WO2024067254A1 - Parallelized circularly-arrayed plaftform for high-speed cell imaging - Google Patents

Parallelized circularly-arrayed plaftform for high-speed cell imaging Download PDF

Info

Publication number
WO2024067254A1
WO2024067254A1 PCT/CN2023/119758 CN2023119758W WO2024067254A1 WO 2024067254 A1 WO2024067254 A1 WO 2024067254A1 CN 2023119758 W CN2023119758 W CN 2023119758W WO 2024067254 A1 WO2024067254 A1 WO 2024067254A1
Authority
WO
WIPO (PCT)
Prior art keywords
platform
cell
chambers
ultrahigh
assay
Prior art date
Application number
PCT/CN2023/119758
Other languages
French (fr)
Inventor
Man Dik Dickson SIU
Kin Man TSIA
Original Assignee
The University Of Hong Kong
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Hong Kong filed Critical The University Of Hong Kong
Publication of WO2024067254A1 publication Critical patent/WO2024067254A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N35/00069—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
    • 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
    • B01L3/502707—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 characterised by the manufacture of the container or its components
    • 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
    • B01L3/502715—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 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • 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
    • G01N15/1425—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement
    • 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
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • 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

Definitions

  • the present invention relates to the field of multiple bioassay chambers, and in particular, to bioassay chambers with rotatory imaging cell assay platforms.
  • a similar rotating platform system is disclosed in US Patent No. 8,420,026. It has a centrifugal force-based microfluidic device that includes a rotation operating unit that rotates a body of revolution, and an external energy source which irradiates a sample with electromagnetic waves.
  • the body of revolution includes the microfluidic structure disposed in the body of revolution, the microfluidic structure including a plurality of chambers, channels connecting the chambers, and valves disposed in the channels to control fluid flow.
  • the microfluidic structure transmits the fluid using centrifugal force due to rotation of the body of revolution; and magnetic beads contained in one of the chambers, which collect a target material from a biomaterial sample flowing into the chamber.
  • the microfluidic structure washes the magnetic beads which collect the target material, and separates nucleic acid by electromagnetic wave irradiation.
  • the design does not take into account microscopic imaging.
  • a different technique disclosed in US Patent No. 10,162,162 uses a microfluidic device to create microvortex that rotates one cell or/clusters around its centre for tomographic imaging.
  • the aim is to rotate the cells so that they can produce 3D images of each cell/cluster one by one.
  • No spinning platform is used so the throughput is limited to single cells or clusters.
  • the chamber has a trapezoidal cross-sectional shape located below the flow channel to create the microvortex.
  • US Application Publication 2022-0195486 also discloses a multiplexable microfluidic culture chamber for imaging monolayer growth of single cells.
  • the cell chamber is divided by a thin PDMS membrane into upper and lower compartments.
  • the lower compartment is used for culturing biological samples.
  • the membrane is also used to "press” and trap the samples in place as they do not necessarily attach to the bottom of the chamber.
  • the movement of the membrane is controlled by the pneumatic pressure in the upper compartment.
  • the design is specified for automatically generating concentration gradients of different chemicals. This design also relies on a flow controller, to provide precise control of the flow rate and the movement of the membrane.
  • Live cells must always be cultured in culture medium. So, for the spinning imaging techniques, the chamber must be sealed to avoid leakage of the liquid medium. These liquid-tight sealings are usually irreversible. Therefore, there is no way to access the cells during or after the assay. It can be important to retrieve the cells after the imaging assay for other genetic and biological assays. Furthermore, more complex experiments that required the addition of chemicals during the assay are not feasible with sealed chambers. Last but not least, the isolation between the chamber and outer environment due to the sealing also disables long time-course assays because it suffocates the cells.
  • US Patent No. 9,612,199 B2 discloses a system for imaging captured cells utilizing an autofocus module to maintain consistent image quality across wells containing the cells. The images are snapshots of each well taken while the wells are stationary. Thus, the process is very slow.
  • US Patent No. 7,709,248 B2 describes a circularly arrayed substrate for fluorescence-based bioassay. Its substrate is intended for detection of fluorescence-labelled substances, such as protein, DNA, lipid molecules. No imaging is used.
  • the present invention is directed to a large-scale single-cell intrinsic morphological profiling strategy using ultrahigh-throughput scanning field of view (FOV) imaging combined with a novel spinning on-the-fly cell-based assay platform.
  • FOV scanning field of view
  • This integrated system demonstrates unprecedented functional assay capability in not only scaling up the assay throughput, but also empowering new cytometric power to perform multiplexed live-cell drug screening (e.g., 96 conditions in a single run) and genetic perturbation assay (by CRISPR) .
  • This platform thus allows for the generation of large cellular quantitative phase imaging (QPI) datasets (e.g., 4.85 TBytes) that could spearhead cost-effective label-free solutions for identifying disease-related or gene-related cellular morphological phenotypes in therapeutic screening.
  • QPI quantitative phase imaging
  • This invention is designed to hold multiple bioassay chambers on a spinning platform to achieve parallel assays similar to commercially available well plates (in 96 wells or more) , but with improved features that make it uniquely compatible with high-resolution and high-throughput spinning imaging assays.
  • the chambers are arranged in circular arrays (or in rings) to allow rapid rotation and imaging.
  • the assay sample can be 2D mono-layer adherent cells, 3D cell cultures, or 3D tissues/organoids.
  • one embodiment features fluidically designed adherent cell chambers concentrically and circularly arrayed for spinning imaging techniques.
  • the platform has chambers that consist of 3 UV-cured adhesive bonded, specially patterned fused silica plates that ensure tight tolerance with regard to substrate flatness for rapid, large field of view, high-resolution optical imaging.
