Automated systems and methods for tissue sectioning, staining, and scanning - Patents.com

Abstract

Disclosed is a plate or film for collecting and analyzing samples. The plate or film includes a surface and multiple slices from the sample immobilized on the surface. Furthermore, the multiple slices are cut from the sample at equal thicknesses, and the multiple slices are placed on the surface of the plate or film according to the order in which the slices are cut.

Description

[Technical field]

[0001] The field of the invention is diagnostic systems and methods. More specifically, the invention presents systems and methods in the field of clinical pathology that automate the process of sectioning, staining and imaging histological specimens and facilitate information analysis and disease diagnosis. [Background technology]

[0002] The importance of pathology to modern medicine cannot be overemphasized. For example, in cancer treatment, accurate pathological reading of invasive areas of cancer on biopsy is a critical step in enabling effective treatment design. Throughout its history of over a century, pathology-based evaluation has always been an essential part of diagnosing disease.

[0003] Despite being a traditional approach, histomorphological evaluation still relies on examining slides mounted with quality tissue sections. However, preparing good tissue slides suitable for clinical use is not only labor intensive but also requires skill from highly trained histological technicians. Currently, the supply of such qualified technicians has not kept pace with the increasing volume of tissue-based testing. Therefore, there is a need to develop new methods, preferably using automated techniques, that can minimize some of the tedious work required to prepare tissue slides.

[0004] Meanwhile, advances in the digital age, especially aided by recent artificial intelligence (AI), have created a nascent but rapidly evolving field called digital pathology. Digital pathology refers to the technologies and techniques of acquiring, storing and interpreting information about pathological specimens with digitally formatted images. Compared to traditional methods working through optical microscopes, digital pathology allows for the observation of images through computer interfaces and the transmission of tissue images through the Internet. Leveraging the rapid development of digital pathology, the field of AI opens up so-called cognitive technologies that can simulate human reasoning and perceptual abilities to analyze medical imaging to improve disease prediction and diagnosis. While digital pathology transforms static images into searchable data, facilitating the retrieval of an increasing amount of information from histological specimens, the field of AI promises to facilitate subsequent qualitative and quantitative analysis of that information.

[0005] Digital pathology, growing in partnership with AI technology, demands ever-increasing amounts of data. However, beyond the above-mentioned production challenges for a given quality of tissue section, current image scanning / acquisition also lags behind. Typical microscope-based reading of pathology images takes a given amount of time for processing and is not amenable to high-throughput approaches. Moreover, for any given tissue sample, the image is limited by current standards to only 2D views of a few random sections. How to improve current standards to obtain more information from that given tissue sample, and more importantly, how to dramatically improve scanning technology to achieve high-throughput image acquisition, becomes a challenge in the era of AI-focused digital pathology.

[0006] A need also exists for a system or method that streamlines the process from preparing a tissue slide to acquiring a digital image. Summary of the Invention

[0007] The present invention provides a system and method that streamlines the process from preparing tissue slides to acquiring digital images to meet the demands of generating multi-layer 3D images for AI-focused digital pathology. At the same time, the present invention promises to obtain more holistic information from tissue samples to enhance the coverage of histological areas for better pathology diagnosis.

[0008] One aspect of the invention relates to a method of processing a sample, the method comprising the steps of: (i) providing a sample and defining for the sample an imaginary horizontal plane and an imaginary vertical axis perpendicular to the plane; (ii) cutting the sample along the imaginary horizontal plane into a plurality of sample slices, the cuts being substantially parallel to the imaginary horizontal plane, the thicknesses of the plurality of sample slices being substantially the same, and the plurality of sample slices being sequential along the imaginary vertical axis according to a cutting order; and juxtaposing the plurality of sample slices on a support in succession according to the cutting order.

[0009] The sample in the above method may be a biological sample. In particular, the sample is a tissue, such as a formalin-fixed, paraffin-embedded (FFPE) tissue.

[0010] Similarly, in the above method, the thickness of the plurality of sample slices may be adjustable, while the support may be a membrane.

[0011] The method may further include fixing a plurality of sample slices on a support.

[0012] Another aspect of the present invention relates to a method of diagnosing a disease in a patient, comprising the steps of: (i) obtaining a sample of interest from said patient; (ii) performing the method described above; (iii) performing histological processing on the sample; (iv) obtaining imaging data from the sample; and (v) comparing and / or correlating existing histological and imaging data to assess the health of said patient.

[0013] Yet another aspect of the invention relates to a film for collecting and analyzing a sample, the film having a long and narrow surface and a number of slices from the sample fixed on the surface and aligned along the long edge of the surface, the slices being cut from the sample with equal thickness and juxtaposed on the surface of the film according to the cutting order of the slices.

[0014] Multiple slices on the surface of the film just described are simultaneously treated with one or more reactions, including histological stains, fluorescent-based stains, or other stain types.

[0015] The described film can be a plastic film. Similarly, the film is optically transparent and heat resistant up to at least 60° C. The film can have a series of holes or notches along the long edge of the surface of the film.

