Automated tissue section capture, indexing and storage system, and method

Abstract

To provide a plurality of imaging and processing modalities to characterize sectioned tissue samples.SOLUTION: A preferred embodiment includes the following four main elements: (1) a vibrating knife microtome 64 capable of sectioning a thin (10 to 1,000 micron) slice from a tissue embedded in a tissue block, for example an agar block; (2) a suction manifold 110 capable of drawing buffer liquid with an equipped pump and refluxing it in a buffer tank, in which fluid force attracting the tissue slice 120 is generated by sucking buffer; (3) a material for transfer such as a porous conveyor belt 114, a tape, or a flexible material for transferring samples being driven around the suction manifold, the manifold operating like a pulley to change a moving direction of a conveying material or a belt; and (4) a collection station where tissue slices are placed.SELECTED DRAWING: Figure 4E

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 424,270, filed November 18, 2016, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Tissue sections are used in a variety of histology and pathology assays, as they facilitate staining, labeling, and imaging of tissues. These assays often cannot be performed directly on thick samples. The sectioning process involves embedding the tissue in a support material such as wax, resin, ice, or gel, and then slicing it thinly with a microtome or vibrating blade microtome to a thickness of a few microns to a few hundred microns. The difference between a microtome and a vibrating blade microtome is that the vibrating blade microtome uses a vibrating blade that is submerged in a fluid. This allows the vibrating blade microtome to controllably slice softer materials such as tissues and gels. Microtomes usually require a harder embedding medium to infiltrate the tissue, and extensive post-processing may be required to remove the infiltrated wax / resin. This post-processing may affect the sample and interfere with secondary assays. After sectioning, retrieval, handling, storage, and mounting of the tissue sections must be performed manually by a skilled technician, even with commercial automated systems. This is a costly and tedious task from a labor and throughput standpoint, and many steps of the process are prone to human error. To reduce costs and increase throughput and consistency of histology assays, there is a demand for robust systems to automate the slicing and storage of tissue sections. Furthermore, these capabilities would facilitate easier integration with existing and new automated laboratory systems. One technology that offers automated capture and imaging of tissue sections is sequential two-photon computed tomography (STPT).

[0003] STPT allows for rapid imaging of thick tissue samples ex vivo at submicron resolution, even across whole organs such as mouse brains. Advanced molecular histology assays can annotate tissue slices with a wealth of molecular information. However, currently no methods are capable of producing molecularly annotated 3D maps of thick tissues and whole organs. STPT lacks the necessary multiplexed biochemical specificity and is generally limited to light microscopy methods. Meanwhile, most molecular assays lack the requisite throughput and 3D spatial extent. Thus, crucial gaps remain in understanding the structure and composition of thick tissues and whole organs. Without such understanding, it is difficult, if not impossible, to develop effective treatments for many diseases and disorders.

[0004] In histopathology, attempts to automate the tissue slicing and archiving process have been largely unsuccessful. Although "automated" microtomes exist commercially, they tend to imply only semi-automated or assisted slicing processes and do not include features to automate the collection and handling of the tissue sections. These devices still require a skilled technician to manually or semi-manually manipulate and collect the slices.

[0005] Existing systems that perform automated microtomy are not compatible with soft specimens and are often not compatible with imaging between slices. These systems have been designed with several different specific target applications in mind. For example, one system uses a microtome to slice paraffin-embedded specimens to produce slides for slide scanning applications. Another system produces thin ultramicrotome sections of resin-embedded specimens (usually Caenorhabditis elegans) for serial electron microscopy. This specimen preparation is fed into a custom-made electron microscope specimen holder.

[0006] Previously, methods have been developed to capture agarose tissue slices using a six-axis robotic arm in a pick-and-place configuration. For example, International Patent Application PCT / GB2014 / 051899 (International Patent Publication WO2014 / 202998) by Troy et al., the entire contents of which are incorporated herein by reference, describes a system in which an arm lowers an end effector into a bath liquid and applies suction to attach the slice to a suction cup on the end effector, then lifts the slice out of the solution and places it in a container while dispensing buffer liquid. The container is then placed on a rotating carousel for storage. Aside from cost and complexity, this system is likely to have reliability issues, especially with thinner slices, because it does not accommodate structural differences between tissue slices well and does not provide good control over the structure of the deposited slice. As researchers move to thinner slices, there is a need to gently capture the slices they handle onto a support substrate. This is because thinner sections can be damaged by direct handling. Summary of the Invention

[0007] The present invention relates to the field of automated histopathology, and in particular to automated methods for the study of biological tissue samples or the pathology of diseases and abnormalities in those tissues, which may involve the processing, handling and execution of various methods on tissue samples of interest.

[0008] Preferred embodiments of the present invention describe vibrating blade microtome technology that improves slicing with a vibrating blade, and multiple modalities for automating the capture, indexing, and storage of histological tissue sections produced by multiple applications of these technologies. The mechanism for capturing the tissue section applies a force to move the tissue section or slice to, for example, a transfer material, plate, or slide. In one preferred method, an adhesive force is provided to pull the tissue slice into contact with a support substrate or pallet as it is sliced ​​from the specimen block in the water bath. The substrate and adhesive force help guide the structure and movement of the tissue slice so that it can be transported from the water bath to a storage location. Once outside the water bath, surface tension between the wet tissue section and the support substrate can be used to keep the slice strongly attached to the surface during transport without losing structure. Surface treatments can also be used to promote adhesion of the slice.

[0009] The slices can be peeled off from the support substrate using fluid inertia forces that overcome the adhesive surface tension for controllable transfer to storage, or other handling systems and handling modalities. The slices are stored in wells or tubes, substrates, or some other containers, with or without buffer liquid depending on the application. The sections can also be stored sequentially on film reels, or coated with thin films for a more automated process. Indexing is accomplished in the control software. Storage coordinates can be loaded into the software to specify how and where the slices are stored, and a metadata file tracks the physical location of each slice. The system configuration is designed to be fully compatible with many imaging modalities, including sequential two-photon tomography (STPT), a method of 3D tissue imaging, so that the physical tissue slices can be tracked to their 3D imaging data obtained from STPT. In other embodiments of the invention, other imaging block face modalities and whole specimen modalities can be used. Multiple wavelengths can be detected by corresponding multiple detector elements. A data processor can be used to process the spectral data generated by the detector.

[0010] Other imaging techniques beyond STPT that would benefit from automation of slice and collection include Coherent Anti-Raman Stokes (CARS) Imaging, Stimulated Raman Scattering (SRS) Optical Coherence Tomography (OCT), Oblique Light Sheet, Light Sheet Theta Microscopy, Inverted Light Sheet Microscopy, Swept Confocal Arranged Planar Excitation (SCAPE), Selective Plane Illumination Microscopy (SPIM), Confocal Raman, Confocal, Spinning Disk Confocal, Block Face Structured Illumination, Block Face Imaging, and Optical Frequency Domain Imaging (OFDI). In preferred embodiments, these imaging modalities are combined with physical slice of tissue.

[0011] In other embodiments, other methods such as magnetic resonance imaging (MRI), positron emission tomography (PET), optical projection tomography (OPT) and ultrasound can be used, since these methods can be used to create 3D volumes of specimens such as tissues (including plant tissues), organs or whole animals. In some embodiments of the invention, the specimens can be cleared using various methods (CLARITY, CUBIC, ScaleS, Sacel, 3Disco, UDisco) or labeled using various methods for whole animal and organ labeling, such as whole mount procedures, e.g. iDisco, or in vivo labeling. The imaged specimens can then be sliced ​​and the collected sections can be analyzed using various methods, both optical and biochemical, and the information obtained from these analyses of the sections can then be organized and registered back to the original 3D volume.

[0012] Thus, the present system removes a major barrier to obtaining 3D molecular maps by enabling the integration of automated tissue capture devices into both 2D and 3D imaging devices. In particular, the sequential block-face imaging approach allows for alternating optical imaging and mechanical slicing to build a 3D data set of a macroscopic sample. The top part of the tissue is imaged, for example to a depth of tens of microns, and then removed with a vibrating blade microtome or other slicing device. The process is then repeated until the entire tissue is imaged. Currently, the tissue slices generated must be randomly distributed in a buffer bath and manually collected, sorted, and individually mounted for secondary assays. This approach is not practical for large numbers of fragile slices. Furthermore, thin tissue slices are difficult to handle because they tend to curl and fold. The system described herein automatically captures tissue slices while maintaining their orientation and storing them for downstream analysis. The resulting detailed molecular information from these analyses can then be mapped back onto high-resolution 3D data sets to build 3D molecular maps of tissues and whole organs. These secondary assays may be specifically targeted to a particular region of interest, may be performed on a subset of the sample, for example every Nth section, or may be performed on the entire sample for more exploratory assays. These secondary assays do not need to be known a priori and can be performed depending on the results of future assays. [Brief description of the drawings]

[0013] [Figure 1A] 1 shows sequential two-photon tomography of mouse brain. [Figure 1B] 1 shows sequential two-photon tomography of mouse brain. [Figure 1C] 1 shows sequential two-photon tomography of mouse brain. [Figure 1D] 1 shows sequential two-photon tomography of mouse brain. [Figure 1E]Figure 1A shows sequential two-photon tomography of a mouse brain. Figure 1A shows an example of the device setup. Figure 1B shows coronal (top), horizontal (middle), and sagittal (bottom) views of a mouse brain after 3D reconstruction with a scale bar of 2 mm. Figure 1C shows a 3D view of the coronal section. Figure 1D and Figure 1E show enlarged areas of the regions marked in Figure 1C. These areas show dendritic spines (1D) and axonal fibers (1E). Scale bars are 25 μm for Figure 1D and Figure 1E and 5 μm for the inset of Figure 1D.

[0014] [Figure 1F] 1 illustrates a data processing and control system for a preferred embodiment of the present invention.