  • the chamber is designed to allow direct contact with the outer environment, which flavors cell growth at all times in order to ensure long-term cell health and continuous access to the medium and cells within, while remaining leak-free during high-speed spinning motion (No upper speed limit) .
  • the design does not require complete sealing while spinning to avoid leakage, so it allows for the retrievability, accessibility and viability of the cells.
  • the platform contains specially designed chambers with a shape that contains two channels for inlet and outlet, which lead to openings located at an inner radius of the platform.
  • This design prevents the liquid medium inside the chamber from spilling out at high-rotational speed even if the openings are unsealed.
  • the liquid medium inside the chamber is allowed to remain in contact with the outer environment.
  • the gaseous components e.g. O 2 and CO 2
  • the medium can remain balanced indefinitely. It ensures the cell health during the assay and allows long time course cell studies. Further, the cells can also be retrieved afterwards.
  • the design utilizes 3 fused silica substrates or plates to define the base, roof and sides of the chamber arrangement.
  • the substrates are glued together using an optical UV-cured adhesive (NOA61) .
  • NOA61 optical UV-cured adhesive
  • This fabrication process allows for tighter tolerance and thus minimizes inconsistent image quality across the chambers. Consistent image quality is the essential element for quantitative cell analysis, so that the change of cell images across chambers can be ensured to be based upon the biophysical phenotypes of the cells themselves (e.g. morphology and dry mass) , instead of fabrication variations.
  • the invention has the following features:
  • the design allows the medium inside to be in contact with air at all times, including the time during spinning, to ensure cell health. It further allows long-term, time-course cell studies.
  • the assay sample can be extended from 2D mono-layer adherent cells to 3D cell cultures and 3D tissues/organoids.
  • the number of the chambers is highly scalable to 384 wells, and 1536 wells or beyond, depending on the fabrication technology.
  • the wells can be patterned in 2D in a single layer or in 3D in multiple layers.
  • This new platform uses three laser patterned fused-silica wafers as its substrate. This ensures tight thickness and flatness tolerances of the platform, i.e., within ⁇ 100 ⁇ m. This essentially narrows down the imaging focal plane variations across chambers, reduces the tuning distance of the focus and thus allows for more rapid imaging workflow.
  • the whole design is tailored for high-throughput, parallelized, long-term image-based cell assays that are largely utile (rare) for chemical (drug) screening, genetic (e.g., by CRISPR technology) screening and cell profiling applications.
  • FIG. 1A is an overview of a high-speed rotational imaging substrate with sample wells or chambers
  • FIG. 1B is an enlarged view of a specified region of the substrate to detail the dimensions of a chamber
  • FIG. 1C is an exploded view of a region with a chamber, detailing the patterns on each fused-silica wafer
  • FIG. 1D is an assembled illustration of the exploded view of FIG. 1C;
  • FIG. 2A is a scaled-up example of the design of FIG. 1A with additional wells or chambers, a larger diameter substrate and two layers of chambers
  • FIG. 2B is an enlarged view of a specified region of the substrate in dash-line to detail the dimensions of a chamber in FIG. 2A
  • FIG. 2C is a sectional side view at the line specified in FIG. 2B illustrating a double-deck design
  • FIG. 2D is a 3D enlarged view of the region specified in FIG. 2A in dash-line;
  • FIG. 3 is a prospective illustration of the platform of a high-speed rotational imaging system according to the present invention including part of an imaging system;
  • FIG. 4 is a flow chart of the overall workflow for fabricating the high-speed rotational imaging platform of the present invention.
  • FIG. 5A is a prospective illustration of an experimental configuration of the present invention including the imaging system
  • FIG. 5B shows an image of the spinning platform
  • FIG. 5C shows and enlarged part of the chamber showing living cells in quantitative phase imaging contrast
  • FIG. 5D shows further enlarged areas of the chamber containing cell clusters from designated portions of FIG. 5C;
  • FIG. 6A shows the experimental conditions at the outermost ring of a spinning disk, the inner most ring of the spinning disk and a stationary disk and FIG. 6B shows the viability of living cells under the conditions of FIG. 6A..
  • the cell assay platform features fluidically designed cell chambers 20 concentrically and circularly arrayed on a substrate 10 as shown in FIGS. 1A and 2A.
  • the rotating substrate in FIG. 1A is designed to have, for example, a diameter of 120mm with 96 wells or chambers. These cell chambers are used in spinning imaging techniques, e.g. QPI, as shown in FIG. 3.
  • the cell chambers 20 are designed to achieve the following conditions while remaining leak-free during high-speed spinning motion: (1) allow direct contact with the outer environment at all times to ensure long-term cell health, (2) allow continuous access to the sample fluid medium and cells within the chamber and (3) allow retrieval of cells after the assay.
  • the whole design is tailored for high-throughput, parallelized, long-term image-based cell assays that are largely utile (rare) for drug or CRISPR screening and cell profiling applications. The conditions mentioned above are not achievable in any of the prior art products and designs.
  • the chambers 20 utilize a complete sealing strategy (often irreversible) to avoid leakage during high-speed spinning.
  • the chambers 20 can be arranged in concentric circular rows (four rows 11, 12, 13, 14 in the case of FIG. 1A) .
  • Each chamber 20 is divided into 3 main parts: the assay region 22, a pair of inlet/outlet channels 24 and an opening 25 (FIG. 1B) .
  • the assay region 22 of the chamber 20 has the cell sample attached to its bottom, i.e., the cell’s have the intrinsic ability to attach to the bottom surface. In order to aid this the bottom surface is coated with a layer of protein (e.g., fibronectin) .
  • the inlet/outlet channels 24 deliver the fluid medium to the assay region 22.
  • the arms that form the channels with the assay region 22 can be uniform and in FIG. 1B the arms are 1mm in width.
  • the openings 25 allow micropipettes to pump liquid through the channels 24 so as to deliver the fluid into the assay region 22.
  • the openings are designed to face toward an inner radial position of the whole platform compared to the assay region. This keeps the liquid in the assay region from being forced out by centrifugal force during spinning of the substrate 10, while maintaining the open-to-outer environment condition before, during and after spinning.
