Tissue Chamber

Abstract

The present invention relates to a system and method for tissue processing and analysis. The tissue chamber is configured to enable single-container chemical processing, imaging, and wax embedding of a tissue sample within a single container without manipulation between steps. A tissue chamber with features for supporting the tissue sample and allowing fluid flow between the tissue and the tissue chamber surface is disclosed. The features may be index-matched to sample structures of interest or dissolvable in a clearing fluid to enable in-chamber imaging with minimal distortion.

Description

[Technical Field]

[0001] The present disclosure generally relates to histological systems and methods for simplified positioning, chemical processing, and / or imaging of tissue samples, including imaging, staining, fixing, dehydrating, and embedding in a single chamber. [Background technology]

[0002] Histology and histopathology involve the study of cells and tissues under a microscope to diagnose and monitor diseases such as cancer. Many of the basic techniques involved in cytological analysis are 100 years old or more and require trained medical professionals.

[0003] With respect to standard histological methods, the current steps include placing a tissue sample inside a plastic cassette with perforated walls to allow fluid access, and exposing the cassette to a fluid environment of changing composition over time to provide chemical fixation of the tissue.

[0004] The tissue is finally dehydrated and infiltrated with paraffin before removing the embedded sample from the cassette through melting of the paraffin. After paraffin infiltration and remelting, the sample is repositioned in the molten paraffin and allowed to cool again, allowing the sample to be fixed in an orientation that allows for sectioning slices of the sample in selective planes optimized for clinical interpretation. The slices are then mounted on slides, stained, and cover slipped, and then either viewed directly by a pathologist or presented to an imager for digitization.

[0005] The above approaches require manipulation of the tissue during processing, resulting in labor costs and significant processing time. Furthermore, small samples, including small skin biopsies and biopsies from the gastrointestinal tract, or elongated core biopsies where orientation is important for interpretive examination of layers or for complete cross-sectioning, may rotate and flex freely while the cassette is submerged in fluid and exposed to agitation or flow. These samples may therefore require significant manipulation and processing time for optimal histological analysis.

[0006] In addition, the series of processing steps described above must be completed before the pathologist can begin substantive analysis of the sample, thereby incurring labor costs and delaying diagnosis.

[0007] When considering methods to mitigate these technical and manual challenges of tissue preparation, modifications that allow for direct imaging of tissues without physical sectioning are feasible and highly desirable. The greatest benefit of this approach, for minimizing processing time, labor, and costs, would be achieved if a single, low-cost tissue holder / cassette could be utilized for both processing and imaging. However, several important considerations exist. First, there are multiple fluids involved in processing, which must be exchanged in a timed sequence. Second, imaging typically requires the sample to be placed flat against a sealed surface, which may reduce chemical access during incubation. A third issue, particularly noticeable when imaging samples in imaging fluid, is managing potential air or gas bubbles within the imaging cassette. In particular, it is important to minimize gaseous bubbles during incubation, since some reagents are costly (e.g., dyes) and therefore constant addition of reagents is undesirable, which could eliminate problems created by bubbles. Also, gas bubbles reduce the contact area for reagent diffusion, and bubbles highly distort imaging. Therefore, filling the tissue chamber with these reagents requires that special effort be made to avoid or disperse bubbles. These and other practical considerations create considerable design challenges. Summary of the Invention [Means for solving the problem]

[0008] The present invention provides a single-chamber solution for sample processing (e.g., dehydration, fixation, staining, and embedding), thereby reducing the cost, labor, and time required for histological analysis. Furthermore, the present invention allows for the efficient incorporation of intermediate imaging steps during tissue processing, thereby providing faster access to diagnostic information and eliminating the need for costly steps (manual cutting, staining, and slide distribution) when such work can be eliminated based on initial imaging results. ) and other benefits such as the potential to avoid

[0009] The single-chamber solution provided herein allows the user to first orient the sample within the chamber at the desired position for imaging and / or sectioning, and then perform all sample processing, including fixation and / or dehydration, initial staining, imaging, and optionally paraffin embedding, without the need to touch the sample again or otherwise manually transport it, reducing errors, manufacturing costs, and processing labor requirements. Thus, the tissue chamber of the present invention enables automated sample processing using the various processing devices described herein. The container aspects, such as specific geometries, materials, and other configurations, allow samples to be successfully processed (e.g., dehydrated, fixed, stained, cleared, and optionally embedded in a solid medium) without the need for direct sample manipulation. In some embodiments, features on the container surface can aid in fluid access, exchange, and / or flow to all sides of the sample. Sealable ports, such as "self-sealing" and "self-repairing" syringe injection ports (needle and needleless variants exist), allow fluid exposure, movement, and exchange while preventing potential gas bubble formation and entrapment that could affect imaging. A key aspect is that the resulting chamber is airtight, thereby preventing highly volatile agents (e.g., alcohol) that are part of the process from rapidly evaporating during incubation, which would both uncontrollably alter the dye concentration in the remaining fluid and potentially dry out the sample, resulting in irreversible morphological changes. It is also important in practice to design a chamber with a durable seal that allows for long-term storage of samples in either liquid, wax (e.g., paraffin), or polymer embedding materials.