[0016] Yet another aspect of the invention relates to a device for collecting and processing samples. The device includes a sample cutter having a blade and a cradle configured to hold the sample. Furthermore, the blade or the cradle is movable to ensure that the blade makes a series of cuts in the sample into multiple slices having the same thickness. The device also includes a collection film having a long and narrow surface. By its design, the device is configured to apply a force to the slices after the sample cutter creates them from the sample to transfer and mount them on the collection film, and the device is also configured to align the multiple slices along the long edge of the surface and juxtapose the multiple slices in succession according to the cutting order of the multiple slices.

[0017] The device may further include a film roller that supplies the collection film.

[0018] Yet another aspect of the present invention relates to an apparatus for processing a sample, comprising the device just described and one or more sample processing modules. In the apparatus, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue, and examples of the one or more sample processing modules include a deparaffinization module, a sealing module, a sample staining module, and a sample imaging module. Preferably, the sample imaging module is configured to simultaneously capture imaging data of multiple slices of tissue.

[0019] For the sealing module, the sealing module can provide a sealant droplet or provide a transparent cover for the collecting film to cover the collecting film or to provide a transparent cover for the collecting film.

[0020] Yet another aspect of the invention relates to a device for collecting and processing samples. The device includes a sample cutter, a conveyor belt, and a collection plate. The sample cutter has a blade and a cradle configured to hold the sample. The blade or the cradle can be movable to ensure that the blade makes a series of cuts in the sample into multiple slices having the same thickness.

[0021] The device can be configured to immediately remove the slices from the sample by the conveyor belt after the sample cutter creates the slices from the sample, and thereafter unload and mount the slices on the collection plate. Further, the device can be configured to juxtapose the slices on the collection plate in succession according to the cutting order of the slices.

[0022] In one embodiment of the device, the conveyor belt is a conveyor film belt made of one layer of film. In this embodiment, the device may further include a film roller that supplies the layer of film.

[0023] Yet another aspect of the present invention relates to an apparatus for processing a sample. The apparatus includes the above-mentioned device and one or more sample processing modules. In the apparatus, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue, and the one or more sample processing modules can include a deparaffinization module. Examples of the one or more sample processing modules can also be a plate sealing module, a sample staining module, a histology staining module when the sample is a tissue, or a sample imaging module. The sample imaging module can be configured to simultaneously capture imaging data of multiple slices of tissue. Meanwhile, the plate sealing module can provide a sealant drop covering the plate, a film covering the plate, or a transparent cover for the plate, such as a glass cover.

[0024] Yet another aspect of the present invention relates to an imaging device, the imaging device including: a plate having a plurality of specimens fixed on a surface of the plate; a carrier for holding the plate; an image capture means having an image capture area; and a moving mechanism for changing a relative positional relationship between the carrier and the image capture means. In the imaging device, the image capture means is configured to simultaneously capture images of all specimens on the plate that fall within the image capture area; the imaging device is configured to divide the plate surface into a plurality of image capture areas. Furthermore, the moving mechanism moves the image capture means sequentially to the plurality of image capture areas, thereby capturing images of the plurality of specimens.

[0025] The imaging device may further include an artificial intelligence unit configured to process the digital output of the imaging device.

[0026] Another aspect of the present invention relates to a method for processing a biological sample, the method including: (i) cutting the biological sample into a plurality of slices having equal thickness; (ii) sequentially transferring the plurality of slices on a collection film having a long and narrow surface, thereby aligning the plurality of slices along the long edge of the surface; (iii) winding the collection film to form a roll having equal thickness spaces between the wound layers; (iv) staining the plurality of slices on the rolled collection film; (v) unwinding the rolled collection film; and (vi) imaging the plurality of slices on the collection film. Moreover, the imaging is performed one slice at a time as the collection film is moved through an imaging device, and includes the following features: the entire slice is captured immediately in the imaging, and the imaging is performed at a speed of 10 slices / min to 60 slices / min.

[0027] In the above-mentioned methods, the biological sample may be FFPE tissue.Similarly, the device may be configured to perform the above-mentioned methods.