  • the diameter of the openings (1mm and 1.5mm in the embodiment of FIG. 1B) is designed according to the diameter of the pipette tips in order to allow effective pumping of liquid through the channels to the assay region. This is illustrated in FIG. 1B, where one hole is designed for medium tips (100-200 ⁇ L) as well as large tips (1000 ⁇ L) , so both tips can access the chamber with ease.
  • the chamber wall is designed to avoid sharp corners and sudden changes in channel widths.
  • the liquid front will progress and fill the chamber in a controlled manner without forming air bubbles.
  • FIG. 1C is an exploded view of a region of the substrate with a chamber, detailing the patterns on each fused-silica wafer.
  • the top layer 31 is 0.5mm in thickness and has two holes in it.
  • Middle layer 33 is 1mm thick and has the shape of the assay region 22 and the channels 24 cut in it.
  • the bottom layer is 0.5mm thick and is flat without any cut-outs.
  • the assembled chamber of this embodiment is illustrated in FIG. 1D. It has an overall thickness of 2mm.
  • FIG. 2A is a scaled-up example of the design of the platform of FIG. 1A with 384-wells and a double-deck design with the light-colored chambers being on the lower layer and the dark-colored chamber being on the upper layer. See FIG. 2C. Its platform is 158mm in diameter. Depending on the fabrication technology it can be further scaled-up to 1536 wells or beyond.
  • FIG. 2B is an enlarged view of a specified region of the platform of FIG. 2A in order to detail the dimensions of a chamber. In this embodiment the assay regions are 5mm and the arms that form the channels are 0.5mm.
  • FIG. 2C is a sectional side view at the line specified in FIG. 2B.
  • FIG. 2D is a 3D enlarged view of the region specified in FIG. 2A and shown in FIG. 2B. Note that the upper and lower chambers are staggered with respect to each other so that each assay region can be illuminated as shown in FIG. 3.
  • FIG. 3 The optical system includes an objective lens 42 to relay light from a light source to focus onto the cells and another objective lens 44 to relay the light to an image receptor.
  • the optical system includes an objective lens 42 to relay light from a light source to focus onto the cells and another objective lens 44 to relay the light to an image receptor.
  • the imaging system images the outer ring 11 of the chambers only. However, it can be moved over all of the other rings 11-14. Because of the tight tolerance of the substrate flatness, rapid, large field of view, high-resolution optical imaging there is little need for autofocusing.
  • step 502 the patterns for the design are implemented in CAD software. Then a laser is used to cut the patterns on the fused silica wafers as shown in FIG. 1C in step 503. Next, in step 504 the bottom wafer 35 and the middle wafer 33 (FIG. 1C) are bonded using bonding steps 602-607.
  • step 602 the wafers are sequentially washed in mild detergent, acetone and IPA, and then are assembled. The wafers are then dried thoroughly and gently wiped using Kimwipe in step 603.
  • step 604 a UV-cured adhesive is applied onto the bonding surface of the middle wafer 33.
  • step 605 the wafers are aligned and the middle wafer 33 is attached to the bottom wafer 35. The attached wafers are then sandwiched between 2 glass slabs and uniform compressive stress is applied to the assembly to hold the wafers together (e.g., using clamps) in step 606.
  • step 607 the assembly is illuminated with a 365 nm light source to pre-cure the adhesive used in the structure. The pre-cure, final cure and aging times depend on the UV-cured adhesive used.
  • step 505 the bonding steps 602-607 are repeated for adhering the top wafer 31 to the middle-bottom assembly.
  • the top-middle-bottom assembly is cleaned with an acetone-soaked Kimwipe to remove excess glue in step 506.
  • step 507 the assembly is illuminated with a 365nm light source for a full cure of the adhesive.
  • the precure, final cure and aging times depend on the UV-cured adhesive used.
  • the assembly is stored in order to age the adhesive, thereby creating stronger bonds and chemical resistances.
  • the present invention utilizes a chamber design with a main assay region and a pair of channels leading to an opening positioned at an inner radial position of the platform. This physical difference makes the present invention stand out compared to the other existing platforms of the same type.
  • a drug assay was performed on two lung cancer cell lines (NCI-H1975 and NCI-H2170) .
  • This QPI-based cell assay exhibits reasonably high specificity/sensitivity to four different drugs with diverse mechanisms of action, i.e., microtubule depolymerizer (docetaxel) , DNA disturber (cisplatin and gemcitabine) , and epidermal growth factor receptor (EGFR) targeted drug (erlotinib) .
  • microtubule depolymerizer docetaxel
  • DNA disturber cisplatin and gemcitabine
  • EGFR epidermal growth factor receptor
  • a CRISPR-based cell-cell fusion assay was developed in which human cells expressing the receptor ACE2 (with gene knock out) were co-cultured with cells expressing SARS-CoV-2 spike.
  • the large QPI datasets that were generated helped to provide deeper assessments of how the underlying machinery of viral entry correlates/impacts the intrinsic cell morphology.
  • This profiling strategy can open up new potentials in identifying novel disease-gene-related morphological phenotypes that serve as cost-effective probes for therapeutics development.
  • FIG. 5A-5C show the setup and images obtained with the optical system of the present invention.
  • the images are of living cells in quantitative phase imaging (QPI) contrast, a contrast typically used to show cells features.
  • FIG. 5A is a prospective illustration of an experimental configuration like that of FIG. 3 but showing the entire platform.
  • FIG. 5A the 120 mm diameter platform is spun at 1300 rpm.
  • FIG. 5B shows an image of the spinning platform which illustrates the large area in which high resolution imaging is achieved with the present invention.
  • FIG. 5C shows an enlarged part of the chamber showing the living cells in QPI contrast and
  • FIG. 5D shows further enlarged areas of the chamber related to designated portions of FIG. 5C.
  • QPI is a microscopy method that quantifies the phase shift that occurs when light waves pass through a more optically dense object like translucent living human cells. The shift can be indicated by a color change as shown in FIG. 5D.
  • FIG. 6A & 6B show the viability of living cells under various conditions of the spinning platform of the present invention. These conditions are at the outermost ring of the spinning platform for one hour, the innermost ring of the spinning platform for one hour and a static or stationary disk for one hour..
  • FIG. 6B shows bar graphs that demonstrate that there is no significant difference in viability and thus the spinning motion does not cause cell death.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Signal Processing (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

