for further scrutiny.
  • look up table 700 includes values at columns 741-74N corresponding to certain characteristics that may indicate the intensity of excitation of nuclear stains, biomarkers, and/or fluorescent dyes present in captured cells.
  • User-defined criteria may indicate values for each characteristic (e.g., size, fluorescence intensity, etc.) such that the look up table can automatically identify locations of captured cells that satisfy the user-defined criteria.
  • the look up table is one means of identifying a hit in embodiments of the present disclosure.
  • systems described herein include means for transmitting or communicating hit locations from look up table 700 to the microscope platform.
  • the microscope platform can move an imager to the identified location and switch to a higher magnification objective for re-imaging and verification of the object at the hit location.
  • Look up table 700 may be configured to provide the filter location (e.g., Al , A2, etc.) and an X, Y coordinate within the individual filter for each identified hit.
  • the microscope platform can be configured to receive the locations for each hit, move to one of the received locations, switch to a higher magnification objective (e.g., switching from l Ox to 20x, 40x, 60x, etc.), and obtain a second, higher resolution image of the object at the hit location.
  • a higher magnification objective e.g., switching from l Ox to 20x, 40x, 60x, etc.
  • the microscope platform can be configured in some aspects to automatically and sequentially move to the next received location, re-imaging each location of an identified hit sequentially.
  • embodiments of the microscope platform described herein can re-image each hit and verify that the captured cell that generated the hit is in fact a cell of interest.
  • the distinct, precisely-defined location of through holes in filters described herein permits the microscope platform to return to the precise location of a hit without having to completely re-image the entire population of cells captured on the filter.
  • imaging cytometry methods that spread cells on a slide are unable to readily identify and/or return to the exact position of potential cell of interest for verification or further analysis, because each cell in the sample is randomly positioned at an imprecise, irregular location on the slide and may be in close proximity to another cell or positioned on top of another cell.
  • Micromanipulator/cell selecting device to the confirmed cell of interest for cell plucking for downstream genetic analysis a microscope platform, micromanipulator, or similar device is moved to the precise location of the confirmed cell of interest and the captured cell at that location is manipulated (for example, removed from the through hole).
  • a micromanipulator or other cell selecting device may extract or pluck the cell from the through hole at the identified location.
  • An exemplary micromanipulator can comprise a needle-like apparatus configured to extract and/or pluck the cell from the through hole and move the cell for storage and subsequent genetic and/or diagnostic analysis.
  • Figure 1 1 illustrates one embodiment of an example of a process 1 100 of controlling a microscope platform for determining cells of interest in a sample containing a plurality of cells, each cell being disposed in a through hole on one of a plurality of filters on a microfluidic chip, each of the plurality of filters having an active area including a plurality of through holes arranged in a pattern.
  • process 1100 generates one or more first images of the plurality of filters on a microfluidic chip, each of the first images having a first resolution and each of the one or more first images depicting at least a portion of a filter of the microfluidic chip.
  • a microfluidic chip may have a plurality of filters disposed thereon.
  • a first image, at a first magnification (or resolution), of each of the plurality of filters can be generated, that is, each of the first images may depict one of the plurality of filters.
  • the process 1100 generates the first images with an imaging device, for example, an imaging device that is part of the microscope platform.
  • Figure 6 illustrates one example of a scan pattern that can be used for imaging the multiple filters on the microfluidic chip.
  • an imaging component may be moved to perform such a scan and/or a part of the platform that holds the microfluidic chip may be moved to perform the scan such that each filter is depicted in an image.
  • two or more of the plurality of filters are depicted in a first image.
  • the one or more first images may be stored in memory and later retrieved for image processing, for example to determine features (e.g., fluorescing cells) in the first images and the location of such features in the first images.
  • the process 1100 may use one or more electronic hardware processors to determine, using the one or more first images, location information for candidate cells of interest that are disposed in one of the through holes.
  • the determined location information may include information indicating a particular filter of the plurality of filters, and information indicating the location of the through hole of the particular filter where each candidate cell of interest is located.
  • a data structure represented by the table 700 may include the location information, which may identify a filter (e.g., square # 750), and coordinates of the cell 710, 720 (e.g., corresponding to a through hole location of the particular filter).
  • Specific information about each cell can also be determined and stored in a data structure (e.g., table 700), either as a result of image processing of the first images or a subsequently generated image that depicts the same through hole/cell (e.g., a second image generated using a higher magnification to produce a higher resolution).
  • the specific information about each of the cells can be used to determine which of the cells are candidate cells of interest and should be further examined.