[0028] The details of the invention are set forth in the drawings and description that follow. Other features, objects, and advantages of the invention will become apparent to those skilled in the art from a study of the drawings and description, and from the appended claims. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 is a diagram of the sectioning and mounting process for one variation of the high-throughput, reusable plate design. Panel (A) shows sample sectioning and transfer onto thin film. Panel (B) shows strips of film. Panel (C) shows multiple strips mounted onto a processing plate. [Diagram 2] FIG. 1 is a diagram showing the sectioning mechanism. Panel (A) shows the relative positioning and movement of the tissue block with respect to the blade. Panel (B) shows how the sample slices are transferred to the film. [Diagram 3] The movement of the block and blade during sectioning is shown. Panels (A) and (B) show before and after images of a design where the blade is fixed. Panels (C) and (D) show before and after images of a design where both the block and blade are moving during cutting. [Figure 4] 1 is a diagram showing a mechanism for maintaining precise control of positioning for a film while it is moving through a device. [Diagram 5] FIG. 1 shows how the strips are mounted on the plate. [Figure 6] FIG. 13 shows how the strips are locked into place on the plate once the plate is full. [Figure 7] FIG. 1 is a diagram showing a design that allows for continuous processing of samples on a single ribbon of film. [Figure 8] 1 is a diagram of two dye module variants. [Figure 9]Figure 1 shows an alternative design of the continuous processing module. Panel (A) shows that once the sections are mounted, the film is wound into a staining spiral. Panel (B) shows the reagent bath staining module. Panel (C) shows the scanning module, which amplifies the analog image before digital scanning and storage. [Figure 10] Figure 1 shows a quality control method for the correction of rotational errors during scanning by the use of a reference scan. Panel (A) shows the reference image. Panel (B) shows the same sample slice post-processing with errors. Panel (C) shows the corrected image of the same sample slice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Methods are provided for acquiring sequential images of a series of tissue sections. Aspects of the invention include preparing a series of parallel slices of a tissue sample, the parallel slices being substantially the same thickness. Also provided are devices and apparatus for carrying out the subject methods.

[0031] Before the method or system is described, it is understood that the present invention is not limited to the particular method described, as it may of course vary. Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings to enable those skilled in the art to practice the present invention. However, it should be understood that the present invention may be implemented in various forms, and is not intended to limit the exemplary embodiments of the present invention. Similarly, in the drawings, the description of parts that are not related to the detailed description is omitted in order to clearly describe the present invention.

[0032] The subject systems and methods are primarily useful for diagnosis, however, successful implementation of these systems and methods offers the potential to transform the practice of pathology, moving it more rapidly towards a quantitative science.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can be used in the practice or testing of this invention, the preferred method and material are described here.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material in which the publication is cited in connection therewith.

[0034] Embodiments of the present invention provide advantageous features and characteristics in the areas of tissue sections, tissue staining, cameras, image identification, and / or automated image analysis, including manufacturing methods, systems, and / or devices. It should be noted that the terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0035] The sample processed and analyzed by the present invention is a biological sample. In particular, the sample can be any tissue collected by biopsy or dissection that requires histological analysis. Before being processed by the present invention, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

[0036] A major aspect of the present invention relates to an integrated method of sample processing, including: (i) serial sample sectioning; (ii) transfer of sample sections onto a support, such as a film, which may include subsequent transfer to a multi-sample grouping and / or processing stage; (iii) deparaffinization by thermal and chemical application; (iv) tissue staining, such as, but not limited to, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) staining, immunofluorescent-based (IF) staining, or other specialized staining protocols applied by batch staining in successive reagent baths or dropwise application of reagents; (v) mountant application; (vi) analog image amplification, digital scanning, and image acquisition / storage; (vii) 3D image stacking and reconstruction; and (viii) AI analysis of processed samples. The method just described is accomplished in such a way that each step can be designed as a module or component of an integrated device, and multiple configurations of devices can implement the basic aspects of the invention depending on the particular needs of the user. Multiple and alternative configurations of the invention are detailed herein.

[0037] The present invention is designed to utilize a conventional microtome design with a cradle and blade configured to hold the sample to perform standard sectioning protocols. Traditionally, the cradle and sample are moved such that a paraffin block is pressed against the cutting edge of the blade to produce slices of uniform thickness. Alternatively, the blade can move perpendicular to the motion of the cradle to facilitate slicing; a vibratome can also be incorporated. The thickness of the sample can be adjusted by the user.

[0038] A camera can also be incorporated into the microtome design. This camera will be positioned perpendicular to and centered on the center of the surface of the tissue sample block. The camera is present to aid in quality control. The camera will take a reference picture of the block face before each slice. A scale bar or markings of known dimensions will be present on the cradle or cassette of the sample. Images can be later referenced to aid in alignment and error correction when reconstructing the stack of scanned images.

[0039] The present invention processes the sample sequentially, transferring each slice sequentially and equidistantly onto a plastic film or tape. The film is positioned close to the blade so that the slices contact and adhere to the film; the film moves at a consistent speed with the slices to minimize wrinkling or tearing of the sections during transfer to the film. By design, the net negative charge of the plastic film and the net positive charge of the FFPE tissue sample aids in the transfer. Additionally, the sample block surface and film can be sprayed with an aqueous solution prior to slicing; water coagulation will aid in the transfer. A mechanism for applying force to the slices can be included to aid in attachment. The film can also be coated with a chemical layer or adhesive that promotes attachment of the tissue slices. Multiple sample slices are mounted on the film according to the cutting order. The microtomy proceeds until the entire or desired thickness of the sample has been sliced, or in some cases, until the sample mounting stage is full.

[0040] The plastic film or tape is stored on a roll or spool and unwound at a rate consistent with the rate of specimen slicing. To maintain precise control of the film and the location of the sections on the film, the film is designed with a series of holes spaced equidistant down the length of the film and along the side edges. Sprockets are used to transport the film between modules throughout the device. The teeth of these sprockets align with the holes in the film, providing precise control of the film's movement. This is important when aligning the sample with the sealant or stain applicator and, in some variations, during scanning. The film is optically clear and heat resistant up to at least 60° C.; this temperature is adequate for deparaffinization.

