Patent Application
20220364961
Some embodiments of the present disclosure are directed to processing biological specimens. More particularly, certain embodiments of the present disclosure provide systems and methods for chemically processing an array of biological specimens simultaneously. Merely by way of example, the present disclosure has been applied to process the array of biological specimens to improve throughput. But it would be recognized that the present disclosure has much broader range of applicability.
It is often desired to chemically process biological specimens for subsequent analysis. For example, the biological specimens may be stained for various imaging purposes. Conventional methods rely on the user of glass slides which limit throughput. Accordingly, there exists a need to develop techniques that can improve the processing of biological specimens.
Some embodiments of the present disclosure are directed to processing biological specimens. More particularly, certain embodiments of the present disclosure provide systems and methods for chemically processing an array of biological specimens simultaneously. Merely by way of example, the present disclosure has been applied to process the array of biological specimens to improve throughput. But it would be recognized that the present disclosure has much broader range of applicability.
According to certain embodiments, a method for processing biological specimens includes loading a plurality of biological specimens on a continuous substrate at a plurality of spaced-apart locations on the continuous substrate. Also, the method includes attaching a plurality of wells to the continuous substrate corresponding to the plurality of spaced-apart locations. Each well of the plurality of wells is configured to surround a respective biological specimen of the plurality of biological specimens on the continuous substrate. Further, the method includes performing one or more chemical processes on the respective biological specimen contained in the each well of the plurality of wells. Moreover, the method includes analyzing results of the one or more chemical processes on the respective biological specimen contained in the each well of the plurality of wells.
According to some embodiments, a system for processing biological specimens includes one or more processors and a memory storing instructions for execution by the one or more processors. The instructions, when executed by the one or more processors, cause the system to load a plurality of biological specimens on a continuous substrate at a plurality of spaced-apart locations on the continuous substrate. Also, the instructions, when executed by the one or more processors, cause the system to attach a plurality of wells to the continuous substrate corresponding to the plurality of spaced-apart locations. Each well of the plurality of wells is configured to surround a respective biological specimen of the plurality of biological specimens on the continuous substrate. Further, the instructions, when executed by the one or more processors, cause the system to perform one or more chemical processes on the respective biological specimen contained in the each well of the plurality of wells. Moreover, the instructions, when executed by the one or more processors, cause the system to process results of the one or more chemical processes on the respective biological specimen contained in the each well of the plurality of wells.
Depending upon the embodiment, one or more benefits may be achieved. These benefits and various additional objects, features and advantages of the present disclosure can be fully appreciated with reference to the detailed description and accompanying drawings that follow.
Some embodiments of the present disclosure are directed to processing biological specimens. More particularly, certain embodiments of the present disclosure provide systems and methods for chemically processing an array of biological specimens simultaneously. Merely by way of example, the present disclosure has been applied to process the array of biological specimens to improve throughput. But it would be recognized that the present disclosure has much broader range of applicability.
In some embodiments, the substrate 102 includes a first surface 108 and a second surface 110. For example, the first surface 108 is disposed on the base 104, and the second surface 110 is configured to support a biological specimen 112 (e.g., a tissue section, a blood sample, etc.).
In various embodiments, the substrate 102 is continuous. For example, the substrate 102 is formed by a continuous sheet. In some embodiments, the substrate 102 may be flexible, foldable, rollable, stretchable, etc. In certain embodiments, the substrate 102 may be rigid. In some embodiments, the substrate 102 includes an adhesive film such as a tape. For example, the second surface 110 of the substrate 102 includes the adhesive film that allows the biological specimen 112 to be attached to the second surface 110. In certain embodiments, the substrate 102 includes a non-adhesive film.
In
In certain embodiments, the compressive element 106 and the base 104 cooperate to secure the compressive element 106 to the substrate 102. For example, the base 104 includes a first magnetic material (e.g., a permanent magnet) and the compressive element 106 includes a second magnetic material (e.g., a metal). As an example, the second magnetic material of the compressive element 106 is magnetically attracted to the first magnetic material of the base 104 such that the compressive element 106 is secured on the substrate 102. For example, the magnetic attraction between the compressive element 106 and the base 104 allows the well 116 to be securely held in place. In some examples, other suitable means (e.g., pneumatic, mechanical, or hydraulic mechanism) can be used to create and secure the well 116 on the substrate 102. In certain examples, mechanisms that use neither magnetic nor pneumatic/hydraulic forces can be used to create and secure the well 116 on the substrate 102.