[0015] [Figure 2A] It shows the conveyor concept. [Figure 2B] The conveyor concept is shown in FIG. 2A, which shows a schematic side view. During slicing, tissue slices are attracted by suction to the conveyor's capstan suction manifold (CIM). The slices are transported out of the solution after being attracted to conformal contact with the conveyor belt, where they adhere to the belt by surface tension. FIG. 2B shows a 3D CAD model of the preliminary conveyor frame design. The inset in FIG. 2B shows a close-up of the CIM with an NPT threaded fluid fitting. Multiple holes are sized and spaced to obtain approximately uniform flow along the length of the CIM.

[0016] [Figure 2C] 1 illustrates an end view of a capstan inlet manifold (CIM) according to various embodiments of the present disclosure. [Figure 2D] 1 illustrates a capstan inlet manifold (CIM) with alternative hole layouts according to various embodiments of the present disclosure.

[0017] [Figure 2E]FIG. 2 illustrates a side view of a vibratome blade holder and a positionable suction manifold according to various embodiments described herein. [Figure 2F] 1 illustrates an end view of a vibratome blade holder and a positionable suction manifold according to various embodiments described herein. [Figure 2G] FIG. 2 illustrates a perspective view of a vibratome blade holder and a positionable suction manifold according to various embodiments described herein.

[0018] [Figure 3A] 1 illustrates a cross section of an alternative conveyor capstan suction manifold (CIM) having a wedge shaped geometry. [Figure 3B] Shown is a cross section of an alternative Conveyor Capstan Suction Manifold (CIM) with an elongated geometry. The CIM cross section is shown as a dark grey shape. The red (inner) outline indicates the area of ​​the surface to which suction will be applied, and the green (outer) curve represents the path of the conveyor belt, thin film or flexible substrate. The arrows indicate the direction of motion.

[0019] [Figure 4A] It illustrates the reel-to-reel concept. [Figure 4B] The reel-to-reel concept is illustrated in Figure 4A. In Figure 4A, a porous or perforated thin film is advanced around a capstan suction manifold (CIM) in a reel-to-reel arrangement. Suction is applied to the manifold header to draw fluid through the thin film and apply an adhesive suction or fluid drag force to the tissue slices as they are sliced. The red box indicates the area shown in Figure 4B. Figure 4B illustrates the storage concept where tissue slices are punched out of the thin film with an automated die cutter. The tissue slices on the perforated thin film substrate are placed in a well array containing a buffer solution.

[0020] [Figure 4C]13 illustrates tissue sections adhering to and detaching from a belt by adjusting fluid density, according to various embodiments described herein.

[0021] [Figure 4D] 1 illustrates a first section capture system and a second section capture system according to embodiments described herein.

[0022] [Figure 4E] 1 shows a first slice capture system functioning simultaneously with a second slice capture system.

[0023] [Diagram 5] Shown is the coating and imaging of a tissue section. The tissue section is captured or transferred to a thin film (shown with a hole) and then coated with a secondary film. A spring roller mechanism is shown to help attach the secondary film. The spring roller mechanism acts like an idler pulley, bringing the two films into contact around it. Using certain optical properties, imaging can be done through the thin film.

[0024] [Figure 6] Figure 6a-d shows a tissue slice peeling mechanism that transfers the slice from the conveyor belt to another object. Figure 6a shows a schematic side view of the mechanism. A jet of compressed inert gas is applied to the tissue slice from the back side of the conveyor belt while the tissue slice is inverted. Fluid inertia and gravity are sufficient to overcome the surface tension of adhesion, allowing the slice to be transferred to an adjacent substrate. Figure 6b shows a side view of the CAD model with the peeling area shown in the dashed box. Figure 6c shows a close-up orthogonal view of the peeling area shown in the dashed box in Figure 6b. A gas outlet with an NPT fitting is shown. The outlet is designed to direct the flow from the NPT fitting down through the belt to the tissue slice. Figure 6d shows a 100 μm coronal tissue slice of mouse brain transferred to a processed glass microscope slide.

[0025] [Figure 7A] FIG. 1 shows the electrostatic adhesion of tissue sections to a functionalized substrate. The substrate is shown in green and is functionalized with a cationic surface treatment to impart a positive charge that attracts the tissue's natural negative charge.

[0026] [Figure 7B] 1 illustrates a fluid flow system for transferring tissue sections, according to various embodiments of the present application.

[0027] [Figure 8A] 1 shows a tissue slice storage configuration based on gas jet tissue slice stripping. [Figure 8B] 1 shows a tissue slice storage configuration based on gas jet tissue slice stripping. [Figure 8C] 8A shows a tissue section storage configuration based on gas jet tissue section peeling. Figure 8A shows a tissue section peeled against a substrate such as a microscope slide shown in green. Figure 8B shows a tissue section peeled against a well containing a buffer solution. Figure 8C shows a tissue section peeled against another conveyor, film or tape.

[0028] [Figure 9A] 1 shows an automated storage system. [Figure 9B]9A shows an automated storage system. Figure 9A shows well plate storage. Tissue sections are placed in a well plate (shown) or slide array (not shown) on the xy stage. When a plate / array is full, the gantry robot transfers it to a storage rack on a motorized carousel, then transfers an unused plate / array from that rack to the xy stage. When a rack is full, the carousel rotates the next rack into position. Figure 9B shows an independent slide storage system. Sections can be transferred to individual slides on the carousel. The gantry robot transfers tissue sections on slides from a single pick-up position to a storage rack, then replenishes the carousel with unused slides from a supply rack.

[0029] [Figure 10A] 1 shows the concept of automated tissue section storage. [Figure 10B] This shows a concept for automating tissue section storage. Figure 10A shows a schematic side view of the storage concept. A flight conveyor belt catches slides from a dispenser and places them on a belt. Tissue sections can be transferred from the section capture conveyor onto individual slides. A robotic end effector with a suction lift (as shown) or a mechanical gripper can place the slides into a rack for storage. Figure 10B shows a schematic top view of the system in Figure 10A.

[0030] [Figure 11] 1 shows a flow chart of a method for performing 3D mapping of histology data using a section capture system in conjunction with STPT and secondary assays.

[0031] [Figure 12]Automated 3D histology mapping is shown, in which STPT is performed on a tissue sample. Tissue sections are captured in the STPT and proceed to secondary assays, which may include staining, imaging, sequencing, or other molecular analyses. The STPT data is combined with secondary assay data as well as other reference information such as organ atlases and animal lineages, and the data is passed to image registration software. The software maps the multiplexed data stream to a high-resolution STPT template, resulting in a molecularly annotated 3D dataset.

[0032] [Figure 13A] 1 shows an improved slicing technique. [Figure 13B] 1 shows an improved slicing technique. [Figure 13C] 1 shows an improved slicing technique. [Figure 13D] Improved slicing techniques are shown. FIG. 13A shows a sample block embedded in a harder secondary material that can be sliced. This secondary material provides additional support to the block during slicing. FIG. 13B shows the use of supports during slicing. If the section is thin, a support can be added to the top of the block to slice, resulting in a substantially thicker section. The support can then be removed. FIG. 13C shows a vibrating blade microtome slicing at an angle θ to add an additional transverse velocity component to the cut. The sample motion is shown by the red arrows. FIG. 13D shows the motion of the sample block in addition to slicing with a regular vibrating blade microtome. The sample trajectory is shown in red to show the rotational or sinusoidal motion of the block. This motion also adds additional transverse velocity to the cut.

[0033] [Figure 14A] 1 shows automated 3D histology mapping. [Figure 14B]Automated 3D histology mapping is shown. STPT is performed on a tissue sample. Tissue sections are captured in the STPT and proceed to secondary assays that may include staining, imaging, sequencing, or other molecular analyses. The STPT data is combined with secondary assay data as well as other reference information such as organ atlases and animal lineages, and the data is passed to image registration software. The software maps the multiplexed data stream to a high-resolution STPT template, resulting in a molecularly annotated 3D dataset.

[0034] [Figure 15] A method is presented to automate the imaging of thin sections of a tissue sample to generate a 3D model of the tissue sample.

[0035] [Figure 16A] 1 illustrates the use of fiducial markers to identify and analyze tissue sections, according to various embodiments described herein.

[0036] [Figure 16B] 1 illustrates a photoelectric sensor array according to various embodiments described herein.

[0037] [Figure 17] 1 shows a rendered view of a slice capture system according to various embodiments described herein.

[0038] [Figure 18A] FIG. 1 illustrates a partially exploded view of a slide storage carousel according to various embodiments described herein. [Figure 18B] FIG. 1 illustrates a partially exploded view of a slide storage carousel according to various embodiments described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] A preferred embodiment of the invention can include four main elements: (1) a vibrating blade microtome capable of cutting thin (10-1,000 micron) sections from a tissue block, e.g., tissue embedded in an agar block; (2) a suction manifold that can draw buffer liquid with an attached pump and return it to a buffer reservoir. This manifold is positioned close to the vibrating blade microtome, also known as a vibratome, and the suction of the buffer creates a hydrodynamic force that attracts the tissue sections. (3) A transfer material for carrying the sample, such as a porous conveyor belt, tape, or flexible material, is driven around the suction manifold. The manifold acts like a pulley to redirect the movement of the conveying material or belt. The belt slides over the surface of the manifold, carrying the tissue sections out of the buffer (Figure 2A), where the tissue sections are strongly attached to the belt by surface tension; and (4) a collection station (e.g., Figure 4B) where the tissue sections are deposited. There are several beneficial aspects of this design. The tissue slices are gently and controllably pulled to the sample transport device or conveyor during slicing, resulting in consistency in slice location and orientation. Pulling the tissue slice into good contact with the surface of the tape or belt eliminates structural variability due to curling or folding, and helps preserve the integrity of the tissue slice by providing uniform support. When transported out of the buffer, surface tension keeps the slice positioned on the belt in a flat configuration. Finally, the system is mechanically simple, has a high degree of flexibility in positioning components, and is driven by simple rotational motion, reducing cost and complexity compared to alternative options such as multi-axis robots. The system is designed to be fully compatible with STPT and other imaging techniques, but can be used independently in general histopathology applications or in similar configurations using whole organ or whole organism imaging modalities. Further details regarding slicing, imaging, and processing of tissue samples can be found in U.S. Pat. No. 8,771,978, the entire contents of which are incorporated herein by reference.