  • one or more of the size, DAPI Intensity, FITC Intensity and PE Intensity information, or other information may be evaluated against user-defined thresholds to determine which cells are candidate cells of interest.
  • information about the filters and the through cells can be used to help determine the location of the cells of interest.
  • one or more of the size of the filter, the size of the through holes in each of the filters, the number of columns and rows of through holes can be used during image processing of the first images to determine the location of through holes and/or cells in the first images.
  • process 1 100 may store the location information (and other determined information about the cells) in memory, for example, in a data structure that can be represented as a table (for example, look up table 700 in Figure 7).
  • the process 1100 can use the information about the cell locations to move the microscope platform to a position to capture another image (e.g., a second image) of each candidate cell of interest using the stored location information, that is, to capture an image at the location of the through hole where the candidate cell of interest is disposed.
  • an imaging system is moved, or optically or electrically aligned to a desired location to capture a second image at the location of a candidate cell of interest.
  • a structure supporting the filter may be moved to perform this alignment.
  • the stored location information can be used to align the microscope platform (e.g., an optical system of the microscope platform) to the location of each of the identified candidate cells of interest in the first images.
  • the process 1100 can use the imaging device to generate a second image depicting each candidate cell of interest, the second image having a resolution (e.g., magnification) higher than the first resolution.
  • the one or more electronic hardware processors can be further configured to perform image processing on the second images to refine the determined cell information, e.g., for size, DAPI Intensity, FITC Intensity, PE Intensity, etc., and determine which of the candidate cells of interest are actual cells of interest that should be sampled (e.g., removed from the though hole for further testing).
  • image processing e.g., for size, DAPI Intensity, FITC Intensity, PE Intensity, etc.
  • the user-defined criteria may be indicative of cells not of interest, e.g., cells that are captured in the through holes but are not desired for downstream analysis, such as maternal blood cells.
  • the hit location can be conveyed to the microscope platform and operation 370 of Figure 3 may be performed to destroy, fragment, and/or remove the captured cell from the through hole, thereby permitting capture of cells of interest in the now-cleared through hole that was previously occupied by the cell not of interest.
  • operation 310, operation 320, operation 330, and operation 340 are repeated after cells that are not of interest are removed during operation 370.
  • Repeating method 300, or certain operations in method 300, in this manner can result in a microfluidic chip having a large number and/or density of cells of interest captured in filters of the microfluidic chip.
  • operation 460 in which confirmed cells of interest are plucked or removed from the filters, is only performed after a significant number of through holes have captured confirmed cells of interest.
  • Figures 8A - 8C illustrate images taken during one implementation of method 400 performed to detect and identify fetal nucleated RBCs from a maternal blood sample using a filter 800.
  • Filter 800 may be substantially similar to filter 100a of Figure 1A, having rectangular through holes.
  • Figures 8A-8C are actual images of filter 800 taken by a microscope platform, where fluorescent fetal nucleated RBCs from blood (e.g., buffy coated) are retained in precisely-defined and identifiable through holes on the filter 800.
  • Figures 8A and 8B show nucleated cells and non-nucleated cells in bright field and fluorescence illumination, while Figure 8C illustrates nucleated cells in fluorescence illumination only.
  • Figures 8B and 8C are zoomed FOV images of Figure 8 A taken using a lOx objective lens on an Olympus® ⁇ -71 microscope and Hamamatsu Cool Snap® camera.
  • the fetal nucleated RBCs captured in the filter 800 may represent cells of interest in a diagnostic or genetic analysis.
  • the object retained at location 810 in Figure 8 A and the object retained at location 820 in Figures 8B and 8C are fetal nucleated RBCs.
  • Filter 800 has also retained non-nucleated cells which are not fluorescent in the image of Figure 8C (see the object positioned at location 830 in Figure 8A, for example).
  • the object captured at position 830 in Figure 8 A may be a cell not of interest, such as a non- nucleated maternal RBC.
  • the through holes of filter 800 are rectangular in shape and having a long dimension of 10 um and a short dimension of 5 um.
  • Figures 8A-8C were generated during one example embodiment of the method 400 for analyzing captured cells to identify cells of interest described with reference to Figure 4.
  • Figure 8A corresponds to a low resolution image of the filter 800 positioned in an imaging region during one embodiment of quick scan operation 410.
  • Figures 8B and 8C correspond to higher resolution images of the same filter 800.
  • the images in Figures 8B and/or 8C were taken during operation 450 to verify and confirm that cells identified as hits or potential (or candidate) cells of interest during a quick scan (see, for example, operations 410-440 of method 400) of filter 800 are in fact cells of interest.
  • Figures 9A and 9B illustrate images taken during another implementation of method 400 performed to detect and identify fetal nucleated RBCs from a maternal blood sample using a filter 900.
  • Figure 9B is an image of a portion of filter 900 taken at a higher magnification objective.
  • Filter 900 is similar to filter 800, but includes through holes that are 7 um circular through holes.