In some embodiments, contact between the compressive element 106 and the base 104 forms a seal 122. For example, once the well 116 is created, the one or more reagents can be introduced into the well 116 for chemically processing the biological specimen 114. As an example, the seal 122 is configured to retain the one or more reagents in the well 116. In
In
In various embodiments, throughput improvements can be realized with a continuous substrate mounted with multiple wells. In certain embodiments, the multiple wells are configured as a liquid handling station in an addressable array such that each well assumes a known spatial position and is independently available for any chemical processing. In some embodiments, each well is positioned on the continuous substrate in a way that aligns the position of each well to a corresponding biological specimen. For example, N biological specimens are arranged in a row with the same center-to-center spacing D, and each well is positioned onto the continuous substrate with the same spacing. As an example, once N wells are aligned with N biological specimens, chemical processing can be performed in each well. For example, the wells are detached after chemical processing and the continuous substrate is mounted with another set of N biological specimens and N wells to repeat the procedure.
In certain embodiments, each biological specimen is identified virtually (e.g., its position known from prior information of its relative order on the substrate). In some embodiments, each biological specimen is identified physically (e.g., by printing or engraving an identification label such as alphanumeric characters, barcodes, etc.) on at least one layer of the continuous substrate. For example, the identification labels contain metadata such as an absolute position of the biological specimen on the continuous substrate, a relative position of the biological specimen on the continuous substrate to aid image registration, information on the origin of the biological specimen, instructions for the intended chemical process of the biological specimen, etc.
In some embodiments, by recording the address of a well each time that chemical processing is performed and by recording the spatial description of each biological specimen when loaded onto the continuous substrate, the relationship between each biological specimen and its sequence in the chemical processing can be retained for future use. For example, this relationship is recorded in electronic media (e.g., by inserting into a database during each step of a workflow). As an example, this relationship is useful when interpreting stained features of multichannel Z-stacks or in the correlation of specific transcript signals with spatial position in tissues.
At the process 210, the base is disposed on a first surface of a substrate according to certain embodiments. In some embodiments, the substrate includes a second surface. In certain embodiments, the substrate is formed by a continuous piece of tape that is flexible, foldable, rollable, stretchable, etc.
At the process 220, the biological specimen is disposed on the second surface of the substrate according to certain embodiments. In some embodiments, the second surface of the substrate includes an adhesive film that allows the biological specimen to be attached to the second surface.
At the process 230, the compressive element is secured on the second surface of the substrate that surrounds the biological specimen according to certain embodiments. In some embodiments, the compressive element includes an opening opposite the second surface of the substrate on which the compressive element is placed. For example, the opening is configured to form a well that allows one or more reagents to be introduced into the well for chemically processing the biological specimen.
In certain embodiments, the compressive element and the base cooperate to secure the compressive element to the substrate to form a seal which is configured to retain the one or more reagents in the well. In some examples, the seal is formed by a section of the compressive element in direct contact with the second surface of the substrate. In certain examples, the seal is formed by an elastomer element disposed between the compressive element and the second surface of the substrate.
At the process 310, a plurality of biological specimens are loaded on a continuous substrate according to certain embodiments. For example, the continuous substrate is a strip of tape that is flexible, foldable, rollable, stretchable, etc. In some embodiments, the plurality of biological specimens are loaded at a plurality of spaced-apart locations on the continuous substrate. For example, the plurality of biological specimens are placed at designated locations on the continuous substrate. In certain embodiments, machine vision can be employed to automatically align and verify the placement of the plurality of biological specimens at the plurality of spaced-apart locations on the continuous substrate.
At the process 320, a plurality of wells are attached to the continuous substrate corresponding to the plurality of spaced-apart locations according to certain embodiments. In various embodiments, each well is configured to surround a respective biological specimen on the continuous substrate. In some embodiments, attaching the plurality of wells includes sealing each well so that reagents can be introduced into the well. In certain embodiments, each well may be protected by receiving a cover. In some embodiments, a gripper can be employed to automatically attach the plurality of wells to the continuous substrate as well as to cover or uncover each well.