[0040] Figures 1A to 1E show an example of serial two-photon tomography of a mouse brain. Figure 1A shows an example of the device setup. Figure 1B shows the coronal (top), horizontal (middle), and sagittal (bottom) views of a mouse brain after 3D reconstruction with a scale bar of 2 mm. Figure 1C shows a 3D view of the coronal section. Figures 1D and 1E show enlarged areas of the region marked in Figure 1C. These areas show dendritic spines (1D) and axonal fibers (1E). Scale bars are 25 μm for Figures 1D and 1E and 5 μm for the inset of Figure 1D.

[0041] 1F shows an imaging and processing system 40 according to the present invention. A processor 42 is connected to system components including a light source 48, a first PMT channel 50, a second PMT channel 52, a third PMT channel 54, an (x,y) scanner 56, a vertical scanner 58 for translating an objective lens 60 relative to a tissue sample 65 (such as a whole organ mouse brain), a motorized sample stage 62, a microtome 64 with a cutting tool 66, a tissue slice transport system 68 for moving each tissue slice to a storage system 70, a processing system 72 for further processing the slices, and a second imaging system 74 that can be used in combination with the first imaging system 45 to generate images of the processed tissue. Images of the tissue, both before and after slicing, can be stored in memory 46 and displayed on a display 44 in various formats as described herein. Further details regarding imaging systems and methods of using these systems are described in U.S. Pat. Nos. 7,372,985 and 7,724,937, as well as U.S. patent application Ser. No. 11 / 442,702, filed May 25, 2006, and published as U.S. Patent Publication No. 2007 / 0057211, the entire contents of which are incorporated herein by reference. Further details of the systems and methods for imaging sliced ​​samples and detecting time-resolved data used in conjunction with preferred embodiments of the present invention are described in International Patent Application No. PCT / US2014 / 072368, filed December 24, 2014 and published as International Patent Publication No. WO2015 / 100421, International Patent Application No. PCT / US2016 / 022106, filed March 11, 2016 and published as International Patent Publication No. WO2016 / 145366, and International Patent Application No. PCT / US2011 / 060831, filed November 15, 2011 and published as International Patent Publication No. WO2012 / 068142, the entire contents of which are incorporated herein by reference. Processor 42 can be programmed with software to operate the system components and process image data as further described herein. A transport system 68 can move the tissue sample for further processing and imaging as described herein.The processor 42 can then analyze the pre- and post-slicing image data to correlate and quantify data detailing tissue characteristics. Slicing can change the surface morphology of the tissue, which can complicate the process of correlating tissue structure details after further processing and imaging. The preferred embodiment provides a method for combining and analyzing pre- and post-slicing image data to detail tissue characteristics.

[0042] FIG. 2A illustrates a section capture system 100 according to various embodiments described herein. To generate an attractive fluid force, a combined fluid suction manifold is used as a capstan around which the transfer material slides. In some embodiments, the transfer material is a porous conveyor belt 114. For simplicity, this will be referred to herein as a capstan suction manifold 110 or CIM. Fluid is drawn through holes 112 on the exterior surface of the CIM 110 and thus through the porous conveyor belt 114, and then back into the bath liquid 116 to maintain the liquid level. This fluid flow provides a drag force against the tissue section 120, which is drawn by suction towards the belt 114. A positive displacement pump works well for this application because it is self-priming and can provide and suction a consistent flow over a range of pressures. FIG. 2B illustrates a 3D CAD model of the conveyor concept, with the belt 114 removed for clarity. The close-up inset shows one CIM design. Single-threaded fittings (e.g., NPT) are used to make the fluid connections to the manifold header 122. To ensure near-uniform suction along the length of the CIM 110, the size and number of holes 112 per axial position can be adjusted so that uneven forces do not damage tissue or change the orientation of the tissue on the belt 114.

[0043] The use of transfer materials such as the porous conveyor belt 114 described above, or various films described below, advantageously allows the tissue sample 120 to be quickly attracted to and fixed to a physical substrate. By fixing the tissue sample 120 to a physical substrate, the tissue sample 120 is less likely to roll up or become damaged during prolonged transport under fluid or suction forces. In addition, the continuous transfer materials described herein provide the ability to fix a series of tissue samples 120 in an orderly manner (e.g., linearly along a film, tape, or belt). This overcomes typical problems with slicing, where loose tissue samples floating in a fluid may adhere to each other or move rapidly in a random order.

[0044] FIG. 2C illustrates a capstan suction manifold 110 according to various embodiments of the present disclosure. In this embodiment, the holes 112 are shown as circular, which may be beneficial for machinability. However, the size and shape of the holes 112 in the CIM 110 may be arbitrary to better control the local flow characteristics relative to the sample block. In some embodiments, the holes 112 in the CIM 110 may be slot shaped, as shown in FIG. 2D. Using computational fluid dynamics (CFD) software, the size, shape and distribution of the suction ports can be parametrically optimized to obtain optimal flow characteristics.

[0045] In some embodiments shown in Figures 2E and 2F, the suction manifold may be positionable. In some embodiments, the vibrating blade holder 470 may be modified to allow the suction manifold 110 to be positioned in the gap between the blade holder supports 471. In some embodiments, the suction manifold 110 may be positioned arbitrarily relative to the cutting edge of the blade 66. In some embodiments, the positioning of the suction manifold 110 may be adjustable or even dynamic. For example, the suction manifold 110 may be moved to accommodate different sample types or may be moved during capture to improve the capture mechanism of the tissue section 120.

[0046] In some embodiments, a gap between the support arms 471 of the vibrating blade holder 470 can reduce the weight of the arm and also allow for fluid flow through. This gap provides a more direct fluid path between the bath liquid 116 and the CIM 110 to apply a more consistent fluid force at a gentler flow rate to the tissue section being captured. In this arm design, the pitch angle or "angle of attack" of the blade 66 is fixed to allow for increased stiffness within the arm and also ensure consistent positioning of the blade 66 relative to the conveyor 114. To adjust this angle, the blade holder components can be replaced with ones that have a different mounting angle. This is in comparison to alternative designs where the entire arm can be rotated to adjust the pitch angle. Such designs introduce variability in the mounting position and local fluid dynamics around the blade, which can result in inconsistencies in capturing tissue sections.

[0047] The simplest geometry for CIM is a cylinder, but non-axisymmetric designs can be used to better control the force distribution on the tissue slice and the belt motion. Two examples are shown in Figures 3A and 3B.

[0048] In some embodiments, the conveyor belt 114 may travel around the CIM 110 at a steep initial angle to clear the tub walls and bend pulley 126 before changing direction of motion to horizontal. The conveyor may be powered by a drum-type drive pulley 128. The drive pulley 128 may be crowned to keep the belt properly advanced and may be attached to a tension bracket 127 to allow for tension adjustment of the belt 114.

[0049] The use of the capstan 114 rather than a rotating pulley avoids the need for submerged bearings and rotary fluid couplings, which add to the size, cost, complexity and failure modes of the system. The capstan 114 can provide consistency in the suction applied since the suction hole 112 is in a fixed position relative to the vibrating blade microtome 64. This design is applicable due to the light load of the conveyor. The additional belt tension required to overcome the friction on the capstan is a factor of 2 or less and is not critical to this system. Low friction polymeric materials such as Teflon or nylon may be used as the CIM 110, and in some embodiments, the bend pulley 126 can be replaced with a similar capstan. Metal or ceramic can be used if wear or durability of the CIM 110 is a concern for a particular application. The simplicity of the components means that they can be replaced more easily and less expensively for maintenance or when cross contamination of biological samples is a concern. The belt material may be biocompatible and corrosion resistant and may be a weakly hydrophilic polymer such as nylon or polyethylene terephthalate (PET / polyester) mesh, which is inexpensive and durable.

[0050] To prevent vibrations from interfering with the slicing process, the conveyor and its motor-driven components (motors, pumps, etc.) may be mounted separately from the vibratome 64 and imaging device, or may be isolated by vibration-damping material.

[0051] As shown in FIG. 4A, instead of the conveyor belt 114, the same tissue section capture system configuration can be used with a different transfer material in the form of a porous film 130 in a reel-to-reel configuration with an output reel 132 and a storage or take-up reel 133. This is desirable when cross-contamination from the belt 114 is a concern or when it is desirable to capture and store a linear array of sections directly on the film 130. This also allows for easy replacement of the film 130 for different samples. As shown in FIG. 5, a secondary coating film 135 can be used to completely cover the tissue sections 120 to preserve them for storage or secondary imaging, assay or analysis. This film 135 can be selected to have specific properties such as refractive index, porosity or chemical functionalization depending on the secondary assay. For example, the refractive index can be selected so that a film strip / reel of sections can be fed directly into a microscope system for further imaging to play the same role as a cover glass. The film reel 130 used in the concept in Figures 4A and 4B may be disposable so that an automated die cutter 401 including a punch press 402 and die 404 can perforate the tissue section-containing portion of the film (Figure 4B). In this way, by using the die cut film as a pallet, the sections 120 are well supported by the film 130 and can be handled more easily. The same fluid mechanism can be used instead of a conveyor mechanism to capture the sections directly onto a porous substrate. An end effector on a robotic arm can have a fluid inlet that includes a peelable mesh substrate through which the fluid will flow. The tissue section 120 is captured by this substrate and removed from the water bath by a similar fluid drag or suction force. The surface tension will then be sufficient to hold the tissue section 120 in place. The substrate with the attached tissue section can then be placed in storage or a secondary handling system and an unused substrate can be attached to the end effector.

