Automated systems and methods for tissue sectioning, staining and scanning

Abstract

A plate or film for collecting and analyzing samples is disclosed. The plate or film comprises a surface and a plurality of slices from the sample fixed on the surface. In addition, a plurality of slices are cut from the sample having equal thickness, and the plurality of slices are placed on the surface of the plate or film in the order in which they are cut.

Description

Technical Field

[0001] The field of the present invention is diagnostic systems and methods. More specifically, it presents systems and methods for automating the process of histological specimen sectioning, staining and imaging and facilitating information analysis and disease diagnosis in the field of clinical pathology. Background Art

[0002] The importance of pathology to modern medicine cannot be overstated. In cancer treatment, for example, accurate readings of the pathology of cancer-infiltrated areas in biopsies are a critical step in enabling effective treatment design. Pathology-based assessments have been an integral part of diagnosing disease for more than a century.

[0003] Although a traditional method, histomorphometric assessment still relies on the examination of slides containing good quality tissue sections. However, the preparation of good tissue slides suitable for clinical use is not only labor intensive, but also requires the skills of well-trained histotechnicians. Currently, the supply of such qualified technicians cannot keep pace with the increasing volume of tissue-based testing. Therefore, there is a need to develop new methods that minimize some of the tedious tasks required to prepare tissue slides, preferably using automation.

[0004] On the other hand, the advent of the digital age, especially with the help of artificial intelligence (AI) today, has given rise to an emerging and rapidly developing field called digital pathology. Digital pathology refers to the science and technology that captures, stores, and interprets information about pathological specimens using images in digital format. Compared to the traditional work through optical microscopes, digital pathology enables the viewing of images through a computer interface and the transmission of tissue images through the Internet. Taking advantage of the rapid development of digital pathology, the field of AI explores the so-called "cognitive technologies" that can simulate human reasoning and perception capabilities to analyze medical imaging to improve disease prediction and diagnosis. Digital pathology's conversion of static images into retrievable data helps to obtain more and more information from histological specimens, and the field of AI heralds the advancement of subsequent qualitative and quantitative analysis of this information.

[0005] As digital pathology develops in tandem with AI technology, it requires more and more data. However, in addition to the above-mentioned challenges in the production of high-quality tissue sections, current image scanning / acquisition also lags behind. Typical microscope-based pathology image reading requires processing time and is not suitable for high-throughput methods. In addition, for any given tissue sample, according to current standards, its image is limited to 2-D views of several random slices. How to improve existing standards to obtain more information from a given tissue sample, and more importantly, how to significantly improve scanning technology to achieve high-throughput image acquisition, has become a challenge in the AI-centric digital pathology era.

[0006] There is also a need for a system or method that simplifies the process from preparing tissue slides to capturing digital images. Summary of the invention

[0007] The present invention provides systems and methods to simplify the process from preparing tissue slides to capturing digital images to meet the needs of generating multi-layer 3D images for AI-focused digital pathology. At the same time, it promises to obtain more overall information from tissue samples to enhance the coverage of histological areas, thereby better pathological diagnosis.

[0008] One aspect of the present invention relates to a method for processing a sample. The method comprises the following steps: (i) providing a sample, and defining a virtual horizontal plane and a virtual vertical axis perpendicular to a plane for the sample; (ii) cutting the sample into a plurality of sample slices along the virtual horizontal plane, wherein the cutting is substantially parallel to the virtual horizontal plane, the thicknesses of the plurality of sample slices are substantially the same, and the plurality of sample slices are arranged in sequence along the virtual vertical axis according to a cutting order; and (iii) juxtaposing the plurality of sample slices on a support member in a continuous manner according to the cutting order.

[0009] The sample in the above method can be a biological sample. Specifically, the sample can be a tissue, for example, a formalin-fixed paraffin-embedded (FFPE) tissue.

[0010] Also in the above method, the thickness of the plurality of sample slices is adjustable, and the support is a membrane.

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

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

[0013] Yet another aspect of the present invention relates to a film for collecting and analyzing a sample. The film has a long and narrow surface and a plurality of slices from the sample, the plurality of slices being fixed on the surface and aligned along the long edge of the surface. In addition, a plurality of slices are cut from a sample having equal thickness, and the plurality of slices are juxtaposed on the surface of a plate or film in the order in which they were cut.

[0014] Multiple sections on the surface of the just described film are processed simultaneously in one or more reactions. The one or more reactions include histological staining, fluorescence-based staining, or other types of staining.

[0015] The film described may be a plastic film. Furthermore, the film is optically transparent and heat-resistant up to at least 60° C. It may also have a series of holes or indentations along the long edges of the surface of the film.

[0016] Another aspect of the present invention relates to a device for collecting and processing samples. The device includes a sample cutter having a blade and a bracket, and the bracket is configured to hold the sample. In addition, the blade or the bracket is movable to ensure that the blade cuts the sample into a series of slices with equal thickness. The device also includes a collection film with a long and narrow surface. Through its design, the device can be configured to apply force to the slice after the sample cutter cuts the slice from the sample to transfer and mount the slice on the collection film, and the device is configured to align multiple slices along the long edge of the surface and juxtapose multiple slices in a continuous manner according to the order in which they were cut.