[0041] Design Variations In one embodiment, the sections are transferred from the film directly onto a mounting plate. The plate stage is controlled by a CNC so that the sections are transferred to the plate in a predetermined order and within a defined area. The plate is an oversized glass or plastic slide designed to hold hundreds of serial sections from a single specimen block; however, these numbers can vary depending on the size of the specimen. Once mounted on the plate, the specimens proceed en masse to deparaffinization, staining, sealing, and scanning. The plate is transferred to storage with the entirety of the specimens mounted on the plate.

[0042] In another embodiment, the film is divided into strips, each containing multiple sections mounted on the strip. In this embodiment, the strips can be processed in two ways. In one variant, the strips are transferred to a mounting plate. Pegs in the mounting plate align with holes in the film, allowing for discrete placement of the strip(s) on the plate. Once the plate is filled with strips, a locking mechanism will hold the strips in place. The plate and mounted strips undergo deparaffinization, staining, sealing, and scanning as in the embodiment described above. However, once scanned, the strips can be removed from the plate and stored separately. The plate can then be reused as a mounting stage. Alternatively, rather than transferring the strips to the plate, they proceed directly to deparaffinization, staining, and sealing using an alternative processing module. This version is not conducive to high throughput analysis and is then more suitable for clinical or research use. Current clinical protocols process approximately 5-10 serial sections of a given tissue sample; this number of sections can fit snugly onto one strip. Studies requiring heterogeneous staining of adjacent sections can utilize alternative staining modules that individually and variably process samples along the strip with H&E, IHC, IF, or SS. In any of these alternative applications, the strip can be transferred back to the plate for collective imaging.

[0043] In yet another embodiment, the film with the mounted samples is transported sequentially through each processing module as one continuous ribbon. Once processed, the samples are wound into a spool for storage. This embodiment has two variants. The first is a continuous design where the sample sections move along a conveyor belt, in this case a carrier film between each module. This version will be time consuming since each sample is processed individually. The second design has the carrier film with the sections wound into a processing spiral. This spiral contains the samples so that they undergo collective deparaffinization and staining. The spiral is designed such that there will be spaces between the layers of the film, allowing liquid reagents to wash each sample section unhindered. The samples will be unwound from the spiral for sealing and scanning, and then rewound into a spool for storage.

[0044] Modular Components The deparaffinization module only serves to dissolve the paraffin wax by heating the sample; all other liquid reagent washes typically used for deparaffinization, such as ethanol solutions and xylene, can be applied by the staining module because these modules are designed for liquid chemical application. The deparaffinization module must heat each sample to about 55° C. for up to 10 minutes. A common design can be that of an oven or heated surface. If a mounting plate, strip, or processing spiral is used, the sample can be transferred into the insulated heated section via a robotic mechanism. If a continuous ribbon of film is utilized, the sample can pass under or through a heated corridor as it moves through the device.

[0045] As mentioned, the staining module serves to finish the deparaffinization process as well as to apply liquid reagents that stain the tissue sample. Two variants of the staining module can be integrated into the device. The first embodiment of the staining module utilizes a reagent bath design, in which the sample is immersed and incubated in a series of liquid reagent baths as required for a specific staining protocol. This variant is preferred when combined with a section mounting plate (with the use of film strips or direct section mounting) and a processing spiral. Thus, the sample is stained as a collective, increasing uniformity. The second variant of the staining module can be that of an automated reagent dispenser, in which an automated applicator drops a specific reagent as a pipette or nozzle onto a specific sample. In this embodiment, the reagents can be stored in a tank within the module, and the reagents can be pumped to the applicator through dedicated tubing to prevent contamination. Discrete application of a specific staining reagent reduces the volume of reagent required, which is essential when adjacent samples require different staining. Chambers or barriers can be placed around individual samples to prevent sample spillage and cross contamination. Variations of this staining module can be integrated into systems for use with: 1) section mounting plates with the use of film strips or direct section mounting, 2) individual strips independent of plates, and 3) conveyor belt designs for continuous processing embodiments for which variations in the reagent baths would be impractical.

[0046] In all embodiments of the staining module, the method of staining will be specified by the user. Both the reagent bath variant and the automated dispenser variant can be capable of performing 1) standard H&E staining, 2) IHC, 3) IF, and 4) various special staining protocols. The specific reagents for each can be manually loaded into the module and the protocol can be indicated by the user via the user interface. Due to the cost of antibodies and non-standard staining reagents, the reagent bath variant is suitable for standard H&E staining, while other staining protocols can benefit from the more precise application performed by the automated dispenser.