[0052] Thin tissue sections have a tendency to curl up into a cylindrical structure when cut from a sample block, which can make it difficult to automatically disentangle them for applications such as microscopy that require the sections to be flattened. This phenomenon can be avoided by drawing the sections into a stable flat structure during the slicing process before the sections are allowed to curl up. This can be accomplished by running the pump and conveyor belt 114 during slicing to cause the sections 120 to adhere to a transfer material such as a porous conveyor belt 114. The periodicity of the conveyor belt 114 movement and pore spacing can help smooth out the pulsation of flow from the positive displacement pump to prevent suction forces from concentrating at specific points on the tissue section.

[0053] The porosity of the conveyor belt can be carefully selected so that the pores are large enough not to significantly impede fluid intake into the CIM110, yet small enough to provide sufficient support for the fragile tissue sections. Plastic filtering meshes with pore sizes on the order of 0.1-1 mm tend to work well for this application, with the added benefits of being corrosion resistant, durable, biocompatible, and inexpensive. Polyethylene terephthalate (PET) and nylon can work well. Ultrasonic welding can be used to join the ends of the belt.

[0054] Instead of adhesive fluidic forces that attract the tissue slices using suction / drag, alternative physical forces can be used. These include electrical forces such as electrophoresis or dielectrophoresis, thermal diffusion, magnetic forces, chemical attraction / attachment, and direct physical contact such as with a gentle mechanical gripper, or any combination of these. For example, after the tissue slice 120 is attracted by fluidic forces, chemical surface functionalization promotes short-range attachment to the belt 114 or film 130. This functionalization involves chemical bonding or electrostatic adhesion. The ionic composition and pH of the buffer solution can be modified, for example using low ionic strength and low pH, to facilitate longer-range and stronger electrostatic interactions between the negatively charged tissue and the positively charged substrate based on tuning the Debye length and zeta potential, to adjust the distance and strength of the interaction between the tissue slice 120 and the substrate. As shown in FIG. 4C, the density of the bath fluid 116 can also be modified, for example to adjust the buoyancy of the slices to make them float at the fluid interface. This is desirable as it allows the slices to remain well spread at the interface. Here, the sections can be easily scooped out due to the flat structure. Depending on the section capture configuration, the embedding medium can be modified to control the magnitude and distribution of the force applied to the section, for example by embedding dielectric or magnetic beads. Such beads can also be used as markers for image registration. The embedding medium can also be adjusted based on the desired assay. For example, low melting agarose (LMA) can be used as the embedding medium because it melts easily at temperatures that do not damage the fixed tissue. This mechanism can be used to separate the captured tissue section from the embedding medium in applications where an embedding medium is not desired. For example, if multiple tissue sections are to be placed on a slide, the embedding medium can be removed to maximize the amount of sections per slide, reducing the cost of reagents or imaging services that are cost per slide. Other embedding methods can be used to improve sectioning, including embedding methods that penetrate the tissue, using hydrogels as well as agar.Additionally, these embedding methods may help cross-link the tissue with the surrounding agar / hydrogel block to prevent the tissue from popping out of the agar block during sectioning or transfer.

[0055] To improve tissue binding to the embedding medium and ensure intact tissue sections for the section capture system, fixed tissue samples can be embedded in sodium periodate-oxidized agarose (typically 4.5-6%) to promote bond formation with proteins on the tissue surface. The sample block can then be soaked overnight in a solution of acrylamide and bis-acrylamide monomers to infiltrate the block and tissue. The next day, acrylamide copolymers can be thermally polymerized to form polyacrylamide by heat treatment at 40°C for 2 hours, resulting in 4-10% polyacrylamide. HEMA copolymers can be added in similar proportions as polyacrylamide to reduce swelling of the sample block in buffered aqueous solutions due to water absorption. These polymers cross-link through the tissue, resulting in more homogenous blocks and improved section quality.

[0056] 4D shows the section capture system 150 positioned on the opposite side to the previous embodiment. The section capture system 150 can include a capstan suction manifold 180 that can attract the tissue section 120 from any desired orientation. In some embodiments, the section capture system 150 can allow the tissue section 120 to be loaded with a different side facing up. In various embodiments, different configurations can allow better access in terms of loading and removal depending on the configuration and integration of the system.

[0057] 4E shows a first section capture system 100 functioning simultaneously with a second section capture system 150. The second section capture system 150 can include a second manifold 180. If two or more suction manifolds 110, 180 with different geometries or suction parameters are used, these manifolds can be used simultaneously to better control the capture mechanism and transport of the tissue sections 120. For example, the second section capture system 150 can be used to selectively divert some tissue sections 120 to different storage areas or analysis lines. In some embodiments, the two section capture systems 100, 150 can function simultaneously to divert the tissue sections 120 in a controlled manner.

[0058] The capstan design is simple and effective, but if required for a particular belt / CIM material or loading conditions, the capstan 110 can be replaced with a pulley 410 with a similar suction manifold integrated therein. This pulley system requires a rotary fluid coupling and submerged bearings or bushings to allow it to rotate with the belt underwater, and may need to be made of a more frictional material or have timing belt grooves integrated to ensure the belt does not slip (in this case the timing belt strips can be integrated into the porous belt / film).

[0059] The system configuration described above uses a vibrating blade microtome 64 in a buffered aqueous solution to cut hydrogels such as agar, agarose or polyacrylamide, or soft tissues embedded in hydrogels. The cryostat embodiment of the section capture system can use the same capture mechanism, except that it may be cutting frozen tissue with the vibrating blade microtome 64 or microtome. The liquid buffer can be selected to hold the frozen tissue sample during sectioning, for example, in a liquid nitrogen / isopentane mixture or some other supercooled bath liquid. This liquid buffer is necessary to utilize fluid forces to gently capture the tissue section. The tissue can be flash frozen in standard cryosectioning media such as optimal cutting temperature (OCT) compounds. The system materials can be tuned and insulated to operate in the presence of significant temperature gradients at this lower temperature. The optics can be tuned to image the frozen sample with multiphoton microscopy. The advantages of freezing the sections are that the sections can be cut much thinner, which may be desirable for some assays, and furthermore, it allows the use of fresh tissue (without fixation), which helps to preserve the biochemical composition of the tissue.

[0060] After the section is detached from the sample block, it is conveyed out of the bath liquid. To prevent asymmetric forces acting on the sample and changing its orientation on the conveyor, the CIM can be carefully designed to apply a nearly uniform flow along its length. The symmetric design also ensures that the forces acting on the tissue section are symmetric when the sample is centered.

[0061] Once captured, it is important to be able to transfer the tissue section to a desired substrate for storage, secondary processing, or general handling. A typical example of such a substrate is a microscope slide. Microscope slides are inexpensive, small, and easily integrated with many standard laboratory systems. After the location of the tissue section is identified, it is transferred to the transfer area of ​​the conveyor. In this area, the belt is oriented horizontally by pulleys and the conveyor frame has a cutout that allows access to the belt. This area is designed to be on the underside of the conveyor frame, where the surface tension is sufficient to hold the tissue section upside down. This allows the section to be transferred to the top surface of the slide, maximizing the space for access from the slide handling system, and also means that gravity aids in the detachment of the tissue section due to the force of gravity itself on the section and because gravity acts on any residual liquid on the section to promote the formation of capillary bridges between the section and the slide below.

[0062] Once in the transfer position, the slide handling system moves the slide upwards until it gently contacts the tissue section and the section completely conforms to the surface of the slide. Because microscope slides are highly hydrophilic, surface tension and gravity tend to preferentially adhere the section to the slide compared to plastic mesh, which is usually weakly hydrophilic or even hydrophobic depending on the material. At this point, a gentle jet of gas, usually air, is applied to the backside of the porous conveyor to help release the section from the belt. As the gas stream hits the section, the slide is slowly lowered until the section has completely released from the belt and is flat on the slide.

[0063] To transfer the tissue section from the section capture system 100 for storage or handling, surface tension can be used to steer the tissue section 120 into position, and a jet of gas can apply gentle fluid inertial forces to transfer the tissue section 120 to an adjacent well, tube, container, substrate, slide, or film without damage. This is shown diagrammatically in FIG. 6a. The tissue section 120 on the conveyor can travel around the drive pulley 128 until its orientation is held inverted by surface tension. With the help of this mechanism, the tissue section 120 travels under the nozzle or outlet 602 of a controlled gas flow system. Then, once the tissue section 120 is centered under the gas outlet 602, a pulse of air or inert gas, e.g., nitrogen or argon, is applied to the backside of the porous conveyor belt 114 or film 130. The gas flows through the pores of the belt 114 or film 130 and exerts a distributed fluid inertial force on the backside of the tissue slice 120 that is aligned with the gravity vector. The downward force is approximately equal to the weight of the tissue slice 102 combined with the momentum transfer from the impinging gas jet. The gas jet can be fine-tuned by controlling the gas supply pressure or gas velocity. The gas used can be any inert gas, but air is the simplest and safest in most environments. The gas can be compressed or supplied by a blower, and control can be based on pressure or flow. The dynamics of the gas jet can be changed to help with cleaner exfoliation, e.g., increase or decrease the flow, pulse the flow, or move the nozzle to follow a specific pattern. A position sensor can be used to feedback the position of the tissue slice 120 on the belt 114. The position sensor can help guide the position of the slice relative to the gas exit area. In Figure 6a, tissue sections 120 are shown being transferred to both a well 612 and a flat substrate such as a glass slide 614. Figures 6b and 6c are CAD models of gas outlets 602, whose purpose is to direct the gas flow evenly down over the belt 114.Figure 6d is a photograph of a coronal section of a mouse brain that has been transferred from a conveyor with compressed air pulses controlled via a solenoid valve onto a flat substrate 614. As shown diagrammatically in Figure 7A, the flat substrate 614 is a microscope slide that may be treated to promote tissue attachment.