[0017] The apparatus may further enclose a film roll that provides a collected film.

[0018] Yet another aspect of the invention relates to an apparatus for processing a sample, the apparatus comprising the apparatus 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 dewaxing 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 the tissue.

[0019] For the sealing module, it can provide a sealant droplet to cover the collection membrane, or provide a transparent cover for the collection membrane.

[0020] Another aspect of the present invention relates to an apparatus for collecting and processing samples. The apparatus includes a sample cutter, a conveyor belt, and a collection plate. The sample cutter has a blade and a bracket, and the bracket is configured to hold the sample. The blade or the bracket can be movable to ensure that the blade continuously cuts the sample into multiple slices with equal thickness.

[0021] The above-mentioned device can be configured to remove the slices by the conveyor belt immediately after the sample cutter cuts the slices from the sample, and then unload the slices and install them on the collection plate. In addition, the device can be configured to continuously juxtapose multiple slices on the collection plate according to their cutting order.

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

[0023] Yet another aspect of the present invention relates to equipment for processing samples. The device includes the above-mentioned equipment and one or more sample processing modules. In the device, the sample can be a formalin-fixed paraffin-embedded (FFPE) tissue, and the one or more sample processing modules can include a dewaxing module. When the sample is a tissue, examples of one or more sample processing modules can also be a plate sealing module, a sample staining module, a histological staining module, or a sample imaging module. The sample imaging module can be configured to capture imaging data of multiple slices of the tissue at the same time. On the other hand, the plate sealing module can provide a sealant droplet for covering the plate, a film for 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 having a plurality of specimens fixed on a surface of a board; a carrier for holding the board; an image capture device having an image capture area, and a moving mechanism for changing the relative positional relationship between the carrier and the image capture device. In the above-mentioned imaging device, the imaging device is configured to simultaneously capture an image of each specimen on the board falling into the image capture area; and the imaging device is configured to divide the board surface into a plurality of image capture areas. In addition, the moving mechanism sequentially moves the image capture device to the plurality of image capture areas to capture images of the plurality of specimens.

[0025] The imaging device may further comprise an artificial intelligence unit for processing 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 includes: (i) cutting the biological sample into a plurality of slices having equal thickness; (ii) sequentially transferring the plurality of slices to a collection film having a long and narrow surface so that the plurality of slices are aligned along the long edge of the surface; (iii) winding the collection film to form a roll having a uniformly thick space between the winding layers; (iv) staining the plurality of slices on the wound collection film; (v) unwinding the wound collection film; and (vi) imaging the plurality of slices on the collection film. In addition, when the collection film is moved by an imaging device, one slice is imaged at a time, and includes the following features: the entire slice is captured at one time in the imaging, and the imaging is performed at a speed of 10 slices per minute to 60 slices per minute.

[0027] In the above method, the biological sample may be FFPE tissue. In addition, the device may be configured to perform the above method.

[0028] The details of the invention are set forth in the drawings and description. Other features, objects and advantages of the invention will be apparent to those skilled in the art upon reading the drawings and description and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 1 is a diagram of the sectioning and mounting process in a variation of a high-throughput reusable plate design. Panel (A) shows sample sectioning and transfer to film. Panel (B) shows film strips. Panel (C) depicts multiple strips mounted on a processing plate.

[0030] Figure 2 is a diagram depicting 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 piece is transferred to the film.

[0031] Figure 3 Depicted are the motion of the block and blade during sectioning. Panels (A) and (B) depict the before and after images of a design where the blade is stationary. Panels (C) and (D) depict the before and after images of a design where both the block and blade are in motion while cutting.

[0032] Figure 4 is a diagram depicting the mechanism that maintains precise control over the positioning of the membrane as it moves throughout the apparatus.

[0033] Figure 5 Shown is how the strips are mounted to the board.

[0034] Figure 6 Shown is how the strips are locked into position on the plate once the plate is full.

[0035] Figure 7 is a diagram depicting a design that enables continuous processing of samples on a single film strip.

[0036] Figure 8 Schematic diagrams of two dye module variants are shown.

[0037] Fig. 9 Alternative designs for the continuous processing model are shown. Panel (A) shows the membrane that rolls up into a staining spiral once the slide is mounted. Panel (B) shows the reagent bath staining module. Panel (C) shows the scanning module that amplifies the analog image and stores it before digital scanning.

[0038] Fig.10 The quality control method for rotation error correction when scanning with a reference scan is shown. Panel (A) depicts a reference image. Panel (B) shows the same sample slice post-processing with errors. Panel (C) shows a corrected image of the same sample slice. DETAILED DESCRIPTION

[0039] A method for obtaining a series of sequential images of tissue sections is provided. Aspects of the method include preparing a series of parallel sections of a tissue sample, the parallel sections having substantially the same thickness. Also provided are devices and apparatus for practicing the subject method.