In certain embodiments, the plurality of biological specimens are uncovered prior to attaching the plurality of wells to the continuous substrate. For example, each biological specimen may be protected by a cover (e.g., protective liner). As an example, the cover is removed so that subsequent chemical processing can take place. In some embodiments, a tape roller can be employed to automatically uncover the plurality of biological specimens.
At the process 330, one or more chemical processes are performed on the respective biological specimen contained in each well according to certain embodiments. In various embodiments, performing the one or more chemical processes includes adding one or more reagents to the respective biological specimen contained in the each well. For example, each well is filled with a reagent that interacts with the respective biological specimen. In various embodiments, chemically processing a biological specimen involves dispensing the reagent, waiting for the reagent to interact with the biological specimen, and then removing the reagent. In certain embodiments, a robot can be employed to automatically pipette reagents (or other liquids) in and out of each well. For example, the robot is a pipette head on a gantry.
In some embodiments, the plurality of wells are detached from the continuous substrate after performing the one or more chemical processes. In certain embodiments, a gripper can be employed to automatically detach the plurality of wells from the continuous substrate. In some embodiments, following detachment of the plurality of wells, the plurality of biological specimens are covered for protection. For example, each biological specimen is laminated with another cover. In certain embodiments, a tape roller can be employed to automatically cover the plurality of biological specimens.
At the process 340, the results of the one or more chemical processes on the respective biological specimen contained in each well are processed according to certain embodiments. For example, one or more imaging or other techniques are employed to process (e.g., analyze) the results. As an example, processing the results includes an operator reviewing digital images and selecting any number of the one or more chemical processes for manual analysis. In various embodiments, one or more processes of the method 300 may be repeated sequentially for any desired number of times.
In some embodiments, spatial coordinates of each well on the continuous substrate are recorded such that the relationship between the biological specimen in each well and any subsequent chemical processing on that biological specimen in the well can be retained for future use.
In certain embodiments, the spatial coordinates of one or more first wells that surround one or more first biological specimens respectively are identified and recorded. For example, one or more first chemical processes may be performed on the one or more first biological specimens. As an example, the spatial coordinates of the one or more first wells subject to the one or more first chemical processes are identified and recorded.
In some embodiments, the spatial coordinates of one or more second wells that surround one or more second biological specimens respectively are identified and recorded. For example, one or more second chemical processes may be performed on the one or more second biological specimens. As an example, the spatial coordinates of the one or more second wells subject to the one or more second chemical processes are identified and recorded. In certain embodiments, the one or more second wells may not be subject to any chemical processes. For example, the spatial coordinates of the one or more second wells not subject to any chemical processes are also identified and recorded.
In some embodiments, the one or more chemical processes include a deparaffinization (DP) process, a histological staining (HIS) process, an antigen retrieval (AR) process, an immunohistochemical staining (IHC) process, a lysis (LP) process, an immunofluorescence staining (IF) process, an in situ hybridization (ISH) process, a spatial transcriptomics (ST) process, mounting cover film (MT) process, and/or a cell culture development (CC) process. In certain embodiments, each chemical process uses a particular protocol. In various embodiments, only a subset of the one or more chemical processes are performed at a given time and in any desired order.
In some examples, the DP process uses the following protocol: submerge once in xylene for 3 minutes, submerge once in fresh xylene for 4 minutes, submerge once in fresh xylene for 5 minutes, submerge twice in 100% ethanol for 3 minutes each, submerge once in 90% ethanol for 3 minutes, submerge once in 70% ethanol for 3 minutes, submerge once in 50% ethanol for 3 minutes, and submerge once in distilled water for 5 minutes.
In certain examples, the HIS process uses the following protocol: submerge in Mayer's hematoxylin for 5 minutes, submerge in tap water for 5 minutes, submerge in eosin Y for 2 minutes, wash with 70% reagents alcohol for 30 seconds to remove excess eosin, and submerge in distilled water for at least 3 minutes and up to 3 hours. In some embodiments, other staining types include trichrome, pentachrome, Verhoeff-Van Gieson stain and/or periodic acid-Schiff.