[0064] In some cases, a gas jet may not be required and surface tension / gravity alone may be sufficient to cleanly detach the tissue section to the substrate. Surface tension can also be altered to tune section adhesion, for example by adding surfactants or adjusting the ionic composition of the capillary bridges.

[0065] The attachment of tissue sections can also be broken by eliminating the liquid interface between the tissue section and the belt. This can be done by submerging the belt and section in liquid to eliminate the interface. At this point, the section will sink or float depending on the density (buoyancy) of the section and the liquid, as previously described in connection with Figure 4C. Typically, agarose-embedded samples will sink because they are denser than water. This detachment mechanism is very gentle and may be desirable for thinner or more fragile samples. If the density of the liquid is higher than that of the section, the section will float and spread out on the surface. This may also be useful when manipulating fragile sections.

[0066] Instead of gas jets to transfer the tissue sections, the user can also use a liquid jet or fluid flow system 705 as shown in FIG. 7B. This may be desirable to serve a dual purpose, for example to simultaneously transfer the tissue section 120 to a well 612, tube, or other container or substrate 710 and dispense a stain or buffer. A mechanical gripper or suction lift on a robotic end effector can be used to pick up the section 120 from the belt 114 or film 130 and place it in storage. The section 120 can also be placed in direct contact with a surface or substrate 710 that is more hydrophilic than the belt 114 or film 130 on which the section 120 is captured. In various embodiments, this can be achieved by a moving substrate or a moving belt. In particular, with the aid of gravity as the section is inverted on the belt 114 or film 130, the capillary bridges formed between the tissue section and this substrate 710 can cause the section to adhere more strongly than they would without the capillary bridges. This may be sufficient to delaminate the segment 130 to the secondary substrate 710, and a gentle pulse of gas can aid in further delamination.

[0067] In some embodiments, the substrate 710 can be covered with a thin film of liquid and moved upward until the section is submerged in the thin film of liquid. This substrate 710 can be patterned with hydrophilic or hydrophobic surface functionalization so that the liquid film has a consistent and controlled size and shape, or can be constrained by shallow wells. Additional liquid can be applied to the backside of the conveyor belt to help cover the sections in the liquid and eliminate capillary bridging. This additional liquid can be used to apply fluid inertia forces to the sections to aid in detachment. This fluid composition can be tailored to the application, including, for example, surfactants, histological stains, or various buffers, and does not have to be an aqueous solution. One of the potential advantages of capturing the tissue section in a thin film of fluid is that the fluid interface can help constrain the structure of the tissue section, preventing it from rolling up or folding.

[0068] As an alternative to the transfer system described, the previously mentioned reel-to-reel arrangement shown in Figures 5 and 6 can be used. In this configuration, the sections 120 are stored directly on the reel 133 or perforated by the die cutter 401, eliminating the need for a technique to transfer the sections from the porous substrate / film / belt on which they are initially captured.

[0069] The tissue sections 120 can be stored in a variety of different containers or substrates depending on the application of interest. The two most widely favored storage methods are directly on glass microscope slides 614 or well plates 612. The gas jet transfer technique is very robust as it can reliably transfer the tissue sections 120 to flat substrates 614 without damaging the structure, but it is also general enough to place the sections in open containers 612. Alternatively, a fluid flow system can be used to peel the tissue sections 120 from the conveyor to the slides 614 or collection chambers 612. As shown in FIG. 8A, the sections 120 can be transferred to microscope slides 614. Microscope slides 614 are ubiquitous, compactly stored, easily functionalized, and compatible with automated systems such as coverslippers, stainers, and slide scanning microscopes that automate the entire process of slicing, slide mounting, staining, and imaging, in addition to the 3D imaging data obtained using STPT. As shown in Figure 8B, well plates 612 are desirable for thicker tissue sections (>50 microns) because many secondary assays use staining techniques that require the sections to be freely suspended in solution. As shown in Figure 8C, tissue sections 120 can also be stored in a secondary linear film strip 132 that can be fed into other systems. Multiple sections can be stored per substrate, well, tube, or container as needed.

[0070] In addition to slides and well arrays, tissue sections can be placed into tissue storage tubes or custom well arrays. Tissue storage tubes are desirable if sections are to be frozen for future analysis. If a researcher wishes to perform a secondary assay on a single tissue section, they need not thaw the entire tissue sample population, but only the specific tube of interest. Custom wells, substrates, or arrays of wells / substrates may be desirable if custom storage features are desired, such as integrated fluidic channels, higher storage density, custom geometries, or compatibility with specific systems.

[0071] The storage process can be further automated, some examples are shown diagrammatically in Figures 9 and 10. Figure 9A shows a layout for depositing tissue sections into well plates 902. The well plates 902 are on a motorized xy stage 910 that positions the wells under the transfer area of ​​the section capture conveyor belt 114. The tissue sections are deposited in the target wells. After the plate 902 is full, the xy stage 910 moves it to a pick-up position 920. At the pick-up position 920, a gantry robot 925 picks up the well plate 902 using a mechanical gripper 927 and deposits it in a rotating carousel 930. The gripper 927 then grabs an empty well plate 902 from the carousel 930 to replenish the xy stage 910. The system in Figure 9A can also be used with racks of tissue storage tubes or arrays of microscope slides. Racks of tissue storage tubes or arrays of microscope slides can be manipulated in the same manner using the xy stage 910 and gantry robot 925 and stored on shelves in a rotating carousel 930. FIG. 9B shows such a system designed to handle individual microscope slides. An empty slide 904 is placed on the carousel 930 and a tissue section 120 is placed on it. The slide with the tissue section is then picked up by the gantry robot 925 with a mechanical gripper 927 and placed in a slide storage rack 940. An unused empty slide 904 is then removed from the supply rack 940 and replenished to the carousel 930 at the unloading position 921. This system can also be used with individual tubes, substrates or storage containers of the same configuration, as long as the storage rack is adjusted accordingly. FIGS. 10A and 10B show an alternative embodiment of slide storage using a flight conveyor belt. FIG. 10A is a schematic side view of the system, and FIG. 10B is a schematic top view of the system. A slide dispenser 1002 uses a passive mechanism to place slides 904 on a conveyor 1004, which travels beneath the conveyor 114 of the section capture system 100. The tissue sections 120 are transferred to the slides 904 on this secondary conveyor 1004, which is then moved incrementally.A gantry robot with a mechanical gripper 927 or suction lift 929 (as shown) picks up the slide 904 with the tissue section 120 thereon and places it in a rack 1006 for storage.

[0072] This section capture technology is designed to work directly with STPT to capture tissue sections that are created during the process of 3D tissue imaging. These sections can be used in secondary assays to map multiplexed biomolecular information, such as protein distribution, onto the subcellular 3D datasets created by STPT. This 3D molecular mapping concept is illustrated in the flow chart in Figure 11 and infographic in Figure 12. A variety of secondary assays can be performed on these tissue sections, including: ●Histological staining o Immunohistochemical staining ·DAB (3,3'-diaminobenzidine) protocol Immunofluorescence (IF) Protocol Isotope-labeled antibody staining with Imaging Mass Cytometry (IMC) or Multiplexed Ion Beam Imaging (MIBI) oDAPI (4',6-diamidino-2-phenylindole) Hematoxylin and eosin (H&E) O Nissle In situ hybridization (ISH) Fluorescence in situ hybridization (FISH) ●Mass spectrometry Tradition oMass spectrometry imaging (IMS / MSI) IMC, MIBI Genomics Next-generation sequencing (NGS) of oDNA Proteomics Transcriptomics NGS of oRNA Fluorescence in situ sequencing (FISSEQ) Metabolic Assay ● Targeted engagement Colocalization studies Chemical and molecular analysis Electron microscopy ●Spectroscopy ●Spectroscopy Microdissection Expansion Microscopy ●ISH ●MerFish ●smFish

[0073] These assays can be semi-automated or fully automated in conjunction with the output of a section capture and storage system. For example, well plates can be fed into an automated liquid handling system to perform free-floating staining / labeling and rinsing. Microscope slides can be used in automated staining, coverslipping and slide scanning systems for fully automated secondary labeling and imaging.

[0074] One of the more interesting applications of this molecular mapping technique is the ability to perform expansion microscopy on specific regions of tissue identified with STPT. After imaging the whole organ and identifying the section of interest, the user can infiltrate and embed the tissue in expansion microscopy medium, and then expand the tissue to image the region of interest at high resolution, for example to look at the dendritic spines of neurons.

[0075] As a stand-alone system, section capture and storage is critical for any histology or pathology laboratory working with tissue samples that require a vibrating blade microtome for sectioning. Manual slicing and handling of tissue sections is a major source of human error and inter-laboratory variability. This technology helps standardize sectioning with greater consistency and throughput and can serve as a technical bridge to further automation of histopathology methods.

[0076] It is important that the thin-sectioning technique used in conjunction with the section capture system herein ensures the production of intact, high-quality sections and allows for the consistent production of thinner sections that are desirable for many secondary assays. For example, thinner sections may be stained more quickly or may be used with imaging modalities that are not compatible with thicker sections. There are several approaches to improving the quality of sections produced with this thin-sectioning system.