[0040] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary.

[0041] Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can perform the present invention. However, it should be understood that the present invention can be implemented in various forms and is not intended to limit the exemplary embodiments of the present invention. In addition, in the accompanying drawings, descriptions of parts not related to the specific embodiments are omitted to clearly describe the present invention.

[0042] The subject systems and methods are primarily intended for diagnostic purposes. However, successful implementation of these systems and methods holds the promise of transforming the practice of pathology, further elevating pathology into a quantitative science.

[0043] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials are now described. All disclosures mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials cited in these disclosures.

[0044] Embodiments of the present invention provide advantageous features and characteristics in the fields of tissue sectioning, tissue staining, cameras, image recognition and / or automatic image analysis, including manufacturing methods, systems and / or devices. Note that the terms "determine", "measure", "evaluate" and "analyze" are used interchangeably and include both quantitative and qualitative determinations.

[0045] The sample processed and analyzed by the present invention is a biological sample. Specifically, the sample can be any tissue collected via a biopsy or dissection that requires histological analysis. Before using the present invention to process, the sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample.

[0046] The main aspects of the present invention relate to an integrated method of sample processing, including: (i) serial sample sectioning; (ii) transfer of sample sections to a support (such as a film), and may include subsequent grouping and / or transfer of multiple samples to a processing station; (iii) dewaxing via heat and chemical use; (iv) tissue staining via batch staining in a sequence of reagent baths or reagent dripping, such as but not limited to hemotoxin and eosin (H&E), immunohistochemistry (IHC), immunofluorescence (IF)-based staining or other special staining protocols; (v) sealant application; (vi) analog image magnification, digital scanning and image collection / storage; (vii) three-dimensional image overlay and reconstruction; (viii) AI analysis of processed samples. The method just described is implemented so that each step can be designed as a module or component of an integrated device, and multiple configurations of the device can perform the basic aspects of the present invention according to the specific needs of the user. Various configurations and alternative configurations of the present invention are described in detail herein.

[0047] The present invention is designed to perform standard sectioning protocols using a conventional ultramicrotome design, which includes a carriage and a blade configured to hold a sample. Traditionally, the carriage and sample are moved so that the paraffin block is pressed against the cutting edge of the blade to produce slices of uniform thickness. Alternatively, the blade can move orthogonally to the movement of the carriage to facilitate sectioning; a vibrating microtome can also be incorporated. The thickness of the sample can be adjusted by the user.

[0048] A camera may also be incorporated into the design of the ultramicrotome. The camera is positioned perpendicular to and facing the center of the tissue sample block surface. The presence of a camera aids in quality control. The camera will take a reference photo of the block face before each sectioning. A scale bar or markings of known size are present on the specimen's carrier or cassette. This image can be referenced later to aid alignment and error correction when reconstructing a superposition of scanned images.

[0049] The present invention processes samples continuously and transfers each slice continuously and equidistantly to a plastic film or tape. The film is positioned near the blade so that the slice will contact and adhere to the film; the film moves at a speed consistent with the slice to minimize wrinkling or tearing of the slice 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 facilitate transfer. In addition, the sample block surface and the film can be sprayed with an aqueous solution before slicing; the cohesive force of the water will aid transfer. A mechanism can be included to apply force to the slice to aid adhesion. The film can also be coated with a chemical layer or adhesive that promotes adhesion of tissue slices. Multiple sample slices are mounted on the film in the order of cutting. Ultrathin sectioning is performed until the entire or desired thickness of the sample is sectioned, or in some cases, until the sample mounting station is full.

[0050] The plastic film or tape is stored in rolls or spools and unwound at a rate consistent with the rate at which the sample is sliced. In order to maintain precise control over the film and the location of the slices on the film, the film is designed with a series of equally spaced holes at the lateral edges along the length of the film. Sprockets are used to transport the film between modules throughout the equipment. The gears of these sprockets align with the holes in the film, allowing precise control of the movement of the film. This is very important when aligning the sample with the sealant or stain applicator and, in some variants, when scanning. The film is optically transparent and heat resistant up to temperatures of at least 60°C; this temperature is relevant for dewaxing.

[0051] Design variants

[0052] In one embodiment, the sections are transferred directly from the film to the mounting plate. The plate stage is controlled via CNC so that the sections are transferred to the plate sequentially within a defined area. Such plates are oversized glass or plastic slides designed to hold hundreds of serial sections from a single specimen block; however, these numbers may vary depending on the size of the specimen. Once mounted on the plate, the specimen is dewaxed, stained, sealed, and scanned together. The plate with the entire specimen mounted on it is transferred to storage.