  • Figures 9A and 9B show nucleated and non-nucleated cells captured from buffy coat of blood in the 7 um circular through holes.
  • the object positioned at location 910 and the object positioned at location 930 are fetal nucleated RBCs.
  • the object positioned at location 920 is a non-nucleated cell, such as a maternal RBC.
  • FIG. 10 illustrates an embodiment of a system 1000 for analyzing cells of interest captured in a microfluidic device according to the present disclosure.
  • analyzing cells of interest includes detecting the presence and location of a cell captured in the microfluidic device, and identifying the detected cell as a cell of interest.
  • the system may be implemented as a software solution contained in microscope platform and/or the user computer, for example in one or more processors executed by a logic device in the microscope or computer. Further, the software solution may be a portion of the software solution utilized to perform method 300 and/or method 400 or may be separate software solution.
  • the system 1000 may include, but is not limited to, software configured to provide instructions to a processor to perform one or more functions of the microscope platform. As illustrated in Figure 10, the system 1000 can include image analysis software 1010, analysis software 1020, scanning software 1030, and micromanipulator moving software 1040.
  • image analysis software 1010 may be configured to generate a look up table from images taken during the quick scan operation 410 of method 400.
  • One example may include the image analysis software instructing the processor to complete operation 430 and/or operation 440.
  • Analysis software 1020 may be configured to generate hits corresponding to potential cells of interest based on user-defined criteria.
  • One example may include the analysis software instructing the processor to complete operation 420 in conjunction with or separate from operation 430 and/or operation 440.
  • the scanning software 1030 may be configured to move at least a portion of the microscope platform to the location of a hit for re-imaging with a higher magnification objective.
  • One example may include the scanning software instructing the processor to complete operation 450.
  • the scanning software may instruct the processor to move the entire microscope objective. In other embodiments, the scanning software may instruct the processor to move only a portion of the microscope objective, such as for example, the imaging device.
  • Micromanipulator moving software 1040 may be configured to move a micromanipulator to the location of the hit for plucking once the user-defined criteria are confirmed for a potential cell of interest under higher magnification. After the micromanipulator moving software 1040 instructs the processor to move the micromanipulator, the microscope platform, a portion thereof, or a separate mechanical unit may proceed to operation 460 to manipulate, pluck, remove, and/or destroy the enriched cells based on the evaluation of the user-defined criteria in accordance with method 400 of Figure 4.
  • GUI graphical user interface
  • the GUI may be implemented in the microscope platform or a separate user operated computer (e.g., a personal computer, laptop, tablet, mobile smartphone, etc.).
  • the GUI may be viewable as one or multiple displays and may be manipulated and configured by the user for viewing images of captured cells taken by the microscope platform.
  • the GUI may present an image of fluorescent enriched cells viewable to the user for analysis and confirmation.
  • One illustrative aspect of the GUI is that it may be configured to provide a means of identifying initial hits from the low magnification objective scan based on blob analysis.
  • the GUI permits a user to zoom in on a hit from an image taken at low magnification objective.
  • the zoom in is permitted only once a hit is generated on the look up table in accordance with operation 420 and/or operation 430.
  • the GUI may be provided on a display that includes a touch screen display.
  • the GUI may enable a user to zoom in and out of the displayed image via a touch operation of the touch screen (e.g., the user operating the touch screen with a finger, stylus, or other input device).
  • the microscope platform may be configured to receive a zoom in command from the user and return to the location of a hit, change and/or modify the magnification objective, and re- image the captured cell at the hit location for verification based on the received zoom command.
  • the GUI may also be configured to allow a user to zoom in on a hit, as described above, mark the morphology (cell periphery), and generate a histogram of analysis on a selected area of the captured cell (e.g., nucleus or cytoplasm).
  • the GUI may also allow the user to visually or physically rotate the cell for confirmation on biomarker localization in the captured cell. In some embodiments, user rotation rotates the displayed image, while in other embodiments user rotation may cause the physical microfluidic device to rotate relative to the imaging device of the microscope platform.
  • the GUI highlights hits for data tracking.
  • all hits that are confirmed via the high magnification objective analysis are highlighted on the look up table for data tracking.
  • the precise location of the hits for each scan is also saved as a checkmark for data tracking.
  • all hits that are confirmed via high magnification objective analysis are highlighted or indicated on the visual display.
  • a software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of non- transitory storage medium known in the art.
  • An exemplary computer-readable storage medium is coupled to the processor such the processor can read information from, and write information to, the computer-readable storage medium.
  • the storage medium may be integral to the processor.
  • the processor and the storage medium may reside in an ASIC.
  • the ASIC may reside in a user terminal, camera, or other device.
  • the processor and the storage medium may reside as discrete components in a user terminal, camera, or other device.