In some examples, the AR process uses the following protocol: add PBS, mix PBS and trypsin solution from an antigen retrieval kit in a 1:1 ratio, incubate with trypsin solution for 10 minutes at room temperature, and rinse five times with PBS.
In certain examples, the IHC process uses the following protocol: incubate in 3% hydrogen peroxide in (diluted once from 30% PBS) for 10 minutes, rinse in PBS for five times, block at room temperature for 10 minutes in a blocking buffer (5% glycerol, 5% cold water fish skin gelatin, 5% normal serum, 85% PBS), incubate in primary Ab (diluted in blocking buffer) for 1 hour at room temperature in humid chamber, rinse in PBS for five times, incubate in ready-to-use secondary antibody for 30 minutes at room temperature, rinse in PBS for five times, incubate in DAB for 15 minutes, and stop reaction by rinsing for 5 minutes in deionized water. In some embodiments, IHC counterstaining uses the above protocol with two additional steps: incubate in Mayer's hematoxylin for 15 minutes at room temperature and incubate in 0.1% sodium bicarbonate for 5 minutes at room temperature.
In some examples, the LP process uses the following protocol: use a lysis kit, pipette in 50 μL of lysis buffer, seal in humid environment provided by RNAse-free water, hold at 60° C. for 90 minutes, hold at −20° C. for 2 minutes, pipette out lysis buffer and into LoBind RNAse-free microfuge tubes, and proceed with rest of lysis kit as outlined in kit manual.
In certain examples, the IF process uses the following protocol: transfer to magnetic staining plate, apply PBS liberally to keep wet, transfer off PBS, apply a blocking buffer, incubate for 30 minutes, dilute primary antibody to appropriate dilution in blocking buffer, transfer off blocking buffer, apply primary antibody, incubate in the dark with primary antibody for 1 hour at room temperature or overnight in a humid environment at 4° C., remove primary antibody, washed five times with PBS, apply secondary antibody, incubate for 1 hour at room temperature in the dark, remove secondary antibody, and wash five times with PBS in the dark.
In some examples, the ISH process uses the following protocol: perform DP and AR processes, wash five times in distilled water, immerse in ice cold 20% acetic acid for 20-30 seconds, apply a 1 minute wash of 70% ethanol, apply a 1 minute wash of 95% ethanol, apply a 1 minute wash of 100% ethanol, air dry, immerse in hybridization solution (formamide, salts, Denhardt's solution, dextran sulphate, heparin, SDS), incubate in humid environment at 60° C. for 1 hour, dilute probes in hybridization solution, heat at 95° C. for 2 minutes, chill on ice, remove hybridization solution, add diluted probes, incubate in humid environment at 65° C. overnight, wash in Wash 1 (formamide in saline sodium citrate) three times for 5 minutes each at 37-45° C., wash in Wash 2 (saline sodium citrate) three times for 5 minutes each at 25-75° C., wash twice in MABT (maleic acid buffer Tween 20) for 30 minutes at room temperature, dry and place back in humid environment, add blocking buffer (MABT+BSA/milk/serum), incubate for 1-2 hours, remove blocking buffer, add secondary antibody, incubate for 1-2 hours at room temperature, wash five times for 10 minutes each with MABT at room temperature, wash twice for 10 minutes each with prestaining buffer (Tris/NaCl/MgCl2), place back in humid environment, and perform color development.
In some examples, the ST process uses the following protocol: coat substrate with spatially encoded DNA barcode array, lyse and capture onto barcode array, optionally ligate mRNA transcripts with array, and dissociate barcoded mRNA transcripts from substrate and extract.
In certain examples, the MT process uses the following protocol: cover in distilled water, drop one drop of mountant solution (30 g fructose in 13 mL distilled water), leave meniscus of mountant solution, laminate with cover film.
In some examples, the CC process uses the following protocol: optionally dissociate cells, maintain in controlled environment, and extract cells for additional analysis.
In various embodiments, the computing device 402 is communicatively coupled to the roller system 404 for implementing the methods described in the present disclosure. For example, the computing device 402 executes one or more operations to implement the method 200 and/or the method 300. In certain embodiments, the computing device 402 includes a processor 406 and a memory 408. For example, the processor 406 is configured to execute instructions to perform the various operations associated with the method 200 and/or the method 300. As an example, the instructions are stored in the memory 408.