[0077] The first approach involves double embedding of tissue samples as shown in FIG. 13A. Slicing with a vibrating blade microtome typically involves embedding tissue in a material with similar viscoelastic properties to create a more uniform material for slicing. This reduces problems due to mismatches in stiffness or damping that can result in differences in vibration amplitude, frequency, or phase between the tissue and the embedding medium. These mismatches can cause stress concentrations at the interface between the tissue and the embedding medium, resulting in tissue delamination or tearing. However, harder embedding media are desirable as they tend to result in better slicing. This approach involves first embedding the tissue sample in one medium 1302 and then embedding the block in a second harder medium 1304, such as a more concentrated agarose, that can also be sliced. This harder medium provides an outer support to the sample block that helps to constrain the flexure of the sample block and improve section quality. Since fracture mechanics dictates that cracks will easily propagate from harder to softer materials, the interface between the sample block and the outer medium should not impede the quality of the cut. At the trailing edge of the block, this additional material will also support the last part of the cut to ensure that it is cleanly severed from the block. Currently, thinner sections may not be completely severed from the trailing edge, leaving them hanging down where they can obstruct the objective lens and cause imaging problems. This is the same concept as drilling a hole in the workpiece to create a scrap piece of material. The scrap piece provides support at the end of the cut to prevent spalling and fraying, improving the quality of the cut.

[0078] Another method to create thinner sections, shown in Figure 13B, may be to add a support layer to the top of the sample block before slicing. For example, if a 10 micron thick section is desired, 40 microns of support material can be added to the top of the block and a 50 micron section can be obtained. The support material can be removed after slicing leaving only a 10 micron tissue section.

[0079] Another simple way to improve section quality is to increase the relative velocity of the sample in the lateral direction with respect to the blade. In a typical vibrating blade microtome, the sample is moved towards the blade at a constant normal velocity while the blade oscillates sinusoidally in the lateral direction to apply shear forces. Optimal slicing requires a good balance between the normal and lateral cutting velocity components (Reyssat E, Tallinen T, Le Merrer M, Mahadevan L. Slicing Softly with Shear. Physical Review Letters. 2012; 109(24):244301, the contents of which are incorporated herein by reference). The lateral velocity component of the present invention is sinusoidal. Therefore, there are moments along the velocity versus time curve where the lateral velocity is close to zero, which is undesirable for the quality of the cut. One way to improve the lateral velocity component is to adjust the angle of the blade with respect to the direction of sample motion (Figure 13C). This results in the motion of the sample block having both normal and tangential velocity components relative to the blade. In addition, the user can move the sample relative to the blade in a way that introduces additional lateral velocities (Figure 13D). For example, the sample can be pushed toward the blade at an angle, rotated, moved back and forth in a triangular or sinusoidal pattern, or any combination of these motions. These motions can be synchronized with the blade vibration for more robust cutting. One synchronization method involves placing the sample on a flexure and vibrating it in phase with the vibrating blade microtome. Another configuration would be to integrate a rotation stage and rotate it toward the blade as the sample is also vibrating. Another configuration is to use the Compressotome as a slicing device.

[0080] 14A and 14B show automated 3D histology mapping. STPT is performed on a tissue sample. Tissue sections are captured in the STPT and proceed to secondary assays that may include staining, imaging, sequencing, or other molecular analyses. The STPT data is combined with secondary assay data as well as other reference information such as organ atlases and animal lineages, and the data is passed to image registration software. The software maps the multiplexed data stream to a high-resolution STPT template, resulting in a molecularly annotated 3D dataset.

[0081] FIG. 15 illustrates a method 1500 for automating imaging of a slice of a tissue sample to create a 3D model of the tissue sample. In some embodiments, the method can include an optional step 1502 of imaging a block face of the tissue sample before slicing begins. The method can include a step 1504 of cutting a section from the block face using a vibrating blade microtome or microtome. In various embodiments, cutting a section using a vibrating blade microtome or microtome can be performed as described above in connection with FIG. 2A. The method can include a step 1506 of attaching the cut section to a conveyor belt or transport system around a pulley or capstan suction manifold. The conveyor belt or transport system, pulleys and capstan suction manifold can be substantially similar to those described above in connection with FIG. 2B, FIG. 3A, FIG. 3B and FIG. 7.

[0082] The method may include step 1508 of transporting the cut sections using a conveyor belt or transport system. Transporting the cut sections may be performed using a conveyor belt as described above in connection with Figures 2B, 6, 9, and 10. The method may include step 1510 of transferring the cut sections from the conveyor belt or transport system to a further processing station and / or storage container. For example, the cut sections may be transferred using a puff of gas as described above in connection with Figures 6a-d. In some embodiments, the cut sections may be placed in a storage container or well, such as a substrate, belt, film, or tape, as described above in connection with Figures 8A-8C. In some embodiments, the cut sections may be transferred to a further imaging system as described above in connection with Figures 4A, 4B, 5, 9A, 9B, 10A, and 10B, or to a further processing station, such as a slide carousel.

[0083] The method may include an optional step 1512 of protecting the cut sections by covering them with a film. In some embodiments, the sections may be individually encapsulated as described above in connection with Figures 4 and 5. The method may include an optional step 1514 of applying a secondary assay to the cut sections and imaging the cut sections. In some embodiments, the imaging step may be performed using sequential two-photon tomography or other multi-photon imaging techniques. Staining and imaging may be performed as described above in connection with Figures 5, 11 and 12.

[0084] The method may include step 1516 of iteratively repeating the previous steps to generate a series of images of a plurality of successive cut sections. The successive slicing and transport of the cut sections may be performed, for example, as previously described in connection with Figures 2 to 5. The method may include step 1518 of analyzing the series of images to generate a 3D model of the tissue sample. The 3D model may include morphological image tissue information and information determined with stains or secondary assays, as previously described in connection with Figure 12.

[0085] In some embodiments, the tissue section 120 may include an external fiducial marker 1602, an internal fiducial marker 1604, or a combination of both, as shown in FIG. 16A. In some embodiments, the fiducial markers 1602, 1604 may enable the use of computer vision. Using computer vision, information about the size, position, orientation, and structure of the tissue section 120 can be extracted, which may help position the section and the substrate for transfer, and fed back to the system as an indication of the quality of the section. Computer vision can also be used to image the tissue section 120 after it has been transferred to the substrate to identify the position, orientation, or structure of the tissue section 120 for secondary processing / analysis, such as secondary imaging and image registration. In some embodiments, the embedding medium can be modified to improve the contrast for computer vision, for example using agarose, acrylamide, or other hydrogels or polymers conjugated to a fluorophore or several different fluorophores. This can be present as an external fiducial marker 1602 throughout the block or only in certain areas. These regions can be differently shaped to aid in identifying the location, orientation or structure of the tissue section 120. An LED or other light source 1606 can be used to excite the fluorophores, and a camera 1610 with an absorption filter 1616 can be used to image the tissue section 120 on the belt 114 or after transfer to the substrate. From these images, thresholding and object recognition can be used to determine the size, location, shape and orientation of the tissue section 120 on the conveyor belt 114 or substrate.

[0086] Identifying the location of the fiducial markers 1602, 1604 in the tissue section 120 may help determine whether the tissue section 120 is aligned with the conveyor belt 114 and whether the tissue section 120 is flat or otherwise in a desired configuration. In some embodiments, the conveyor belt 114 may transport the tissue section 120 past an illumination or excitation source 1606. The illuminated tissue section 120 may then be imaged using a camera 1610 attached to a processing unit 1612. In some embodiments, an absorption filter 1616 may be positioned between the camera 1610 and the tissue section 120 to filter light from the tissue section 120. In some embodiments, the processing unit 1612 may include an object recognition module 1610 for isolating and identifying the fiducial markers 1602, 1604 in images acquired by the camera 1610. The processing unit 1612 can then apply an analysis module 1612 to the image to identify the locations of the fiducial markers 1602, 1604 within the image. For example, the centroid of each fiducial marker can be calculated and coordinates in the xy plane can be assigned to the centroid. If the cth marker has coordinates (x c ,y c ) is assigned. The processing unit 1612 may also include an output or system feedback module 1614. The output or system feedback module 1614 may identify or calculate characteristics for each of the imaged fiducial markers 1602, 1604, including area, shape, orientation, center of gravity, and local deformation. The characteristics of the fiducial markers 1614 may be analyzed to determine whether the vibrating blade microtome 64 or section capture system 100 should be adjusted to return to normal operation. For example, if a threshold number of tissue sections 120 include a fiducial marker 1602, 1604 that is deformed, missing, or set to an incorrect size or shape, adjustments to the microtome or section capture system 100 may need to be made.

[0087] Because tissue sections are thin, transparent, and wet, it can be difficult to sense the mesh against the background. For digital detection signals, there are several types of standard automated sensors that work when properly adjusted, including: Diffuse photoelectric sensor Capacitive proximity sensor Gloss sensor

[0088] A custom photoelectric sensor 1660 will also work, but the photoelectric sensor 1660 can be tailored for a particular application as shown in Figure 16B. In particular, the shallow angle reflection of the laser diode 1650 off the phototransistor 1660, output to a relay or comparator circuit 1670, can be used to detect the tissue section 120, so that it is more specular than the mesh background.

[0089] To reduce the occurrence of false positive sensor signals, the detection sensor 1660 can be "gated" so that the sensor state is monitored only when the tissue section 120 is in close proximity. To determine the exact location of the tissue section 120, the initial movement of the conveyor 114 can serve as coarse positioning and the sensor edge detection routine can serve as fine positioning. Multiple redundant sensors can also be used to reduce the occurrence of false positives in various embodiments.

[0090] Several steps can be taken to improve the contrast of the tissue section against the background, including improving the embedding material as described above, and using different mesh materials and different colors.