[0047] The sealing module serves to apply a transparent cover to the stained samples. This cover is designed to prevent drying and damage to the samples throughout the scanning and analysis process as well as during storage. One of three types of sealing modules can be integrated into the device depending on the needs of the user. The first embodiment of the sealing module dispenses a liquid sealant onto each sample. The application of the sealant will be of a controlled and defined volume, in a manner similar to the auto-dispensing staining module. In certain embodiments of the integrated device, a variation of this sealing module can be integrated into the staining module. This variation can be used with all embodiments of the invention. The second and third variations of the sealing module are primarily applied to embodiments of the invention that utilize direct mounting of sections onto the plate. Once the mounting plate and all sections have undergone staining, by whatever means, an adhesive-coated plastic film can be applied over the plate and samples. Alternatively, a large glass cover slip can be applied over the plate. This last variation of sealing will be most applicable for users who may need access to tissue samples, post-processing.

[0048] The scanning module serves to capture and store digital images of all stained tissue samples. One embodiment of this module utilizes a high-resolution digital camera that captures images of one or more samples within a field of view (FOV). For example, for use in a continuous feed variant of the invention, this high-resolution camera can capture images of each section sequentially. Alternatively, when using a mounting plate, the camera FOV can capture an area of ​​some or all of the samples on the plate. If only certain areas are captured, the camera or plate can be built on a CNC scaffold, allowing the camera to capture multiple areas of the plate that will later be stitched together by the reconstruction software. Another embodiment of the scanning module incorporates an array of high-resolution digital cameras; this embodiment is best integrated with a variant of the mounting plate. The FOV of each camera can capture an area of ​​the mounting plate such that each image includes one or more tissue sections. This variant is designed to reduce the time spent scanning the plate when the camera FOV is limited by the desired resolution. In yet another embodiment of the scanning module, a light projector can be used to illuminate one side of the stained tissue section. As the light passes through the sample, the analog image can be amplified through a series of lenses and captured by a high-resolution multicolor digital detector array. The sensor of the detector array would need a suitably large number of pixels so that the effective resolution remains high after digital conversion and binning. The sensor would also need to be large enough to capture the entirety of the projected and amplified image. This just-described embodiment of the scanning module can be integrated into all embodiments of the invention; however, it would be preferred for the single ribbon continuous processing variant.

[0049] Analysis software The remaining aspects of the invention describe the features and functionality of the software, which performs three operations: 1) generating a 3D reconstruction of a tissue sample; 2) differentiating tissue types within the reconstruction, allowing a user to filter specific tissue types from the reconstruction and highlight target tissues within the sample; and 3) using AI for machine learning to facilitate disease diagnosis.

[0050] Reconstruction is accomplished by first identifying and ordering the individual tissue sections. If image capture is performed sequentially, the digital images should be stored in the correct order. When using certain embodiments of the invention, such as a scanning module that captures all samples on a mounting plate, separate algorithms can be implemented to 1) identify and number each section in a given image, and 2) save a new set of images, each containing only one section. In certain embodiments, a stitching function may be required to initially generate a complete image of the plate. Once the individual images have been saved and ordered, a quality control and error correction process can be implemented. By matching the high-resolution image of the processed tissue sample with a reference image taken immediately prior to slicing, changes to the relative position, rotation, and morphology can be observed. To aid in this process, a virtual scale or reference marker can be placed on the high-resolution image. Once all images are stacked in a predetermined order and corrected for errors, the reconstruction process can begin. The reconstruction algorithm will utilize the known thickness of each cut to interpolate between the 2D images and form a 3D model of the processed tissue sample.

[0051] Once the reconstruction is complete, an AI analysis will be performed. By cross-referencing the staining patterns within the reconstruction with a histological image database of known cell types, the AI ​​system will identify and show features of the tissue sample including, but not limited to, the overall structure, both physical and pathological, tissue types and boundaries, vascular networks, etc. The software also generates a dynamic 3D model of the tissue sample where specific tissues and structures can be enhanced or removed depending on the user's objectives.

[0052] A user interface, most likely presented in the form of a computer application, will be used to manage the settings of the integrated device and to edit the dynamic 3D reconstruction. Some features that the user can control include sample thickness, staining protocols, and image resolution. This list is not exhaustive. The user may also be required to indicate which embodiment of the device is in use, including which modules are incorporated.

[0053] The present invention is designed to combine all the processes of histology into one integrated device with high throughput. The modular nature of each component allows for great variation in the final design of the system. Deciding which variation to use depends on the type of sample to be analyzed and the analytical method, especially which staining protocol. Certain modules described above can be used independently or, in certain cases, can be omitted if manual processing is desired; however, the present invention is intended to be developed as an autonomous integrated system with no or minimal user input.

[0054] BEST MODE FOR CARRYING OUT THE PRESENTINVENTION The following examples and figures more specifically explain the present invention, but do not limit the field or scope of the invention.