[0053] In another embodiment, the film is divided into a plurality of strips, each strip containing a plurality of slices mounted thereon. In this embodiment, the strips can be processed in two ways. In one variant, the strips are transferred to a mounting plate. The pins on the mounting plate are aligned with the holes on the film, allowing (multiple) strips to be discretely placed on the mounting plate. Once the plate is filled with strips, a locking mechanism secures the strips in place. As described in the above embodiments, the plate and the attached strips undergo dewaxing, staining, sealing and scanning. However, once scanned, the strips can be removed from the plate and stored separately. The plate can then be reused as a mounting station. Alternatively, instead of transferring the strips to the plate, an alternative processing module is used directly for dewaxing, staining and sealing. This version is not conducive to performing high-throughput analysis and is therefore more suitable for clinical or research applications. Current clinical protocols process approximately 5 to 10 consecutive slices of a given tissue sample; this number of slices can be mounted on one strip.

[0054] Studies requiring non-uniform staining of adjacent sections can utilize an alternative staining module that can process samples individually and variably along the strip using one of H&E, IHC, IF, or SS. In any of these alternative applications, the strip return plate can be used for collective imaging.

[0055] In yet another embodiment, a film with mounted samples is sequentially transported through each processing module as a continuous belt. Once processed, the samples are rolled into a spool for storage. This embodiment has two variations. The first is a continuous design in which sample slices are moved between each module along a conveyor belt (in this case, a carrier film). This version will be time consuming because each sample is processed individually. The second design has a carrier film in which the slices are rolled into a processing spiral. The spiral contains the samples because the samples undergo collective dewaxing and staining. The spiral is designed so that there is space between the layers of the film, allowing liquid reagents to clean each sample slice unimpeded. The samples will be unrolled from the spiral for sealing and scanning, and then rewound into the spool for storage.

[0056] Modular components

[0057] The dewaxing module is used only to melt paraffin by heating the sample; all other liquid reagent cleaning solutions (such as ethanol solutions and xylene) commonly used for dewaxing can be applied by the staining module because these modules are designed for liquid chemical application. The dewaxing module must heat each sample to approximately 55°C for 10 minutes. The general design can be an oven or a heating surface. If a mounting plate, strip or processing spiral is used, the sample can be transferred to an insulated heating chamber via a robotic mechanism. If a continuous film strip is utilized, the sample can pass through or through a heating corridor when passing through the device.

[0058] As mentioned above, the dyeing module is used to apply the liquid reagent that completes the dewaxing process and dyes the tissue sample.Two variants of the dyeing module can be integrated into the equipment.The first embodiment of the dyeing module utilizes a reagent bath design, wherein, as required for a specific staining protocol, the sample is immersed in a series of liquid reagent tanks and cultivated in a series of liquid reagent tanks.When combined with a slice mounting plate (using a film strip or direct slice mounting) and a processing spiral, this variant is very suitable.In this way, the sample is dyed as a collective, increasing uniformity.The second variant of the dyeing module can be an automatic reagent dispenser, wherein an automatic applicator (applicator) (such as a pipette or nozzle) drips a specific reagent onto a specific sample.In this embodiment, the reagent can be stored in a storage tank in the module, and can be pumped to the applicator by a dedicated pipeline to prevent contamination.The discrete application of a specific staining reagent will reduce the volume of the required reagent, and if adjacent samples require different staining, the discrete application of a specific staining reagent is crucial.A chamber or barrier can be placed around a single sample to prevent sample runoff and cross contamination. This stain module variant can be integrated into systems for use with 1) a slice mounting plate (using film strips or direct slice mounting), 2) a separate strip independent of the plate, and 3) a conveyor belt design for continuous processing embodiments where the reagent bath variant is not applicable.

[0059] In all embodiments of the staining module, the method of staining will be specified by the user. Both the reagent bath variant and the automatic dispenser variant are capable of performing 1) standard H&E staining, 2) IHC, 3) IF and 4) various special staining protocols. The specific reagents of each module can be manually loaded into the module, and the user can indicate the protocol via the user interface. Due to the cost of antibodies and non-standard staining reagents, the reagent bath variant is very suitable for standard H&E staining, while other staining protocols can benefit from the more accurate application performed by the automatic dispenser.

[0060] The sealing module is used to apply a transparent cover to the stained sample. The cover is designed to prevent the sample from drying and being damaged during scanning and analysis and during storage. Depending on the needs of the user, one of the three variants in the sealing module can be integrated into the device. The first embodiment of the sealing module dispenses a liquid sealant onto each sample. The application of the sealant will be controlled and have a defined volume, which is similar to the method of automatically dispensing the staining module. In certain embodiments of the integrated device, this sealing module variant can be integrated into the staining module. This variant can be used with all embodiments of the present invention. The second and third variants of the sealing module are mainly applicable to the embodiments of the present invention that utilize the direct mounting of the slices onto the board. Once the plate is mounted and all slices are stained, a plastic film with an adhesive can be applied to the plate and the sample anyway. Alternatively, a large glass coverslip can be applied to the plate. The last variant of the seal is most suitable for users who may need to use tissue samples for post-processing.