[0091] Computer vision can also be used to identify the location and orientation of the tissue slices on the conveyor belt. In some embodiments, a camera can image the belt using various filters, such as standard bandpass absorption filters or polarizing filters, under specific lighting conditions that may include diffuse light, dark field, UV, IR, polarized light sources or low angle light sources (or any combination of these). Once the slices are illuminated with bright light with good contrast, thresholding and object recognition algorithms can be used to identify them. Computer vision can be used as the sensor itself to detect the presence and location of the tissue slices 120, or it can be used in addition to digital sensors to provide additional information to the system. For example, orientation data can be fed back to the system so that it can be corrected in the transfer process. Large deviations in the orientation, size or shape of the tissue slices can be used as an indication of the quality of the slices, which can cause errors in the system.

[0092] FIG. 17 shows a rendered view of a slice capture system according to various embodiments described herein.

[0093] In various embodiments, a slide storage carousel 1800 can be used to store slides, as shown in Figures 18A and 18B. A four-axis SCARA robotic arm can be used to accomplish slide handling and storage. The four-axis SCARA robotic arm can move an end effector in X, Y, and Z Cartesian space, with an additional axis for rotation about the Z axis. The robot can have a force-controlled electric gripper that ensures a defined amount of force is used to grip a slide, which is useful when slides of different sizes or shapes are used. For example, the robot can handle cover slips by reducing the gripper force.

[0094] The gripper tooling is custom designed for handling microscope slides and consists of two elongated fingers with grooves that fit the sides of a microscope slide. These grooves are sized to the tolerances of a typical slide (1.1mm) and have curved edges to guide the edge of the slide in if slightly misaligned without spalling or damage. The grooves terminate at a specific distance along the length of the fingers to provide a hard stop at the edge of the slide.

[0095] Slides can be stored in polyceramic mini-racks that are compatible with commercial staining and coverslipping systems, can be autoclaved and reused. The racks can be oriented vertically with slides stored flat. These racks can be stationary or attached to the robot's envelope or a rotating carousel, actuator or stage to move the racks to a more desirable location for the robot to place the slides or for the user to access the racks. They can also be surrounded by sponges or active humidifiers for humidification. Figures 18A and 18B show an exemplary surrounded rotating slide storage carousel 1800.

[0096] To grab a slide from the rack, the robotic gripper can open wide and move to a position on the slide that is only a few millimeters from the desired gripping position. The gripper can then close until there is a gap just slightly wider than the slide so that the slide remains in the groove but is not subject to gripping force. In some embodiments, this gap can be approximately 1 mm or less. This initial movement serves the purpose of roughly aligning the slide if it is misaligned. The gripper can then be moved to the desired gripping position on the slide. The tooling is closed enough so that if the slide protrudes too far from the rack, the slide hits a hard stop at the end of the groove and is gently pushed into position. Once in position, the gripper can be fully actuated to apply gripping force to the slide, which will push the slide further into the groove in case of any misalignment. This gripper movement and tooling design allows the robot to compensate for any misalignment of the slide in the rack position. Once gripped, the slide is moved into position for transfer of the tissue section.

[0097] Once the sections are transferred to the slide, the robot returns to the rack to insert the slide, opens fully to release the slide, and then retreats. The grippers can be wide enough to handle 25 mm or 50 mm glass slides. The gripper tooling can be slender enough not to interfere with adjacent slides in the rack, ensuring good storage density that reduces the size and footprint of the storage system.

[0098] The robot can be taught the location of the slide rack using a predefined local coordinate system, so that only one point per rack needs to be taught. Capacitive proximity, inductive or photoelectric sensors can be integrated into the end effector and synchronized with the robot to provide semi-automatic positioning feedback for teaching routines or for use in detecting the presence or absence of a slide in the rack. Electric grippers can also provide feedback indicating whether they have reached a target position and a target gripper trigger force. This feedback can be used as an indication of whether the slide has been gripped or not.

[0099] The automated section capture and storage system includes several elements that work in combination to controllably slice tissue sections and deposit them onto a planar substrate such as a microscope slide. These elements include the following functions, which are generally illustrated in Figures 1 through 18: 1. A vibratome, which can slice embedded tissues successively into thin, controllable slices of thickness on the order of tens of microns. 2. An intake manifold connected to a circulating pump that applies a suction fluid force to the tissue sections as they are being sectioned by the vibratome. 3. A porous substrate to which the tissue sections adhere using fluid forces including fluid drag within the bath and surface tension as a three-phase interface forms outside the bath. 4. A conveyor system for transporting the tissue slices out of the bath liquid. The conveyor belt may function as the porous substrate described in (3). 5. A sensor or camera to detect the presence of the tissue section and record its location. 6. The area of ​​the conveyor belt where the transfer of sections takes place. This area consists of: a. Cutout in conveyor frame allowing direct access to conveyor belt b. A nozzle positioned to direct a gentle stream of inert gas through a porous substrate onto the tissue section to aid in peeling. 7. Substrate (microscope slide) handling and storage system. This system comprises: a.SCARA 4-axis robotic arm b. Electric gripper with force control c. Custom-made tooling d. Removable rack for storing and handling boards e. Enclosures that allow humidification of substrates for periods ranging from hours to days 8. A computer or PLC to program, control and synchronize the system, including electronics and hardware such as: A. Operation controller b. Sensor c. Fieldbus with analog and digital I / O d. Cables, terminal blocks, valves, switches, relays, safety devices

[0100] The system shown in FIG. 17 illustrates a transfer system that operates as illustrated in the following example to provide automated control over section processing operations. 1. The user inputs all relevant operating parameters into the software GUI for system control, such as: a. Circulation pump flow rate (0-1000ml / min, typically 350ml / min) b. Conveyor speed (0-50mm / sec, typically 9.5mm / sec) c. Section thickness (10-1000um, generally 50-100um) d. Slicing frequency (0-90Hz, typically 50-80Hz) e. Slicing speed (0-20mm / sec, typically 0.5mm / sec) f. Sample length (0-45mm, typically 22mm) g. Conveyor positioning parameters (depending on system setup) h. Number of sections (depending on storage capacity, typically 240) i. Gas pressure or flow parameters (typically 60 psi compressed air) 2. The slicing system is initiated in the software. 3. The robot picks up the substrate (microscope slide) from a storage rack and moves it into position within the transfer area of ​​the conveyor belt. 4. The stage moves upwards the desired thickness of the sample, the vibratome is activated at the target frequency, and the stage moves the sample towards the blade at a defined slicing speed to slice a thin section of tissue from the sample block. 5. The conveyor and the circulation pump start during slicing. The conveyor is porous, slightly hydrophilic and biocompatible. 6. The tissue section is captured on a conveyor belt and at the end of the slicing motion the pump and vibratome are stopped and the conveyor performs a stepping motion to bring the section into the sensor area. 7. The conveyor motion controller then uses a sensor edge detection routine to locate and detect the edges of the tissue section. 8. Upon detection of a tissue section, the conveyor performs a stepping motion that precisely places the section in the transfer area of ​​the belt. 9. The robotic arm moves the substrate into direct contact with the tissue section. The tissue section preferentially adheres to the hydrophilic substrate by surface tension and gravity. A gas nozzle is then activated to deliver a controlled burst of gas to the backside of the tissue section to aid in cleanly detaching the tissue section to the substrate. 10. The substrate is lowered away from the belt and the gas nozzles are stopped. 11. The substrate with attached tissue sections is then returned to the storage rack. 12. The next slide in the sequence is gripped and put into position for the next section, the stage is reset to its original position and the sequence is repeated until the target number of sections is reached.