An ultrahigh-throughput quantitative phase imaging (QPI) system incorporating a spinning on-the-fly cell-based assay platform with concentric rings of cell chambers (20). Each chamber (20) has an assay region (22) and a pair of inlet/outlet channels (24) with an opening (25) between their ends. The inlet/outlet channels (24) are adapted to deliver a fluid with a cell sample that has been introduced into the opening (25) to the assay region (22). The openings (25) face toward an inner radial position of the spinning platform compared to the assay region (22) so as to keep the liquid in the assay region (22) from being forced out by centrifugal force during spinning of the platform, while being open to the outer environment before, during and after spinning.

Description

PARALLELIZED CIRCULARLY-ARRAYED PLAFTFORM FOR HIGH-SPEED CELL IMAGING
Cross-Reference to Related Patent Applications
This application claims the benefit of priority to U.S. Application No. 63/410,363 filed September 27, 2022, which is incorporated herein by reference in its entirety.
Field of the Invention
The present invention relates to the field of multiple bioassay chambers, and in particular, to bioassay chambers with rotatory imaging cell assay platforms.
Background of the Invention
Cell morphology is well-recognized as an underpinning of the determination of complex cell types/states/functions. Hence, image-based screening assays have been increasingly popular in drug discovery. Current strategies largely involve fluorescence labelling, which however becomes laborious when multiple fluorescence labels are needed. Cell-based assays are currently replacing conventional, population-based assays due to their superiority in analysing cells individually. Optical microscopic imaging is one of the effective means for carrying this out. However, to discover rare subpopulations of cells or produce representable results of the cell population (usually>106) , an enormous number of cells must be analysed. Another requisite of these assays is the ability to analyse multiple conditions at one time.
To attain these criteria, a high-throughput, parallelized imaging system is needed. One technique for increasing the speed by which assays can be imaged is through the use of high-speed spinning-disk cytometry platforms. By leveraging the spinning motion for high-speed scanning, a class of high-throughput imaging techniques were developed to perform parallelized imaging-based assays. See Tang, A. H., et al., “Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay, ” Biomedical Optics Express, 8 (2) , 640-652 (2017) . Similarly, US Application Publication 2021-0381979A1 describes a method that utilizes high-speed spinning motion for cell/tissue imaging assays. The cells in this are irreversibly sealed with UV glue in order to prevent any culture medium leakage during spinning. With the power  of high-throughput imaging, these techniques can potentially be applied in many cell-based assays, including but not limited to drug discovery, CRISPR screening and cellular biophysical profiling. However, several practical issues hinder mainstream use of these techniques.
A similar rotating platform system is disclosed in US Patent No. 8,420,026. It has a centrifugal force-based microfluidic device that includes a rotation operating unit that rotates a body of revolution, and an external energy source which irradiates a sample with electromagnetic waves. The body of revolution includes the microfluidic structure disposed in the body of revolution, the microfluidic structure including a plurality of chambers, channels connecting the chambers, and valves disposed in the channels to control fluid flow. The microfluidic structure transmits the fluid using centrifugal force due to rotation of the body of revolution; and magnetic beads contained in one of the chambers, which collect a target material from a biomaterial sample flowing into the chamber. As a result, the microfluidic structure washes the magnetic beads which collect the target material, and separates nucleic acid by electromagnetic wave irradiation. However, the design does not take into account microscopic imaging.
A different technique disclosed in US Patent No. 10,162,162 uses a microfluidic device to create microvortex that rotates one cell or/clusters around its centre for tomographic imaging. The aim is to rotate the cells so that they can produce 3D images of each cell/cluster one by one. No spinning platform is used so the throughput is limited to single cells or clusters. Further, the chamber has a trapezoidal cross-sectional shape located below the flow channel to create the microvortex.
US Application Publication 2022-0195486 also discloses a multiplexable microfluidic culture chamber for imaging monolayer growth of single cells. The cell chamber is divided by a thin PDMS membrane into upper and lower compartments. The lower compartment is used for culturing biological samples. The membrane is also used to "press" and trap the samples in place as they do not necessarily attach to the bottom of the chamber. The movement of the membrane is controlled by the pneumatic pressure in the upper compartment. The design is specified for automatically generating concentration gradients of different chemicals. This design also relies on a flow controller, to provide precise control of the flow rate and the movement of the membrane.
Live cells must always be cultured in culture medium. So, for the spinning imaging techniques, the chamber must be sealed to avoid leakage of the liquid medium. These liquid-tight sealings are usually irreversible. Therefore, there is no way to access the cells during or after the assay. It can be important to retrieve the cells after the imaging assay for other genetic and biological assays. Furthermore, more complex experiments that required the addition of chemicals during the assay are not feasible with sealed chambers. Last but not least, the isolation between the chamber and outer environment due to the sealing also disables long time-course assays because it suffocates the cells.
Another issue is the image quality variations across chambers of plastic-based substrates that make further analysis difficult. To achieve quantitative analysis, the chambers must have tolerances that minimize aberrations and focus variations that produce inconsistent image qualities across chambers.
US Patent No. 9,612,199 B2 discloses a system for imaging captured cells utilizing an autofocus module to maintain consistent image quality across wells containing the cells. The images are snapshots of each well taken while the wells are stationary. Thus, the process is very slow. US Patent No. 7,709,248 B2 describes a circularly arrayed substrate for fluorescence-based bioassay. Its substrate is intended for detection of fluorescence-labelled substances, such as protein, DNA, lipid molecules. No imaging is used.
International patent application WO2018-045978A1 describes the use of polycarbonate sheets and spacers to construct the substrate. Leakage is prevented by using completely UV-cured glue sealed chambers. However, the patent does not specify any chamber shape.
Summary of the Invention
The present invention is directed to a large-scale single-cell intrinsic morphological profiling strategy using ultrahigh-throughput scanning field of view (FOV) imaging combined with a novel spinning on-the-fly cell-based assay platform. This integrated system demonstrates unprecedented functional assay capability in not only scaling up the assay throughput, but also empowering new cytometric power to perform multiplexed live-cell drug screening (e.g., 96  conditions in a single run) and genetic perturbation assay (by CRISPR) . This platform thus allows for the generation of large cellular quantitative phase imaging (QPI) datasets (e.g., 4.85 TBytes) that could spearhead cost-effective label-free solutions for identifying disease-related or gene-related cellular morphological phenotypes in therapeutic screening.
This invention is designed to hold multiple bioassay chambers on a spinning platform to achieve parallel assays similar to commercially available well plates (in 96 wells or more) , but with improved features that make it uniquely compatible with high-resolution and high-throughput spinning imaging assays. The chambers are arranged in circular arrays (or in rings) to allow rapid rotation and imaging. The assay sample can be 2D mono-layer adherent cells, 3D cell cultures, or 3D tissues/organoids.