[0061] The scanning module is used to capture and store digital images of all stained tissue samples. One embodiment of the 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 present invention, the high-resolution camera can capture images of each slice in sequence. Alternatively, when a mounting plate is used, the camera FOV can capture areas of some or all samples on the plate. If only specific areas are captured, the camera or plate can be built on a CNC bracket, allowing the camera to capture multiple areas of the plate, which will then be stitched together by the reconstruction software. Another embodiment of the scanning module combines an array of high-resolution digital cameras; this embodiment is best integrated with the mounting plate variant. The FOV of each camera can capture an area of ​​the mounting plate so that each image contains one or more tissue slices. If the FOV of the camera is limited by the desired resolution, this variant is designed to reduce the time spent scanning the plate. In another embodiment of the scanning module, a light projector can be used to illuminate one side of the stained tissue slice. As the light passes through the sample, the analog image can be magnified by a series of lenses and captured by a high-resolution multi-color digital detector array. The sensors of the detector array will need a suitably large number of pixels so that a high effective resolution is maintained after digital conversion and binning. The sensors also need to be large enough to capture the entire projected and magnified image. This just described embodiment of the scanning module can be integrated into all embodiments of the present invention; however, it is well suited to the single-strip continuous processing variant.

[0062] Analysis software

[0063] The remaining aspects of the invention describe the features and functions of the software. The software performs three actions: 1) generates a three-dimensional reconstruction of a tissue sample; 2) distinguishes tissue types in the reconstruction, thereby allowing the user to filter certain tissue types from the reconstruction and highlight target tissues in the sample; 3) uses AI for machine learning to facilitate disease diagnosis.

[0064] Reconstruction is accomplished by first identifying and arranging the individual tissue slices. If image capture is performed continuously, the digital images should be stored in the correct order. If certain embodiments of the present invention are used, such as a scanning module that captures all samples on a mounted plate, separate algorithms can be implemented to 1) identify each slice in a given image and number each slice, and 2) save a new set of images, each containing only one slice. In certain embodiments, a stitching function may be required to first generate a complete image of the plate. Once the individual images are saved and sorted, a process of quality control and error correction can be performed. By matching a high-resolution image of the processed tissue sample with a reference image taken before slicing, changes in relative position, rotation, and morphology can be observed. To assist in this process, a virtual scale or reference marker can be overlaid on the high-resolution image. Once all images are superimposed and errors are corrected in sequence, the reconstruction process can begin. The reconstruction algorithm will interpolate between the 2D images using the known thickness of each incision and form a 3D model of the processed tissue sample.

[0065] Once the reconstruction is complete, AI analysis will be performed. By observing the reconstruction of known cell types (both physiological and pathological) and cross-referencing the staining patterns in the histological image database, the AI ​​system will identify and indicate the characteristics of the tissue sample, including but not limited to physiological and pathological overall structure, tissue types and boundaries, vascular networks, etc. The software also generates a dynamic 3D model of the tissue sample, in which certain tissues and structures can be highlighted or removed depending on the user's goals.

[0066] A user interface (most likely in the form of a computer application) will be used to manage the settings of the integrated device and edit dynamic 3D reconstructions. Some features that can be controlled by the user include sample thickness, staining schemes, and image resolution. This list is not exhaustive. The user may also need to indicate which embodiment of the device is being used, including which modules are incorporated.

[0067] The present invention is designed to combine all the processes of histology into one high-throughput integrated device. The modular nature of each component allows for a wide range of variations in the final design of the system. Determining which variant to use depends on the type of sample being analyzed and the analysis method, especially which staining protocol. Some of the above modules can be used independently or, in some cases, can be omitted if manual processing is required; however, it is intended that the present invention be developed as an autonomous integrated system with no or minimal user input.

[0068] Best practices for implementation

[0069] The following examples and drawings explain the present invention in more detail but do not limit the scope of the present invention.

[0070] Figure 1A diagram depicting the sectioning and mounting process in an embodiment of a high throughput, reusable plate of the present invention. As shown in panel (A), a block 1 of a formalin-fixed, paraffin-embedded (FFPE) biological tissue sample is sectioned by an ultramicrotome blade 2. A camera 4 is present and aimed at the block face 24; this captures an image of the block face 24 before each cut. This reference image is saved for quality control purposes. The resulting tissue sections 3 are sequentially transferred to a thin, transparent, heat-resistant mounting film 5. The film 5, which is initially wound onto a spool 6, is moved through the device using a high-precision sprocket system 7. In the embodiment of the present invention depicted in this figure, the leading end length of the film 5 can be extended and tensioned with the aid of a number of clamps 8. A certain length of film 5 collected between these clamps 8 has a plurality of sectioned tissue sections 3 mounted thereon; in the context of the present invention, this length of film is referred to as a strip 10. The strip 10 is cut by a blade 9. The strips 10 of the film are transferred via an automated mechanism to an oversized glass slide designed to hold multiple strips 10 of the film (defined as a mounting plate 11). The mounting plate 11 can hold multiple strips 10. They are arranged so that the maximum number of strips 10 can be mounted on the plate 11. The cutting order of the sample block 1 is also maintained so that the placement of the sample slices 5 on the plate 11 is continuous from left to right and from top to bottom. Once the mounting plate 11 is filled, all mounted strips 10 or directly mounted sample slices (not depicted in the embodiment) will undergo sample processing, including dewaxing, staining, sealing, scanning 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.