[0101] While the present invention has been particularly shown and described in relation to preferred embodiments thereof, it will be understood by those skilled in the art that various equivalents thereof, or changes in form and detail, may be made thereto without departing from the scope of the invention as encompassed by the appended claims. Further, the embodiment includes the following items. [Item 1] 1. A method for processing a biological sample, comprising the steps of: Slicing one or more portions of the biological sample to form one or more sections; applying a force to the one or more sections from the biological sample using a tissue capturing device and transferring the one or more sections to a carrier material; transporting the sections from the slicing station to a processing or storage station; A method for providing the above. [Item 2] 2. The method according to claim 1, wherein the step of transporting includes attaching the sample in the fluid bath to a transport material and moving the material to the processing station. [Item 3] 2. The method of claim 1, wherein the sample is sliced ​​using a vibrating blade microtome. [Item 4] 2. The method of claim 1, further comprising a data processor programmed to control the slicing device and the conveying device. [Item 5] 2. The method of claim 1, wherein the tissue capturing device comprises a suction manifold. [Item 6] 6. The method of claim 5, wherein the suction manifold includes a capstan positioned in a fluid reservoir. [Item 7] Item 13. The method of item 1, wherein the conveying material comprises a moving belt. [Item 8] 6. The method of claim 5, wherein the suction manifold is mounted to a frame having one or more pulleys including a drive pulley. [Item 9] 2. The method of claim 1, wherein the tissue capture device comprises a porous substrate. [Item 10] 2. The method of claim 1, further comprising a sensor for sensing a section on the conveying material. [Item 11] Item 10. The method of item 1, wherein the intake manifold is coupled to a fluid pump. [Item 12] 2. The method of claim 1, wherein the transfer material comprises a porous belt or a porous substrate that applies a fluid force to the tissue section. [Item 13] Item 11. The method of item 10, wherein the sensor includes an imaging device or camera. [Item 14] 2. The method of claim 1, further comprising a nozzle for directing a gas stream to detach the tissue section from the porous substrate. [Item 15] 2. The method of claim 1, further comprising a robotic arm for carrying a section substrate, such as a glass slide. [Item 16] 16. The method of claim 15, further comprising a glass slide storage device. [Item 17] 2. The method of claim 1, further comprising the step of automatically controlling the system using a computer or controller. [Item 18] 2. The method of claim 1, further comprising displaying system operating parameters using a graphical user interface (GUI) and display. [Item 19] 11. The method according to claim 10, wherein the sensor operates with an edge detection program to detect edges of the section. [Item 20] 2. The method of claim 1, wherein the system controller sets system parameters including pump flow rate, belt speed, slice thickness, slicing frequency, slicing speed and gas flow. [Item 21] 2. The method of claim 1, wherein a robotic arm positions the substrate relative to the belt so that the section is placed on the substrate. [Item 22] 22. The method of claim 21, further comprising directing an inert gas at the section to adhere the section to the substrate. [Item 23] 2. The method of claim 1, wherein a camera images each slice and the images are processed to determine its position and orientation on the moving belt. [Item 24] 24. The method of claim 23, wherein a light source illuminates the sample for imaging. [Item 25] a slicer for slicing sections from the block face of the tissue sample for processing; a transport system for transporting each section of the sample; a processing station for receiving the tissue section from the transport system; A storage container for storing the sample; an imaging device for imaging each slice; a data processor for forming a 3D representation of the tissue sample; A method for an automated sample processing system comprising: [Item 26] 26. The system of claim 25, wherein the transport system is a conveyor belt. [Item 27] 27. The system of claim 26, wherein the conveyor belt moves relative to a capstan suction manifold. [Item 28] 28. The system of claim 27, wherein the capstan suction manifold has one of a cylindrical cross-section, a wedge-shaped cross-section, or an elongated cross-section. [Item 29] 26. The system of claim 25, further comprising a secondary assay applied to the cut sections prior to further imaging. [Item 30] 26. The system of claim 25, wherein the imaging device includes a serial two-photon computed tomography system. [Item 31] 26. The system of claim 25, wherein the imaging device includes an optical coherence tomography system. [Item 32] 26. The system of claim 25, further comprising a film covering the cut sections to protect the cut sections. [Item 33] 26. The system of claim 25, wherein the gas source directs a pulse of gas at the cut section. [Item 34] 26. The system of claim 25, wherein further comparing a fluid source to the cut section from the delivery system includes directing a fluid against the cut section. [Item 35] 26. The system of claim 25, further comprising a coating fluid applied to the sample surface. [Item 36] 26. The system of claim 25, wherein attaching the cut piece to the transport system includes functionalizing a portion of the transport system to enhance chemical bonding or electrostatic adhesion. [Item 37] 26. The system of claim 25, wherein the tissue sample is embedded in at least one of agar, agarose or polyacrylamide, or a hydrogel. [Item 38] 26. The system of claim 25, wherein the storage container is one of a well, a substrate, a belt, a tape, or a film. [Item 39] 26. The system of claim 25, further comprising a data processor for processing image data. [Item 40] 26. The system of claim 25, further comprising a two-photon tomography system. [Item 41] 26. The system of claim 25, further comprising a vibrating blade microtome. [Item 42] 26. The system of claim 25, wherein the tissue capture device further comprises a porous cylinder. [Item 43] 26. The system of claim 25, further comprising an indexing system. [Item 44] Item 44. The system of item 43, wherein the indexing system identifies each section image and storage location in a memory device. [Item 45] Item 26. The system of item 25, wherein the slicing device includes a blade positioned relative to the gap between the support arms. [Item 46] 26. The system of item 25, comprising a tissue capture device. [Item 47] Item 47. The system of item 46, wherein the tissue capture device includes an aspiration manifold, a belt, and a porous member. [Item 48] Item 48. The system of item 47, further comprising a light source for illuminating the samples on the belt for imaging. [Item 49] 26. The system of claim 25, further comprising a film covering system. [Item 50] 1. A method for automatically imaging a thin section of a tissue sample to create a 3D model of the tissue sample, comprising: slicing sections from a block face of the tissue sample using a vibrating blade microtome; attaching the cut pieces to a carrier system; transporting the cut sections using the transport system; transferring the cut sections from the transport system to a further processing station or to a storage container; imaging the cut section; repeating the slicing, attaching, conveying, transferring, and imaging steps to generate a series of images of a plurality of consecutive cut sections; analyzing the series of images to generate a 3D model of the tissue sample; A method for providing the above. [Item 51] 51. The method of claim 50, wherein the transport system is a conveyor belt. [Item 52] 51. The method of claim 50, wherein the imaging step comprises recording an image of the section on a belt or film. [Item 53] 52. The method of claim 51, wherein the conveyor belt includes a capstan suction manifold. [Item 54] 54. The method of claim 53, wherein the capstan suction manifold has one of a cylindrical cross-section, a wedge-shaped cross-section, or an elongated cross-section. [Item 55] 51. The method of claim 50, further comprising applying a secondary assay to the cut sections prior to imaging. [Item 56] 51. The method of claim 50, wherein the step of imaging the cut sections comprises sequential two-photon tomography or multiphoton imaging techniques. [Item 57] 51. The method of claim 50, further comprising the step of protecting the cut sections by covering the cut sections with a film. [Item 58] 51. The method of claim 50, wherein transferring the cut section from the transport system comprises directing a pulse of gas at the cut section. [Item 59] 51. The method of claim 50, wherein attaching the cut pieces to the carrier system comprises functionalizing a portion of the carrier system to enhance chemical bonding or electrostatic adhesion. [Item 60] 51. The method of claim 50, wherein the tissue sample is embedded in at least one of agar, agarose, or polyacrylamide. [Item 61] 51. The method of claim 50, wherein the storage container is one of a well, a substrate, a belt, a tape, or a film. [Item 62] 51. The method of claim 50, further comprising processing the spectral data in a data processor. [Item 63] 51. The method of claim 50, further comprising sensing an edge of each slice. [Item 64] 51. The method of claim 50, further comprising processing the sample using a system according to any of claims 25 to 49. [Item 65] Item 51. The method of item 50, further comprising imaging each slice in a plurality of imaging modes, optionally including computed tomography.

Claims

1. 1. An automated sample processing system for processing a biological sample, comprising: a slicing system for automatically slicing one or more portions of a biological specimen in a fluid bath to form one or more sections; a transport system for transporting the one or more sections removed from the biological sample, the one or more sections being transported from the fluid bath on a surface of a transport material; a controller for controlling a speed at which the section is conveyed by the conveying material; at least one of a processing station and a storage station that receives one or more sections transferred from the fluid bath; An automated sample processing system comprising:

2. The system of claim 1 , further comprising a sensor, such as a photodetector, for detecting a sample transported on the transport material, the transport material being porous and fluid in the fluid reservoir being capable of being attracted to the porosity of the transport material.

3. The system of claim 1 , wherein the transport system further comprises a tissue capture device including a manifold within the fluid bath that displaces fluid within the fluid bath to apply a fluid force to each section.

4. The system of claim 3 , wherein the manifold includes a capstan for use in suction to apply the fluid force to the one or more segments.

5. The system of claim 3, wherein the controller is connected to the slicing system including a slicing blade and the tissue capturing device, and the biological sample is mounted on a movable stage connected to the controller so that it can be moved within the fluid bath.

6. The system of claim 1 , wherein each section is stored and processed on a movable substrate.

7. The system of claim 1 , further comprising a fluid pump for transporting fluid in the fluid reservoir, the fluid reservoir comprising water, such as a buffered aqueous solution.

8. The system of claim 1 , wherein the system software includes operating parameters including a slicing speed, a pump rate for fluid flow in the fluid bath, and the speed of the conveyed material.

9. 10. The system of claim 1, wherein a fluid intake manifold provides a fluid flow that draws the pieces located at least partially within the fluid bath toward the conveyor belt.

10. The system of claim 1 , wherein the conveyed material comprises a conveyor belt.

11. 10. The system of claim 9, wherein the fluid intake manifold comprises a manifold header having a plurality of holes that provides a fluid force to the conveyor belt having a porosity, thereby securing each cut section on the conveyor belt for transport.

12. 10. The system of claim 1, wherein a conveying material moves the section to the sensor or camera, and the controller is operative to move the conveying material, including the conveyor, and to employ an edge detection routine to detect edges of the section.

13. 2. The system of claim 1, wherein the system software includes an indexing system that includes storage coordinates for each section in the storage station or a location within the processing station, the indexing system tracks each section for registration within a three-dimensional (3D) volumetric representation of the biological specimen, and the indexing system identifies a subset of sections for further processing.

14. 14. The system of claim 13, wherein the indexing system further comprises at least one of a sensor and a camera for detecting the position of the sections, the camera detecting images of each section before and / or after slicing, such that at least one image of each section is stored in memory.

15. 2. The system of claim 1, wherein the stored image of the slice includes fiducial markers that identify the slice.

16. 10. The system of claim 1, wherein each section is located in a well, in a tube, on a substrate, on a film, on a slide, or on an array thereof for storage and further processing.

17. The system of claim 1, wherein the system software and the controller synchronize the slicing, transport, placement of sections onto a substrate for storage and / or further processing, and one or more sensors, detectors and / or cameras that operate to identify and position sections within the system.

18. 1. A method for automated sample processing of a biological sample, comprising: Operate an automated sample processing system having a controller coupled to a slicing system and a transport system to automatically slice the biological sample in the fluid bath to form one or more sections, the sections being transported from the fluid bath; The method comprises: slicing the biological specimen into one or more sections in the fluid bath at a controlled rate with the slicing system; conveying the one or more sections with the conveying system, the one or more sections being conveyed from the fluid bath at a controlled rate on a conveying material; further processing the transported section or sections in a processing system that generates further data for each processed section. A method for providing the above.

19. 20. The method of claim 18, wherein the carrier material is porous such that the fluid comprises a buffered aqueous solution.

20. and indexing the sections transferred from the fluid bath with the indexing system for indexing each of the one or more sections, the position of each section being stored.

20. The method of claim 19, wherein performing further processing comprises further transporting the indexed sections from a storage location to an imaging location and imaging one or more of the indexed sections.

21. 20. The method of claim 19, wherein performing further processing comprises transporting each selected section on the slide to a processing station where at least one of imaging, staining and analysis is performed to generate further data and where the further data is stored.

Images