While the invention can have a plurality of embodiments, one embodiment features fluidically designed adherent cell chambers concentrically and circularly arrayed for spinning imaging techniques. The platform has chambers that consist of 3 UV-cured adhesive bonded, specially patterned fused silica plates that ensure tight tolerance with regard to substrate flatness for rapid, large field of view, high-resolution optical imaging.
The chamber is designed to allow direct contact with the outer environment, which flavors cell growth at all times in order to ensure long-term cell health and continuous access to the medium and cells within, while remaining leak-free during high-speed spinning motion (No upper speed limit) . The design does not require complete sealing while spinning to avoid leakage, so it allows for the retrievability, accessibility and viability of the cells.
In particular, the platform contains specially designed chambers with a shape that contains two channels for inlet and outlet, which lead to openings located at an inner radius of the platform. This design prevents the liquid medium inside the chamber from spilling out at high-rotational speed even if the openings are unsealed. Thus, the liquid medium inside the chamber is allowed to remain in contact with the outer environment. Particularly, the gaseous components (e.g. O2 and CO2) in the medium can remain balanced indefinitely. It ensures the cell health during the assay and allows long time course cell studies. Further, the cells can also be retrieved afterwards.
As noted, the design utilizes 3 fused silica substrates or plates to define the base, roof and sides of the chamber arrangement. The substrates are glued together using an optical UV-cured adhesive (NOA61) . This fabrication process allows for tighter tolerance and thus minimizes inconsistent image quality across the chambers. Consistent image quality is the essential element for quantitative cell analysis, so that the change of cell images across chambers can be ensured to be based upon the biophysical phenotypes of the cells themselves (e.g. morphology and dry mass) , instead of fabrication variations.
The utilization of fused silica and UV-cured adhesive for fabrication of the substrates minimizes the thickness variations (<10μm) to the minimum and keeps the whole platform as flat as possible (<100μm variation across the 120mm diameter) . These tolerances minimize the aberration and focus variations that produce inconsistent image qualities across chambers compare to plastic-based substrates.
The invention has the following features:
● Accessibility to cell samples and culture medium inside the chamber in the middle or after the rotational imaging assay. The design allows the medium inside to be in contact with air at all times, including the time during spinning, to ensure cell health. It further allows long-term, time-course cell studies. The assay sample can be extended from 2D mono-layer adherent cells to 3D cell cultures and 3D tissues/organoids.
● The design of chamber geometry and channel shapes ensure that the liquid medium for the specimen fills up the well completely without forming air bubbles, which is important for stable spinning imaging.
● The number of the chambers is highly scalable to 384 wells, and 1536 wells or beyond, depending on the fabrication technology. The wells can be patterned in 2D in a single layer or in 3D in multiple layers.
● This new platform uses three laser patterned fused-silica wafers as its substrate. This ensures tight thickness and flatness tolerances of the platform, i.e., within～100μm. This  essentially narrows down the imaging focal plane variations across chambers, reduces the tuning distance of the focus and thus allows for more rapid imaging workflow.
The whole design is tailored for high-throughput, parallelized, long-term image-based cell assays that are largely utile (rare) for chemical (drug) screening, genetic (e.g., by CRISPR technology) screening and cell profiling applications.
Brief Description of the Drawings
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing (s) will be provided by the Office upon request and payment of the necessary fee.
The foregoing and other objects and advantages of the present invention will become more apparent when considered in connection with the following detailed description and appended drawings in which like designations denote like elements in the various views, and wherein:
FIG. 1A is an overview of a high-speed rotational imaging substrate with sample wells or chambers, FIG. 1B is an enlarged view of a specified region of the substrate to detail the dimensions of a chamber, FIG. 1C is an exploded view of a region with a chamber, detailing the patterns on each fused-silica wafer and FIG. 1D is an assembled illustration of the exploded view of FIG. 1C;
FIG. 2A is a scaled-up example of the design of FIG. 1A with additional wells or chambers, a larger diameter substrate and two layers of chambers, FIG. 2B is an enlarged view of a specified region of the substrate in dash-line to detail the dimensions of a chamber in FIG. 2A, FIG. 2C is a sectional side view at the line specified in FIG. 2B illustrating a double-deck design and FIG. 2D is a 3D enlarged view of the region specified in FIG. 2A in dash-line;
FIG. 3 is a prospective illustration of the platform of a high-speed rotational imaging system according to the present invention including part of an imaging system;
FIG. 4 is a flow chart of the overall workflow for fabricating the high-speed rotational imaging platform of the present invention;
FIG. 5A is a prospective illustration of an experimental configuration of the present invention including the imaging system, FIG. 5B shows an image of the spinning platform, FIG. 5C shows and enlarged part of the chamber showing living cells in quantitative phase imaging contrast and FIG. 5D shows further enlarged areas of the chamber containing cell clusters from designated portions of FIG. 5C; and
FIG. 6A shows the experimental conditions at the outermost ring of a spinning disk, the inner most ring of the spinning disk and a stationary disk and FIG. 6B shows the viability of living cells under the conditions of FIG. 6A..
Detailed Description of the Invention
In the present invention, the cell assay platform features fluidically designed cell chambers 20 concentrically and circularly arrayed on a substrate 10 as shown in FIGS. 1A and 2A. The rotating substrate in FIG. 1A is designed to have, for example, a diameter of 120mm with 96 wells or chambers. These cell chambers are used in spinning imaging techniques, e.g. QPI, as shown in FIG. 3. The cell chambers 20 are designed to achieve the following conditions while remaining leak-free during high-speed spinning motion: (1) allow direct contact with the outer environment at all times to ensure long-term cell health, (2) allow continuous access to the sample fluid medium and cells within the chamber and (3) allow retrieval of cells after the assay. The whole design is tailored for high-throughput, parallelized, long-term image-based cell assays that are largely utile (rare) for drug or CRISPR screening and cell profiling applications. The conditions mentioned above are not achievable in any of the prior art products and designs.
The main, novel elements that enable the mentioned features are in the design of the new chamber shape, inlet/outlet channels and the placement of openings. In the existing technologies, the chambers utilize a complete sealing strategy (often irreversible) to avoid leakage during high-speed spinning. Here in the present invention, the chambers 20 can be arranged in concentric circular rows (four rows 11, 12, 13, 14 in the case of FIG. 1A) . Each chamber 20 is divided into 3 main parts: the assay region 22, a pair of inlet/outlet channels 24 and an opening 25 (FIG. 1B) . The assay region 22 of the chamber 20 has the cell sample attached to its bottom, i.e., the cell’s have the intrinsic ability to attach to the bottom surface. In  order to aid this the bottom surface is coated with a layer of protein (e.g., fibronectin) . The inlet/outlet channels 24 deliver the fluid medium to the assay region 22.