In some embodiments, the roller system 404 includes a starting roller 410, one or more driver rollers 412, one or more guide rollers 414, and a finishing roller 416. For example, the roller system 404 operates to move a continuous substrate 420.
In certain embodiments, a plurality of biological specimens are attached to the continuous substrate 420. In some embodiments, the plurality of biological specimens are initially protected by a cover (e.g., protective liner). In certain embodiments, the plurality of biological specimens are uncovered at a location 422 in the roller system 404. In some embodiments, after uncovering the plurality of biological specimens, a plurality of wells 424 and a base 426 cooperate to attach the plurality of wells 424 to the continuous substrate 420, where each well is configured to surround a respective biological specimen on the continuous substrate 420.
In various embodiments, the plurality of biological specimens in the plurality of wells 424 are chemically processed simultaneously. In some embodiments, after chemical processing, the plurality of wells 424 are detached from the continuous substrate 420. In certain embodiments, the plurality of biological specimens are re-covered for protection at a location 428 in the roller system 404.
In certain embodiments, one or more biological specimens 512 are prepared in the cryotome 502. For example, the one or more biological specimens 512 are covered with a protective liner and placed under freezing condition until needed. In some embodiments, the one or more biological specimens 512 may be non-frozen (e.g., paraffin sections).
In various embodiments, the one or more biological specimens 512 are attached to a continuous substrate 514. For example, the continuous substrate 514 is formed by a continuous sheet of tape. In certain embodiments, metadata 516 associated with each of the one or more biological specimens 512 are determined and recorded. For example, the metadata 516 include information such as a position of each biological specimen on the continuous substrate 514, an origin or source of each biological specimen, instructions for an intended chemical process of each biological specimen, etc.
In some embodiments, the roller system 504 moves the continuous substrate 514 to facilitate movement of the one or more biological specimens 512 between different stations. In certain embodiments, visual inspection is performed at a location 518 on the continuous substrate 514 by the imaging station 506. For example, machine vision can be employed to align and verify the placement of the one or more biological specimens 512 on the continuous substrate 514. As an example, the resulting data are digitally registered into a Z-stack dataset 520. In various embodiments, each of the one or more biological specimens 512 may be identified by a respective label 522 (e.g., a barcode).
In certain embodiments, one or more chemical processes are performed at the one or more assay stations 508, 510. For example, at a location 524 on the continuous substrate 514, the one or more biological specimens 512 are stained for imaging by the assay station 508. As an example, whole slide imaging scans 526 are performed on the one or more biological specimens 512 by the assay station 508. For example, at a location 528 on the continuous substrate 514, the one or more biological specimens 512 are masked and lysed by the assay station 510. As an example, the lysed materials 530 are extracted for further analysis 532 by the assay station 510.
In various embodiments, the workflows 600 to 1900 shown in
In certain embodiments, different types of contrast can be combined to support correlative and multi-channel images such that images in adjacent specimens provide complementary knowledge of cell and tissue states (see
In some embodiments, the data are a combination of microscope imagery and molecular assay results (see
According to certain embodiments, a method for processing biological specimens includes loading a plurality of biological specimens on a continuous substrate at a plurality of spaced-apart locations on the continuous substrate. Also, the method includes attaching a plurality of wells to the continuous substrate corresponding to the plurality of spaced-apart locations. Each well of the plurality of wells is configured to surround a respective biological specimen of the plurality of biological specimens on the continuous substrate. Further, the method includes performing one or more chemical processes on the respective biological specimen contained in the each well of the plurality of wells. Moreover, the method includes analyzing results of the one or more chemical processes on the respective biological specimen contained in the each well of the plurality of wells. For example, the method is implemented according to at least
According to some embodiments, a system for processing biological specimens includes one or more processors and a memory storing instructions for execution by the one or more processors. The instructions, when executed by the one or more processors, cause the system to load a plurality of biological specimens on a continuous substrate at a plurality of spaced-apart locations on the continuous substrate. Also, the instructions, when executed by the one or more processors, cause the system to attach a plurality of wells to the continuous substrate corresponding to the plurality of spaced-apart locations. Each well of the plurality of wells is configured to surround a respective biological specimen of the plurality of biological specimens on the continuous substrate. Further, the instructions, when executed by the one or more processors, cause the system to perform one or more chemical processes on the respective biological specimen contained in the each well of the plurality of wells. Moreover, the instructions, when executed by the one or more processors, cause the system to process results of the one or more chemical processes on the respective biological specimen contained in the each well of the plurality of wells. For example, the system is implemented according to at least
For example, some or all components of various embodiments of the present disclosure each are, individually and/or in combination with at least another component, implemented using one or more software components, one or more hardware components, and/or one or more combinations of software and hardware components. In another example, some or all components of various embodiments of the present disclosure each are, individually and/or in combination with at least another component, implemented in one or more circuits, such as one or more analog circuits and/or one or more digital circuits. In yet another example, while the embodiments described above refer to particular features, the scope of the present disclosure also includes embodiments having different combinations of features and embodiments that do not include all of the described features. In still another example, various embodiments and/or examples of the present disclosure can be combined.