[0071] Figure 2 is a diagram depicting a slicing 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 sample block face 24, while the cutting edge of the blade is displaced by the desired slice thickness; 5um in the figure (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 sample slice 3 curls and contacts the film 5; as shown in panel (B). The sprocket 7 that controls 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 movement 25 so that the sample slice 3 is not torn or damaged during the transfer. Prior to cutting, an aqueous solution 26 can be sprayed onto the film 5 and the block face 24 to aid transfer to the film 5.

[0072] Figure 3Depicted are the motion of the sample block 25 and blade 27 during sectioning. Panels (A) and (B) depict designs where blade 2 is stationary and block 1 is forced into the cutting edge. Panels (C) and (D) depict designs where both block 1 and blade 2 are in motion during sectioning. In this case, the cutting edge of the blade translates to the right 27 relative to the sample, cutting through the sample as the block moves downward 25 into the blade. In both cases, the direction of motion of the tissue block 25 remains linear and perpendicular to the cutting edge.

[0073] Figure 4 is a diagram depicting a mechanism for maintaining precise control of the positioning of tissue sample slices 3 as they are transferred between modules on a film 5. Equidistant holes 12 along the lateral edges of the film 5 are aligned with the gears on the sprockets. The rotation of all sprockets throughout the device is coordinated so that the position of all sample slices 3 is precisely and accurately defined.

[0074] Figure 5 It is shown how a strip of film 10 is mounted on a plate 11. In this embodiment of the invention, the mounting plate 11 is designed with protrusions or pins 13 extending through the plate 11, which are orthogonal to its mounting surface. These pins 13 are located along the opposite lateral edges of the plate and align with the holes 12 in the film strip 10. This design provides discrete and consistent placement of the strip 10, optimizes the mounting area, and in certain embodiments of the invention, facilitates scanning and staining (not shown).

[0075] Figure 6 Additional safety measures are shown to prevent movement of the board mounting strip 10 during processing. A mechanical locking bar or clamp 14 can be lowered onto the pins 13 on the board 11.

[0076] Figure 7 is a schematic diagram of an embodiment of the present invention, in which a single continuous film strip sequentially transports sample slices between processing modules. Figure 1 As described, the sample is first sliced ​​and transferred to a carrier film. The embodiment depicted here shows that instead of being cut into strips, the film and mounted sample slices (not shown) move in a conveyor belt manner along through the dewaxing 15, staining 16, sealing and scanning 17 modules. Once processed, the film is rolled into a storage spool 18. The dewaxing module 15 is used to heat each sample and melt the paraffin. Due to the design of this embodiment, the dewaxing module 15 can be designed as a corridor arranged along the heating elements. Chemical cleaning is also commonly used for dewaxing, however, these reagents can be incorporated into and applied by the staining module. The staining module 16 described here is an automatic dispenser design that applies staining reagents 21 to individual sample slices (see Figure 8A). The sealing module (not shown) of this embodiment applies droplets 21 of liquid sealant to each sample; given a similar mechanism, the system can also be incorporated into and applied by a staining module. A scanning module 17 is used to capture a digital image of each sample. There are several possible embodiments of this module (see Detailed Description: Modular Components); a version used with an embodiment of the present invention can continuously take high-resolution images of the sample as the sample enters the camera's field of view. The movement of the carrier film and the positioning of the sample within each module can be controlled by a high-precision sprocket 7.

[0077] Figure 8 Two possible embodiments of the staining module are shown. Panel (A) shows an automatic dispenser design 16. In this design, an automatic applicator 20, which is a pipette or nozzle, drops a specific staining reagent 21 on a specific sample, depicted here as dropping on a strip 10 of a thin film. Staining reagents, dewaxing reagents and liquid sealants can be stored separately in tanks within the body of the module 19. In order to reduce costs and waste, discrete and relatively small volumes of reagents can be used for each sample. Chambers or barriers (not shown) can be placed around each sample to prevent sample runoff and cross-contamination. This module variant can be designed to have multiple applicators 20, which can dispense multiple reagents 21 and process multiple samples simultaneously, as well as variable staining between adjacent sections. An automatic applicator staining module variant 16 is depicted here to process isolated strips 10. However, this design can be integrated into most embodiments of the present invention; when a processing spiral is used, this design is not suitable (see specific embodiments: modular components).

[0078] Panel (B) shows the reagent bath design 22. The variant sample is immersed and incubated in a series of liquid reagent baths 23 as required by the desired staining protocol. The figure depicts an isolated strip being processed; however, the variant is well suited for use with a slide mounting plate 11 (using a film strip ( Figure 1 ) or direct section mounting (not shown) and processing spiral ( Fig. 9 ) for collective sample staining.