The arms that form the channels with the assay region 22 can be uniform and in FIG. 1B the arms are 1mm in width. The openings 25 allow micropipettes to pump liquid through the channels 24 so as to deliver the fluid into the assay region 22. The openings are designed to face toward an inner radial position of the whole platform compared to the assay region. This keeps the liquid in the assay region from being forced out by centrifugal force during spinning of the substrate 10, while maintaining the open-to-outer environment condition before, during and after spinning. The diameter of the openings (1mm and 1.5mm in the embodiment of FIG. 1B) is designed according to the diameter of the pipette tips in order to allow effective pumping of liquid through the channels to the assay region. This is illustrated in FIG. 1B, where one hole is designed for medium tips (100-200μL) as well as large tips (1000μL) , so both tips can access the chamber with ease.
To ensure the whole chamber is filled with the desired liquid medium during pipetting and to prevent the formation of air bubbles, the chamber wall is designed to avoid sharp corners and sudden changes in channel widths. When pipetting liquid into the chamber, the liquid front will progress and fill the chamber in a controlled manner without forming air bubbles.
The chambers consist of 3 UV-cured adhesive bonded, laser patterned fused silica plates 31, 33, 35 to ensure tight tolerance of the substrate flatness for rapid, large field of view, high-resolution optical imaging. FIG. 1C This construction minimizes the thickness variations (<10μm) to the minimum and keeps the whole platform as flat as possible (<100μm variation across the 120mm diameter) . FIG. 1C is an exploded view of a region of the substrate with a chamber, detailing the patterns on each fused-silica wafer. In this embodiment the top layer 31 is 0.5mm in thickness and has two holes in it. Middle layer 33 is 1mm thick and has the shape of the assay region 22 and the channels 24 cut in it. The bottom layer is 0.5mm thick and is flat without any cut-outs. The assembled chamber of this embodiment is illustrated in FIG. 1D. It has an overall thickness of 2mm.
FIG. 2A is a scaled-up example of the design of the platform of FIG. 1A with 384-wells and a double-deck design with the light-colored chambers being on the lower layer and the  dark-colored chamber being on the upper layer. See FIG. 2C. Its platform is 158mm in diameter. Depending on the fabrication technology it can be further scaled-up to 1536 wells or beyond. FIG. 2B is an enlarged view of a specified region of the platform of FIG. 2A in order to detail the dimensions of a chamber. In this embodiment the assay regions are 5mm and the arms that form the channels are 0.5mm. FIG. 2C is a sectional side view at the line specified in FIG. 2B. It illustrates the double-deck design where in this embodiment the upper and lower chambers are 1mm in thickness and are separated by a 0.5mm space vertically. FIG. 2D is a 3D enlarged view of the region specified in FIG. 2A and shown in FIG. 2B. Note that the upper and lower chambers are staggered with respect to each other so that each assay region can be illuminated as shown in FIG. 3.
In conducting rapid, large field of view, high-resolution optical imaging, a general imaging system configuration 40 is employed. FIG. 3. The optical system includes an objective lens 42 to relay light from a light source to focus onto the cells and another objective lens 44 to relay the light to an image receptor. As light is directed onto chambers 20 it passes through the chambers on the substrate 10, which consists of the 3 UV-cured adhesive bonded, specially patterned fused silica chambers, and the specimen. The result is an image of the specimen that can be used for single-cell intrinsic morphological profiling studies. In FIG. 3, the imaging system images the outer ring 11 of the chambers only. However, it can be moved over all of the other rings 11-14. Because of the tight tolerance of the substrate flatness, rapid, large field of view, high-resolution optical imaging there is little need for autofocusing.
A more detailed fabrication protocol or flow chart is shown in FIG. 4. In step 502 the patterns for the design are implemented in CAD software. Then a laser is used to cut the patterns on the fused silica wafers as shown in FIG. 1C in step 503. Next, in step 504 the bottom wafer 35 and the middle wafer 33 (FIG. 1C) are bonded using bonding steps 602-607.
In bonding step 602 the wafers are sequentially washed in mild detergent, acetone and IPA, and then are assembled. The wafers are then dried thoroughly and gently wiped using Kimwipe in step 603. In step 604 a UV-cured adhesive is applied onto the bonding surface of the middle wafer 33. Next, in step 605 the wafers are aligned and the middle wafer 33 is attached to the bottom wafer 35. The attached wafers are then sandwiched between 2 glass slabs and  uniform compressive stress is applied to the assembly to hold the wafers together (e.g., using clamps) in step 606. Then, in step 607 the assembly is illuminated with a 365 nm light source to pre-cure the adhesive used in the structure. The pre-cure, final cure and aging times depend on the UV-cured adhesive used.
After the bottom wafer 35 and the middle wafer 33 are bonded, in step 505 the bonding steps 602-607 are repeated for adhering the top wafer 31 to the middle-bottom assembly. The top-middle-bottom assembly is cleaned with an acetone-soaked Kimwipe to remove excess glue in step 506. In step 507 the assembly is illuminated with a 365nm light source for a full cure of the adhesive. As in step 607, the precure, final cure and aging times depend on the UV-cured adhesive used. Finally in step 508, the assembly is stored in order to age the adhesive, thereby creating stronger bonds and chemical resistances.
None of the existing spinning imaging assay technologies can achieve no-leak and no-seal at the same time. The present invention utilizes a chamber design with a main assay region and a pair of channels leading to an opening positioned at an inner radial position of the platform. This physical difference makes the present invention stand out compared to the other existing platforms of the same type.
Two large-scale functional image-based assays (4.85 TBytes) have been achieved with the present invention using QPI, which has been largely underexploited in the past. First, a drug assay was performed on two lung cancer cell lines (NCI-H1975 and NCI-H2170) . This QPI-based cell assay exhibits reasonably high specificity/sensitivity to four different drugs with diverse mechanisms of action, i.e., microtubule depolymerizer (docetaxel) , DNA disturber (cisplatin and gemcitabine) , and epidermal growth factor receptor (EGFR) targeted drug (erlotinib) . Second, a CRISPR-based cell-cell fusion assay was developed in which human cells expressing the receptor ACE2 (with gene knock out) were co-cultured with cells expressing SARS-CoV-2 spike. The large QPI datasets that were generated helped to provide deeper assessments of how the underlying machinery of viral entry correlates/impacts the intrinsic cell morphology. This profiling strategy can open up new potentials in identifying novel disease-gene-related morphological phenotypes that serve as cost-effective probes for therapeutics development.
FIG. 5A-5C show the setup and images obtained with the optical system of the present invention. The images are of living cells in quantitative phase imaging (QPI) contrast, a contrast typically used to show cells features. FIG. 5A is a prospective illustration of an experimental configuration like that of FIG. 3 but showing the entire platform. In FIG. 5A the 120 mm diameter platform is spun at 1300 rpm. FIG. 5B shows an image of the spinning platform which illustrates the large area in which high resolution imaging is achieved with the present invention. FIG. 5C shows an enlarged part of the chamber showing the living cells in QPI contrast and FIG. 5D shows further enlarged areas of the chamber related to designated portions of FIG. 5C. QPI is a microscopy method that quantifies the phase shift that occurs when light waves pass through a more optically dense object like translucent living human cells. The shift can be indicated by a color change as shown in FIG. 5D.
FIG. 6A & 6B show the viability of living cells under various conditions of the spinning platform of the present invention. These conditions are at the outermost ring of the spinning platform for one hour, the innermost ring of the spinning platform for one hour and a static or stationary disk for one hour.. FIG. 6B shows bar graphs that demonstrate that there is no significant difference in viability and thus the spinning motion does not cause cell death.
While the invention is explained in relation to certain embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. Therefore, it is to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims.