[0055] FIG. 1 shows a diagram of the sectioning and mounting process in an embodiment of a high-throughput reusable plate of the invention. As shown in panel (A), a formalin-fixed paraffin-embedded (FFPE) biological tissue sample block 1 is sliced ​​by a microtome blade 2. A camera 4 is present and oriented to the block face 24; it captures an image of the block face 24 before each cut. This reference image is stored for quality control. The resulting tissue slices 3 are transferred sequentially onto a thin, transparent, heat-resistant mounting film 5. This film 5, originally wound in a spool 6, moves through the device using a system of high-precision sprockets 7. In the embodiment of the invention shown in this figure, the leading length of film 5 can be stretched and held in tension with the help of an assembly of clamps 8. The length of film 5 collected between these clamps 8 has a plurality of sliced ​​tissue sections 3 mounted on the film 5; this length of film is referred to as a strip 10 in the context of the invention. This strip 10 is cut by a blade 9. The film strips 10 are transferred by an automated mechanism to an oversized glass slide designed to hold multiple film strips 10, defined as a mounting plate 11. The mounting plate 11 can hold multiple strips 10. The multiple strips 10 are arranged so that a maximum number of strips 10 can be mounted on the plate 11. The cutting sequence of the sample block 1 is maintained in the same way so that the placement of the sample sections 5 on the plate 11 is uninterrupted from left to right and top to bottom. Once the mounting plate 11 is full, all the mounted strips 10 or directly mounted sample sections (not shown in the embodiment) will undergo sample processing, including deparaffinization, staining, sealing, and analysis. In this embodiment, the strips 10 can be removed and stored separately; the mounting plate 11 can be reused for the next set of strips 10.

[0056] FIG. 2 is a diagram showing the sectioning mechanism. Panel (A) shows the relative positioning of the tissue block 1 with respect to the blade 2. The bevel of the blade is parallel to the specimen block face 24 while the cutting edge of the blade is displaced by the desired slice thickness; in this view 5 μm (not to scale). The movement of the tissue block 25 is linear and parallel to the block face 24. The film 5 is positioned close to the blade 2 so that the specimen slice 3 rolls up and contacts the film 5; shown in panel (B). A sprocket 7 controlling the movement of the film 5 ensures that the speed of the film 5 at the transfer point is equal to the speed of the cutting motion 25, so that the specimen slice 3 is not torn or damaged during transfer. An aqueous solution 26 can be sprayed onto the film 5 and block face 24 prior to cutting to aid in the transfer of the film 5.

[0057] Figure 3 shows the motion of the sample block 25 and blade 27 during sectioning. Panels (A) and (B) show a design where the blade is fixed and the block 1 is pushed into the cutting edge. Panels (C) and (D) show a design where both the block 1 and blade 2 are in motion during cutting. In this case, the cutting edge of the blade 27 translates to the right relative to the sample, slicing the sample as the block 25 moves down to engage the blade. The direction of motion of the tissue block 25 remains straight and perpendicular to the cutting edge in both cases.

[0058] 4 is a diagram showing the mechanism for maintaining precise control of the positioning of the tissue sample sections 3 on the film 5 while they are transported between modules. Equidistant holes 12 along the side edges of the film 5 align with teeth on the sprockets. The rotation of all of the sprockets throughout the device cooperates so that the position of every sample section is precisely and accurately defined.

[0059] 5 shows how a strip of film 10 can be mounted on a plate 11. In this embodiment of the invention, the mounting plate 11 is designed with protrusions or pegs 13 that extend through the plate 11, perpendicular to its mounting surface. These pegs 13 are positioned along opposite side edges of the plate and are spaced to align with holes 12 in the filmstrip 10. This design provides for discrete and consistent placement of the strip 10, optimizes the mounting area, and also aids in scanning and staining (not shown) in certain embodiments of the invention.

[0060] 6 shows a further safety measure to prevent movement of the plate-mounted strips 10 during processing. A mechanized locking bar or clamp 14 can be lowered onto a peg 13 on the panel 11.

[0061] FIG. 7 is a diagram of one embodiment of the invention in which a single continuous ribbon of film sequentially transports sample sections between processing modules. The sample is first sectioned and transferred onto a carrier film as described in FIG. 1. Instead of being cut into strips, the embodiment shown here shows the film and mounted sample sections (not shown) moving in a conveyor belt fashion through a deparaffinization module 15, a staining module 16, a sealing module, and a scanning module 17. Once processed, the film is wound into a storage spool 18. The deparaffinization module 15 heats each sample and helps melt the paraffin wax. Due to the design of this embodiment, the deparaffinization module 15 can be designed as a corridor aligned with a heating element. Chemical washes are generally also used for deparaffinization, however, these reagents can be incorporated into and applied by the staining module. The staining module 16 shown here is an automated dispenser design that applies staining reagents 21 to individual sample sections (see FIG. 8A). A sealing module (not shown) for this embodiment applies a droplet 21 of liquid sealant onto each sample; considering a similar mechanism, this system can be integrated into and applied by the staining module. A scanning module 17 serves to capture a digital image of each sample. There are various possible embodiments of this module (see DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS: Modular Components); the version used with this embodiment of the invention can take high-resolution images of the samples in succession as they come into the camera's field of view. A high-precision sprocket 7 can control the movement of the carrier film and the positioning of the samples within each module.