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Abstract

L'invention concerne des procédés et des systèmes pour commander une plateforme de microscope pour déterminer des cellules d'intérêt dans un échantillon contenant une pluralité de cellules, chaque cellule étant disposée dans un trou traversant sur l'un d'une pluralité de filtres sur une puce microfluidique, chacun de la pluralité de filtres ayant une zone active comprenant une pluralité de trous traversants agencés selon un motif. Un procédé consiste à générer une première image d'un filtre à puce microfluidique, à déterminer des informations d'emplacement pour des cellules d'intérêt candidates qui sont disposées dans des trous traversants de filtre, à aligner la plateforme de microscope afin de capturer une image de chaque cellule d'intérêt candidate à l'aide des informations d'emplacement stockées pour aligner la plateforme de microscope afin de capturer une image à l'emplacement du trou traversant où la cellule d'intérêt candidate est disposée, et à générer une seconde image, avec une résolution supérieure à celle de la première image, représentant chaque cellule d'intérêt candidate.
PCT/US2017/049731 2016-09-13 2017-08-31 Procédés et dispositifs d'imagerie d'objets sur une puce microfluidique WO2018052730A1 (fr)

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CN111513729A (zh) * 2020-05-07 2020-08-11 吉林大学 一种高效的产前诊断间期胎儿细胞检测装置
CN112867916A (zh) * 2018-08-17 2021-05-28 恩里奇疗法公司 选择和隔离颗粒和细胞的系统和方法及其用途

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US20130258091A1 (en) * 2010-12-14 2013-10-03 The Regents Of The University Of California Method and device for holographic opto-fluidic microscopy
WO2015177654A2 (fr) * 2014-05-01 2015-11-26 King Abdullah University Of Science And Technology Dispositif microfluidique qui sépare des cellules
WO2016118484A1 (fr) * 2015-01-23 2016-07-28 Basetra Medical Technology Co. Ltd. Détection de cellules fœtales basée sur la microfluidique et isolement pour des tests prénataux non invasifs

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US20130258091A1 (en) * 2010-12-14 2013-10-03 The Regents Of The University Of California Method and device for holographic opto-fluidic microscopy
WO2015177654A2 (fr) * 2014-05-01 2015-11-26 King Abdullah University Of Science And Technology Dispositif microfluidique qui sépare des cellules
WO2016118484A1 (fr) * 2015-01-23 2016-07-28 Basetra Medical Technology Co. Ltd. Détection de cellules fœtales basée sur la microfluidique et isolement pour des tests prénataux non invasifs

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112867916A (zh) * 2018-08-17 2021-05-28 恩里奇疗法公司 选择和隔离颗粒和细胞的系统和方法及其用途
EP3830546A4 (fr) * 2018-08-17 2022-06-08 Enrich Therapeutics Inc. Système et procédé permettant de sélectionner et d'isoler des particules et des cellules et leurs utilisations
US11994456B2 (en) 2018-08-17 2024-05-28 Enrich Biosystems Inc. System and method to select and isolate particles and cells and uses thereof
CN112867916B (zh) * 2018-08-17 2024-06-07 恩里奇生物系统有限公司 选择和隔离颗粒和细胞的系统和方法及其用途
CN111513729A (zh) * 2020-05-07 2020-08-11 吉林大学 一种高效的产前诊断间期胎儿细胞检测装置

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