Claims (13)

  1. An ultrahigh-throughput scanning field of view imaging system comprising:
    a spinning on-the-fly cell-based assay platform with at least one ring of cell chambers, wherein each chamber has an assay region, a pair of inlet/outlet channels with an opening between their ends, the inlet/outlet channels are adapted to deliver to the assay region a fluid medium for a cell sample that is introduced to the openings, the openings face toward an inner radial position of the spinning platform compared to the assay region so as to keep the liquid in the assay region from being forced out by centrifugal force during spinning of the platform, while being open to the outer environment before, during and after spinning; and
    an imaging system for passing light through the cell chambers and receiving an image of the cell sample.
  2. The ultrahigh-throughput scanning field of view system of claim 1 wherein the chambers are arranged in a plurality of concentric rings and the relative movement between the imaging system and the platform is possible, so as to bring the imaging area of the system from ring to ring.
  3. The ultrahigh-throughput scanning field of view system of claim 1 wherein the platform has at least two layers with chambers on each layer, the chambers on one layer being laterally offset from the chambers in the other layer so as to permit tight arrangement of the chamber assay regions without overlapping, and only the non-assay regions (e.g. the inlet/outlet channels) are overlapped.
  4. The ultrahigh-throughput scanning field of view system of claim 1 wherein the cell chambers are made from multiple stacked UV-cured adhesive bonded, laser patterned fused silica plates, where each chamber has an upper plate with two holes in it, a middle plate has an assay region with a pair of inlet/outlet channels and an opening between their ends formed by a laser, and a bottom plate is flat without any cut-outs, the channels lead from the opening at an inner radius of the platform to the assay region at a more outer radius of the platform; and wherein the silica plates are bound together with UV-cured adhesive.
  5. The ultrahigh-throughput scanning field of view system of claim 1 wherein the assay sample can be 2D mono-layer adherent cells, 3D cell cultures or 3D tissues/organoids.
  6. The ultrahigh-throughput scanning field of view system of claim 1 wherein the chamber is designed to allow direct contact with the outer environment that flavors cell growth at all times in order to ensure long-term sample cell health, continuous access to the medium and sample cells within the chamber so substances can be added or removed and retrieval of sample cells after assay, while remaining leak-free during high-speed spinning motion.
  7. The ultrahigh-throughput scanning field of view system of claim 1 wherein the speed is in the range of 100 rpm to 6,000 rpm for a 120 mm platform. -
  8. The ultrahigh-throughput scanning field of view system of claim 1 wherein the variation across a 120 mm diameter platform is less than 100μm.
  9. The ultrahigh-throughput scanning field of view system of claim 5 wherein the openings are 0.5-1.5 mm in diameter, the top layer is 0.5 mm thick, the middle layer is 1 mm thick and the bottom layer is 0.5 mm thick and the chamber has an overall thickness of 1 mm.
  10. The ultrahigh-throughput scanning field of view system of claim 2 wherein the platform has four (4) concentric rings and a total of 96 chambers evenly spaced.
  11. The ultrahigh-throughput scanning field of view system of claim 4 wherein the platform has twelve (12) concentric rings and a total of 384 chambers evenly spaced.
  12. A method of fabricating a chamber for an ultrahigh-throughput scanning filed of view system comprising the steps of:
    implementing the patterns for the design in CAD software;
    using a laser to cut the patterns on the fused silica wafers;
    bonding the bottom wafer and the middle wafer;
    repeating the bonding steps 602-for adhering the top wafer to the middle-bottom assembly;
    cleaning the top-middle-bottom assembly with an acetone-soaked Kimwipe to remove excess glue;
    illuminating the assembly with a light source for a full cure of the adhesive; and
    storing the assembly in order to age the adhesive, thereby creating stronger bonds and chemical resistances.
  13. The method of claim 13 wherein the step of bonding comprises the steps of:
    sequentially washed the wafers in mild detergent, acetone and IPA;
    assembling the wafers;
    thoroughly drying the wafers and then gently wiping them using Kimwipe;
    applying a UV-cured adhesive onto the bonding surface of one wafer;
    aligning the one wafer with another wafer and attaching the wafers;
    sandwiching the two wafers between 2 glass slabs and applying uniform compressive stress to hold the wafers together; and
    illuminating the wafters with a 365 nm light source to cure the adhesive.
PCT/CN2023/119758 2022-09-27 2023-09-19 Parallelized circularly-arrayed plaftform for high-speed cell imaging WO2024067254A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263410363P 2022-09-27 2022-09-27
US63/410,363 2022-09-27