[0062] FIG. 8 shows two possible embodiments of the staining module. Panel (A) shows an automated dispenser design 16. In this design, an automated applicator 20 drops a specific staining reagent 21 as a pipette or nozzle onto a specific sample, shown here on a strip of film 10. The staining reagent as well as the deparaffinization reagent and liquid sealant can be stored separately in a tank in the body of the module 19. Discrete and relatively small amounts of reagent can be applied to each sample to reduce costs and waste. Chambers or barriers (not shown) can be placed around individual samples to prevent sample spills and cross contamination. Variations of this module can be designed to have multiple applicators 20 capable of dispensing multiple reagents 21, processing multiple samples simultaneously, as well as variably staining between adjacent sections. A variant 16 of an automated applicator staining module that processes a separation strip 10 is shown here. However, this design can be integrated into most embodiments of the invention; this design will not be appropriate while using a processing spiral (see DETAILED DESCRIPTION OF THE EMBODIMENTS: MODULAR COMPONENTS).

[0063] Panel (B) shows the reagent bath design 22. In this variation, the specimen is immersed and incubated in a series of liquid reagent baths 23 as necessary for the desired staining protocol. This figure shows that separate strips are processed; however, this variation is suitable for mass specimen staining using film strips (FIG. 1) or the section mounting plate 11 and processing spiral (FIG. 9) with direct section mounting (not shown).

[0064] FIG. 9 shows an alternative embodiment for single ribbon continuous processing. Similar to the continuous processing shown in FIG. 7, the sample sections are transferred onto a carrier film and processed. However, as seen in panel (A), the film and sample sections are wound into a staining spiral 32. This spiral is designed to contain the entire length of the complete sectioned tissue sample block 1 while maintaining space between the layers of the wound film. Once the entire sample has been sectioned and transferred into the spiral, the film is cut (not shown) and the spiral continues with the processing: deparaffinization (not shown), staining using a reagent bath transformation module 22 (panel (B)), sealing (not shown), scanning (panel (C)), and storage 18. One embodiment of the scanning module 17 shown in panel (C) shows that the analog image of the sample is amplified prior to image capture. Here a projector 33 positioned below the stained sample section 3 projects the analog image of the sample through multiple lenses 34. These lenses amplify and focus the image onto a high resolution, multi-color, digital detector array 35. In this diagram, images from each sample are collected sequentially and consecutively, however, variations of this scanning module can be utilized in all embodiments of the present invention. Once scanned, the samples are transferred into a storage spool 18.

[0065] FIG. 10 displays the quality control method implemented before 3D image reconstruction. As seen in FIG. 1, a camera 4 is present and oriented to the block face 24; it captures an image of the block face 24 before each cut. This reference image is saved. In this reference image (panel (A)), a scale or marking 28 of known dimensions is observable. This scale can be positioned on the microtome cradle or on the specimen cassette. Panel (B) shows an image of a post-processing of the same specimen slice, visible by a high-resolution scanner 29; this specimen has undergone an unintended rotation in this view. The scale 30 in this panel shows a virtual scale placed by the reconstruction software. The reference image and scale 28 are compared to the virtual image 29 and scale 30 to determine unintended changes to the specimen position, rotation, and morphology. After determining the errors, if any, the software can apply corrections to the high-resolution image, as seen in panel (C). The corrected image 31 is saved and incorporated into the 3D reconstruction.

[0066] Example 1 The FFPE tissue sample is manually inserted into the cradle of the microtome of the sectioning device. Serial sectioning is performed while the sample sections are transferred onto the carrier film as mentioned. The sample sections are transferred from the carrier film onto the mounting plate with the help of a CNC; the samples are transferred consecutively by the cutting sequence so that the position of each sample on the plate is restricted within a known area of ​​the plate. The plate and all mounted samples are transferred to a deparaffinization module, designed as an insulating oven. The plate is then transferred to an H&E reagent bath staining module. A coverslip is applied to the plate. A scanning module, equipped with one high-resolution camera capable of scanning the entire plate, captures one high-resolution image. The plate is removed from the device and placed in storage. Using a software algorithm, the high-resolution image is divided into areas, each of which contains only one sample section; new data sets are saved so that each file is an image of a single section. The images are numbered and placed in a predefined order. All sample images undergo quality control and correction for translational, rotational, and / or morphological errors; new image files are generated as necessary. The image datasets are used to reconstruct a 3D model. This reconstruction is analyzed by AI and machine learning algorithms to aid in disease diagnosis and tissue sample presentation.