[0079] Fig. 9 An alternative embodiment of single belt continuous processing is shown. Figure 7The continuous process depicted is similar, with sample slices being transferred and processed on a carrier film. However, as shown in panel (A), the film and sample slices are wound into a staining spiral 32. The spiral is designed to accommodate the entire length of a fully sliced ​​tissue sample block 1 while maintaining the space between the layers of the wound film. Once the entire sample is sliced ​​and transferred to the spiral, the film is cut (not shown), and the spiral continues to process: dewaxing (not shown), staining using a reagent bath variant module 22 (panel (B)), sealing (not shown), scanning (panel (C)), storage 18. An embodiment of the scanning module 17 depicted in panel (C) shows a simulated image of the sample magnified before image capture. A projector 33 positioned below the now stained sample slice 3 projects a simulated image of the sample through multiple lenses 34. These lenses magnify and focus the image into a high-resolution multi-color digital detector array 35. In this figure, images from each sample are collected sequentially and continuously, however, this scanning module variant can be used in all embodiments of the present invention. Once scanned, the sample is transferred to a storage spool 18.

[0080] Fig.10 The quality control method implemented before 3D image reconstruction is shown. Figure 1 As seen in , camera 4 is present and aimed at block face 24; this captures an image of block face 24 before each cut. The reference image is saved. A scale or mark 28 of known size can be observed within the reference image (panel (A)). The scale can be located on the ultramicrotome bracket or sample box. Panel (B) shows an image of the same sample after sectioning as seen by a high-resolution scanner 29; the sample has undergone an unexpected rotation in this figure. The scale 30 in this panel represents a virtual scale overlaid by the reconstruction software. The reference image and scale 28 are compared with the virtual image 29 and scale 30 to determine unexpected changes in sample position, rotation, and morphology. After determining errors (if any), the software can correct the high-resolution image, as seen in panel (C). The corrected image 31 is saved and incorporated into the 3D reconstruction.

[0081] legend:

[0082] 1. Formalin-fixed paraffin-embedded (FFPE) biological tissue samples

[0083] 2. Ultrathin slicer blade

[0084] 3. Tissue Sample Sectioning

[0085] 4. Low-resolution camera

[0086] 5. Carrier film

[0087] 6. Film spool

[0088] 7. Sprocket

[0089] 8. Fixture

[0090] 9. Cutting blade

[0091] 10. Strips of film

[0092] 11. Mounting plate

[0093] 12. Holes in thin films

[0094] 13. Pin

[0095] 14. Locking rod

[0096] 15. Dewaxing module

[0097] 16.Automatic staining reagent dispensing module

[0098] 17. High-resolution camera

[0099] 18. Storage Spools

[0100] 19. Dyeing reagent storage tank

[0101] 20. Automatic dispenser nozzle

[0102] 21. Staining Reagents

[0103] 22.Automatic staining reagent bath module

[0104] 23. Staining reagent bath

[0105] 24. Block

[0106] 25. Block movement direction

[0107] 26. Water spray

[0108] 27. Blade movement direction

[0109] 28. Reference scale

[0110] 29. High-resolution images of processed samples

[0111] 30. Virtual reference scale

[0112] 31. Corrected high-resolution image of the processed sample

[0113] 32. Processing spiral

[0114] 33. Projector

[0115] 34. Lens or lens series

[0116] 35. Detector

[0117] Example 1

[0118] The FFPE tissue sample is manually inserted into the holder of the ultramicrotome of the sectioning device. As described above, serial sectioning is performed when the sample sections are transferred to the carrier film. The sample sections are transferred from the carrier film to the mounting plate by means of CNC; the samples are transferred serially in the order of cutting so that the position of each sample on the plate is confined to a known area of ​​the plate. The plate and all mounted samples are transferred to the dewaxing module, which is designed as an insulated oven. The plate is then transferred to the H&E reagent bath staining module. A cover slip is applied to the plate. The scanning module, including a high-resolution camera capable of scanning the entire plate, captures a high-resolution image. The plate is removed from the device and stored. Using software algorithms, the high-resolution image is divided into regions each containing only one sample section; a new data set is saved so that each file is an image of a single section. The images are numbered and arranged. All sample images undergo quality control and correction for translation, rotation and / or morphological errors; new image files are generated as needed. The image data set is used to reconstruct a three-dimensional model. The reconstruction is analyzed via AI and machine learning algorithms to aid in disease diagnosis and tissue sample presentation.

[0119] Example 2

[0120] The FFPE tissue samples are manually inserted into the holder of the ultramicrotome of the sectioning device. As described above, serial sectioning is performed as the sample sections are transferred to the carrier film. The carrier film is rolled into the processing spiral. Once the entire tissue block is sectioned and all samples are contained in the spiral, the film is cut. The spiral and its contents are transferred to the dewaxing module, which is designed as an insulated oven. The plate is then transferred to the H&E reagent bath staining module. Once stained, the film is unrolled 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, including a projector, a series of lenses and a high-resolution multi-color digital detector array, magnifies the analog image of a single section and focuses the image into the sensor. The sample is scanned continuously as it is unrolled from the spool. Once scanned, the film is rolled into the storage spool. The high-resolution images should be in reverse order. All sample images are quality controlled and corrected for translation, rotation and / or morphological errors; new image files are generated as needed. The image dataset is used to reconstruct the three-dimensional model. The reconstruction is analyzed via AI and machine learning algorithms to aid in disease diagnosis and tissue sample presentation.