Publications (1)

Publication Number Publication Date
WO2024067254A1 true WO2024067254A1 (en) 2024-04-04

Family

ID=90476188

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/119758 WO2024067254A1 (en) 2022-09-27 2023-09-19 Parallelized circularly-arrayed plaftform for high-speed cell imaging

Country Status (1)

Country Link
WO (1) WO2024067254A1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1894586A (en) * 2001-11-19 2007-01-10 长冈实业株式会社 Methods and apparatus for blood typing with optical bio-discs
KR20080071786A (en) * 2007-01-31 2008-08-05 삼성전자주식회사 Centrifugal force-based microfluidic device for cell culture and cell-based assay and microfluidic system comprising the same
US20100317094A1 (en) * 2008-02-04 2010-12-16 Dublin City University milk analysis microfluidic apparatus for detecting mastitis in a milk sample
CN109844542A (en) * 2016-09-07 2019-06-04 港大科桥有限公司 The device and method of rotation imaging bioassay for multiplexing
CN111164412A (en) * 2017-08-17 2020-05-15 雅培医护站股份有限公司 Method of imaging blood cells
CN112424329A (en) * 2018-02-21 2021-02-26 耐克思乐生物科学有限责任公司 Systems and methods for cell imaging, analysis, and measurement

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1894586A (en) * 2001-11-19 2007-01-10 长冈实业株式会社 Methods and apparatus for blood typing with optical bio-discs
KR20080071786A (en) * 2007-01-31 2008-08-05 삼성전자주식회사 Centrifugal force-based microfluidic device for cell culture and cell-based assay and microfluidic system comprising the same
US20100317094A1 (en) * 2008-02-04 2010-12-16 Dublin City University milk analysis microfluidic apparatus for detecting mastitis in a milk sample
CN109844542A (en) * 2016-09-07 2019-06-04 港大科桥有限公司 The device and method of rotation imaging bioassay for multiplexing
CN111164412A (en) * 2017-08-17 2020-05-15 雅培医护站股份有限公司 Method of imaging blood cells
CN112424329A (en) * 2018-02-21 2021-02-26 耐克思乐生物科学有限责任公司 Systems and methods for cell imaging, analysis, and measurement

Similar Documents

Publication Publication Date Title
Vasdekis et al. Review of methods to probe single cell metabolism and bioenergetics
US10488321B2 (en) Devices and methods for high-throughput single cell and biomolecule analysis and retrieval in a microfluidic chip
Liu et al. Microfluidic systems for biosensing
US10190082B2 (en) Multiwell plate
Vitorino et al. Microfluidics for peptidomics, proteomics, and cell analysis
US7238521B2 (en) Microarray hybridization device having bubble-fracturing elements
US20020110839A1 (en) Micro-array evanescent wave fluorescence detection device
Xu et al. Forming a large-scale droplet array in a microcage array chip for high-throughput screening
US20080274451A1 (en) Body for flow-through cells and the use thereof
JP7500560B2 (en) Device and method for microdroplet detection of cells
JP2014514926A (en) Cell culture equipment
CN110437992A (en) A kind of liquid phase sample is extensive, fast digitizing decomposition chip and its application method
WO2005007796A2 (en) Improved multiwell plate
Fu et al. Microfluidic systems applied in solid-state nanopore sensors
Huang et al. Droplet detection and sorting system in microfluidics: a review
Han et al. Photoactivated selective release of droplets from microwell arrays
CH703278B1 (en) Device and platform for multiplex analysis.
Hu et al. Microfluidics on lensless, semiconductor optical image sensors: challenges and opportunities for democratization of biosensing at the micro-and nano-scale
WO2024067254A1 (en) Parallelized circularly-arrayed plaftform for high-speed cell imaging
Walt Fiber optic array biosensors
KR102475225B1 (en) Fabrication of apparatus for real-time monitoring of organoid through R2R technology
EP3892713A1 (en) Multi-well plates for creation of high-density arrays of compartmentalized cell cultures for usage in high capacity applications
Li et al. Inkjet-Printed Bioassays for Direct Reading with a Multimode DVD/Blu-Ray Optical Drive
WO2024084958A1 (en) Detection device and method for detecting target molecules
Trietsch Microfluidic 3D cell culture for high throughput screening

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23870486

Country of ref document: EP

Kind code of ref document: A1