Additionally, the methods and systems described herein may be implemented on many different types of processing devices by program code comprising program instructions that are executable by the device processing subsystem. The software program instructions may include source code, object code, machine code, or any other stored data that is operable to cause a processing system to perform the methods and operations described herein. Certain implementations may also be used, however, such as firmware or even appropriately designed hardware configured to perform the methods and systems described herein.
The systems' and methods' data (e.g., associations, mappings, data input, data output, intermediate data results, final data results) may be stored and implemented in one or more different types of computer-implemented data stores, such as different types of storage devices and programming constructs (e.g., RAM, ROM, EEPROM, Flash memory, flat files, databases, programming data structures, programming variables, IF-THEN (or similar type) statement constructs, application programming interface). It is noted that data structures describe formats for use in organizing and storing data in databases, programs, memory, or other computer-readable media for use by a computer program.
The systems and methods may be provided on many different types of computer-readable media including computer storage mechanisms (e.g., CD-ROM, diskette, RAM, flash memory, computer's hard drive, DVD) that contain instructions (e.g., software) for use in execution by a processor to perform the methods' operations and implement the systems described herein. The computer components, software modules, functions, data stores and data structures described herein may be connected directly or indirectly to each other in order to allow the flow of data needed for their operations. It is also noted that a module or processor includes a unit of code that performs a software operation, and can be implemented for example as a subroutine unit of code, or as a software function unit of code, or as an object (as in an object-oriented paradigm), or as an applet, or in a computer script language, or as another type of computer code. The software components and/or functionality may be located on a single computer or distributed across multiple computers depending upon the situation at hand.
The computing system can include client devices and servers. A client device and server are generally remote from each other and typically interact through a communication network. The relationship of client device and server arises by virtue of computer programs running on the respective computers and having a client device-server relationship to each other.
This specification contains many specifics for particular embodiments. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations, one or more features from a combination can in some cases be removed from the combination, and a combination may, for example, be directed to a subcombination or variation of a subcombination.
Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. In certain circumstances, multitasking and parallel processing may be advantageous. Moreover, the separation of various system components in the embodiments described above should not be understood as requiring such separation in all embodiments, and it should be understood that the described program components and systems can generally be integrated together in a single software product or packaged into multiple software products.
Although specific embodiments of the present disclosure have been described, it will be understood by those of skill in the art that there are other embodiments that are equivalent to the described embodiments. Accordingly, it is to be understood that the present disclosure is not to be limited by the specific illustrated embodiments.
This application claims priority to U.S. Provisional Patent Application No. 63/189,308, filed May 17, 2021, incorporated by reference herein for all purposes. The following two applications, including this one, are being filed concurrently and the other application is hereby incorporated by reference in its entirety for all purposes: 1. U.S. patent application Ser. No. ______, titled “Devices and Methods for Handling Biological Specimens” (Attorney Docket Number 520295.000012); and 2. U.S. patent application Ser. No. ______, titled “Systems and Methods for Processing Biological Specimens” (Attorney Docket Number 520295.000013).
| Number | Date | Country | |
|---|---|---|---|
| 63189308 | May 2021 | US |