[0121] All disclosures and patents cited in this specification are incorporated herein by reference, as if each individual disclosure or patent is specifically and individually indicated as being incorporated herein by reference, and all disclosures and patents are incorporated herein by reference to disclose and describe methods and / or materials cited in conjunction with the publications. Any reference to a disclosure is a reference to the public documents before the filing date, and should not be construed as admitting that the present invention has no right to precede such publications due to prior inventions. Further, the publication date provided may be different from the actual publication date, and the actual publication date may need to be confirmed separately. If the definition of terms incorporated into the document referenced herein conflicts with the definition of terms clearly defined herein, the definition herein shall prevail.

Claims

1. A device for collecting and processing samples, comprising: A sample cutter, comprising a blade and a bracket, the bracket being configured to hold a sample, wherein the blade or the bracket is movable to ensure that the blade performs a series of cuts on the sample into a plurality of slices having equal thickness; a collecting film having a long and narrow surface, the device being configured to apply force to the slices to transfer and mount the slices onto the collecting film after the sample cutter cuts the slices from the sample, the plurality of slices being successively transferred to the collecting film in the order in which they are cut, the device being further configured to wind the collecting film around a winding roller to form a wound collecting film, wherein the collecting film is wound with the plurality of slices on the collecting film; A plurality of sample processing modules, wherein the plurality of sample processing modules comprises: a dewaxing module configured to receive the rolled collection film, the dewaxing module further configured to dewax the plurality of slices while the plurality of slices are contained on the rolled collection film, a sample staining module configured to receive the rolled collection film, the sample staining module further configured to stain the plurality of slices while the plurality of slices are contained on the rolled collection film, and The sample imaging module is configured to sequentially image each of the plurality of slices on the collection film that has been unwound from the rolled collection film.

2. The apparatus of claim 1, further comprising a film roll, wherein the collection film is provided by the film roll.

3. The device according to claim 1, characterized in that: The samples were formalin-fixed paraffin-embedded (FFPE) tissues.

4. The device according to claim 1, characterized in that The plurality of sample processing modules further include a sealing module and a histological staining module.

5. The device according to claim 4, characterized in that: The samples were formalin-fixed paraffin-embedded (FFPE) tissues.

6. The device according to claim 1, characterized in that: The sample imaging module is configured to simultaneously capture imaging data of the plurality of slices.

7. The device according to claim 4, characterized in that The sealing module provides a sealing liquid droplet to cover the collecting membrane.

8. The device according to claim 4, characterized in that The sealing module provides a transparent cover for the collection film.

9. The apparatus of claim 1, further comprising: a film roller for moving the collection film; wherein the sample imaging module comprises an image capturing device having an image capturing area, wherein the image capturing device is configured to image the slices on the collection film falling into the image capturing area, Wherein the film roller is configured to move the plurality of slices on the collection film into the image capture area one at a time.

10. The device according to claim 9, characterized in that The image capture device includes one or more digital cameras.

11. The device according to claim 1, characterized in that The apparatus is configured to align the plurality of slices along the long edge of the surface and to juxtapose the plurality of slices transferred to the collecting film in a sequential order in which they were cut.

12. The device according to claim 1, characterized in that The collecting film comprises a plurality of slices from the sample, the plurality of slices being fixed to the surface and aligned along a long edge of the surface, wherein: cutting the plurality of slices from the sample having the same thickness, and The plurality of slices are juxtaposed on the surface of the collecting film in the order in which they were cut.

13. The device according to claim 1, characterized in that The multiple sections are processed simultaneously in one or more reagents of the sample staining module.

14. The device according to claim 13, characterized in that The one or more reagents include a reagent for H&E staining, a reagent for IHC staining, or a reagent for fluorescence-based staining.

15. The device according to claim 1, characterized in that The collection film is optically clear and heat resistant up to at least 60°C.

16. The device according to claim 15, characterized in that The collection film further comprises a series of holes or indentations along the long edges of the surface of the collection film.

17. A method for processing a biological sample, comprising: cutting the biological sample into a plurality of slices having equal thickness; transferring the plurality of slices sequentially to a collecting film having a long and narrow surface so that the plurality of slices are aligned along the long edge of the surface; winding the collection film to form a roll having uniformly thick spaces between wound layers; staining the plurality of sections on the rolled collection film; unwinding the wound collection film; as well as imaging the plurality of slices on the collection film, in: As the collection film is moved through an imaging device, one section is imaged at a time. The entire slice is captured at once in the imaging, and The imaging was performed at a speed of 10 slices per minute to 60 slices per minute.

18. The method according to claim 17, characterized in that The biological sample is FFPE tissue.

19. The method according to claim 17, characterized in that The plurality of slices are juxtaposed on the surface of the collecting film in the order in which they were cut.

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