[0067] Example 2 The FFPE tissue sample is manually inserted into the cradle of the microtome of the sectioning device. Serial sectioning is performed while the sample slices are transported onto the carrier film, as mentioned. The carrier film is wound into a processing spiral. Once the entire tissue block has been sectioned and all the samples are included in the spiral, the film is cut. The spiral and its contents are transported to a deparaffinization module, designed as an insulated oven. The plate is then transported to an H&E reagent bath staining module. Once stained, the film is unwound from the spiral with the help of a sprocket. The sealing module can apply a drop of liquid sealant to each sample. The scanning module, which comprises a projector, a series of lenses, and a high-resolution multicolor digital detector array, amplifies the analog image of a single slice and focuses the image into the sensor. The sample is scanned continuously as it unwinds from the spool. Once scanned, the film is wound into a storage spool. The high-resolution images should be in reverse order. All sample images undergo quality control and correction for translational, rotational, and / or morphological errors; new image files are generated as necessary. The image datasets are used to reconstruct 3D models. These reconstructions are analyzed by AI and machine learning algorithms to aid in disease diagnosis and tissue sample presentation.

[0068] All publications and patents cited herein are incorporated by reference as if each individual publication and patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed. To the extent that the definitions of terms set forth in documents incorporated herein by reference conflict with the definitions of terms expressly defined herein, the definitions set forth herein control. [Explanation of symbols]

[0069] 1. Formalin-fixed, paraffin-embedded (FFPE) biological tissue samples 2 Mictome Blade 3. Tissue sample sections 4. Low-resolution camera 5. Carrier Film 6 Film Spool 7 Sprocket 8. Clamp 9 Cutting Blades 10 Film Strips 11 Mounting plate 12 Hole in the film 13 Peg 14. Rock Bar 15 Deparaffinization Module 16 Automated Dye Reagent Dispenser Module 17 High Resolution Camera 18 Storage spool 19 Dyeing Reagent Tank 20 Automated Dispenser Nozzle 21 Dyeing Reagents 22 Automated staining reagent chamber module 23 Dyeing Reagent Tank 24 Block Face 25 Block movement direction 26 Aqueous solution spray 27 Blade movement direction 28 Reference Scale Bar 29 High resolution images of processed samples 30 Virtual Reference Scale Bar 31 Corrected high resolution images of processed samples 32 Processing Spiral 33 Projector 34 A lens or set of lenses 35 Detector

Claims

1. 1. A device for collecting and processing a sample, comprising: a sample cutter comprising a blade and a cradle configured to hold a sample, the blade or the cradle being movable to ensure that the blade makes a series of cuts in the sample into a plurality of slices having the same thickness; a collecting film having a long and narrow surface; a plurality of sample processing modules; the device is configured to apply a force to a slice after the specimen cutter creates a slice from the specimen to transfer and mount the slice onto the collection film; the plurality of slices are transferred to the collection film in sequence according to a cutting order of the plurality of slices; the device is further configured to wind the collection film with the plurality of slices about a take-up roll to form a rolled collection film; The plurality of sample processing modules include: a deparaffinization module configured to receive the rolled collection film, the deparaffinization module further configured to deparaffinize the plurality of slices while the plurality of slices are contained on the rolled collection film; a specimen staining module configured to receive the rolled collection film, the specimen staining module being further configured to stain the plurality of slices while the plurality of slices are contained on the rolled collection film; a specimen imaging module configured to sequentially image each slice of the plurality of slices on the collection film unwound from the roll of collection film.

2. The device of claim 1 further comprising a film roller, said collection film being fed from said film roller.

3. The device of claim 1 , wherein the sample is formalin-fixed paraffin-embedded (FFPE) tissue.

4. The device of claim 1 , wherein the plurality of sample processing modules further comprises a sealing module and a histology staining module.

5. 10. The device of claim 1, wherein the device is configured to align the plurality of slices along a long edge of the surface and juxtapose the plurality of slices to be transferred to the collecting film in succession according to a cutting order of the plurality of slices.

6. The device of claim 4 , wherein the sample imaging module is configured to simultaneously capture imaging data for the multiple slices.

7. The device of claim 4 , wherein the sealing module provides a sealant droplet to cover the collection film.

8. The device of claim 4 , wherein the sealing module provides a transparent cover for the collection film.

9. a film roller for moving the strip of film; the specimen imaging module has an image capture area and is configured to image a specimen on the strip of film that enters within the image capture area; The device of claim 1 , wherein the film roller is configured to move the slices on the collection film, one at a time, into the image capture area such that images of the samples are obtained sequentially.

10. The device of claim 9 , wherein the sample imaging module comprises one or more digital cameras.

11. The device of claim 1 , wherein the sample imaging module comprises an imaging device.

12. the collection film further comprising a plurality of slices from a sample immobilized on the surface and aligned along a long edge of the surface; the plurality of slices are cut from the sample at equal thickness; and The device of claim 1 , wherein the plurality of slices are juxtaposed on a surface of the collecting film according to the cutting order.

13. The device of claim 1 , wherein the multiple slices are simultaneously treated with one or more reagents of the sample staining module.

14. The device of claim 13 , wherein the one or more reagents comprise an H&E stain, an IHC stain, or a fluorescent-based stain.

15. The device of claim 1 , wherein the collection film is optically transparent and heat resistant to at least 60° C.

16. The device of claim 15 , wherein the collection film further comprises a series of holes or notches along a long edge of a surface of the collection film.

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