[0010] Our initial attempts to solve the bubble problem focused on ensuring that no air was introduced into the chamber during fluid filling. This approach worked reasonably well in situations where capillary action kept the fluid attached to all cross-sectional chamber channel sides and the chamber dimensions were small enough that air was not a risk of becoming trapped. For horizontally aligned tubular chambers, these conditions relate to the degree of intermolecular attraction of the fluid relative to gravity, as described by a dimensionless ratio known as the Bond number. Relevant parameters are the fluid density, the surface tension of the fluid (and tissue), and the dimensions of the fluid chamber. For water in a glass tube, the fluid will remain attached for a characteristic dimension of approximately 2 mm. Beyond that, the fluid becomes detached from the chamber walls, potentially increasing the risk that some air will remain in the chamber and reach the outlet. The fluid then continues to flow around trapped gas bubbles, which are difficult to remove. Importantly, we observed that tissues incubated with methanol mixtures at temperatures in the range of 40-50 degrees Celsius resulted in the generation of gas bubbles, which in turn affected fluid exchange during the dehydration, coloring, and organic solvent steps of processing, creating additional challenges in reducing the deleterious effects of the bubbles.

[0011] Port location and chamber orientation are relevant for reducing bubble effects. By positioning the outlet port higher than the inlet port and at or near the highest point inside the chamber during filling, gas can escape during the filling process. Any air / gas in the chamber will rise toward the elevated outlet port and be purged as the fluid fills the chamber. One approach we have demonstrated to increase dye access to the large imaging surface is to use either index-matched or solvent-sensitive features between the imaging window and the sample, allowing fluid exposure at the imaging surface during processing. These features can either be transparent during imaging or dissolved prior to imaging, substantially reducing or eliminating optical distortion during imaging. We have also shown that the inclusion of a substantially non-fluorescent and non-reflective sponge between the sample and the non-imaging surface of the chamber can aid in mounting and / or positioning, help ensure that position is maintained, prevent tissue compression artifacts, accelerate global fluid exchange, and allow fluid flow around the non-imaging side of the sample, which may provide a surface for improved "wetting." When optically transparent, this can also enable imaging through multiple surfaces, which is particularly valuable for the use of a multiphoton modality known as second harmonic generation (which can be detected in both transmission or reflection, but best detected in transmission).

[0012] Alternatives (such as those described in U.S. Pat. Nos. 8,796,038 and 2008,022,7144, incorporated herein by reference) have been explored to fix the position of small samples during tissue processing so that repositioning during the wax-embedding step is not necessary. However, none of these conventional methods allow for imaging immediately after the clearing step; i.e., they are all designed for final removal by manual cutting and staining prior to visual interpretation or digital scanning. In addition, they are typically best imaged in very specific orientations and would not work for orienting many common types of biopsies, including gastrointestinal or skin biopsy samples, which have irregular geometries.

[0013] The present invention offers significant advantages over existing techniques by allowing samples to be placed in a container for histological analysis prior to full chemical processing (i.e., either before or after exposure to formalin fixative) and undergo chemical processing through the clearing stage in an oriented location within a single container device that can be used for all steps of dehydration, staining, clearing, and imaging. Furthermore, the tissue chamber of the present invention can then optionally be used for paraffin or polymer embedding and, optionally, for subsequent automated or machine-assisted removal of the embedded sample using a removal device configured to cooperate with the tissue chamber of the present invention.

[0014] As noted above, the tissue chamber can include features operable to minimize the contact area between the chamber floor and / or walls and the sample, thereby allowing good fluid access to all areas of the sample for fixatives, stains, dehydration solutions, molten embedding materials, and / or other processing fluids. The chamber walls and features can be optically clear and / or index-matched to the clearing fluid and / or structures of the sample to be examined (e.g., organelles, membranes, or proteins). The features can comprise irregular surfaces, which effectively function as microchannels with dimensions in the range of tens of micrometers to 500 micrometers. In some embodiments, the surface irregularities form channels with heights of 100 micrometers. In other embodiments, the channels are 200 micrometers deep. In other embodiments, the channels are 50 micrometers deep. In still other embodiments, the surface features are bumps of similar dimensions. In certain embodiments, the features may comprise a material that dissolves in the presence of certain solutions (e.g., clearing fluids) used in sample processing, such that the features space the sample from the container walls and provide good fluid contact with all portions of the sample for processing, but dissolve prior to any initial imaging of the sample within the chamber and therefore do not interfere with the imaging process. The walls of the tissue chamber itself (or the imaging window portion therein) are, in preferred embodiments, substantially optically clear and / or index-matched to the clearing fluid and / or the structure of the sample to be examined. In some embodiments, the index of refraction is exactly the same as or close to that of a common coverslip, which is typically made from glass with a refractive index of about 1.515 at specific visible wavelengths.

[0015] Single-chamber processing as described herein can offer the added benefit of minimizing reagent usage. Processing samples in a chamber allows for strict control over the volume of the chamber and reagents used in processing. Instead of placing a tissue-containing cassette openly in a fluid environment that may be hundreds of times the volume of the tissue, as in conventional techniques, a single sample processing vessel as described herein has a volume that is minimized relative to the target tissue size, reducing reagent expenditures. The optimal fluid-to-tissue ratio can vary depending on the desired compromise between processing speed and imaging depth. As a general rule, the reagent volume should be at least 11 times the volume of the tissue, but imaging depths of hundreds of microns requires a lower ratio. Reagent conservation is particularly important for controlling dye costs, which can otherwise be prohibitive, especially for certain fluorescent markers. Thus, the sample preparation reagent vessel is particularly suitable for staining of unembedded samples, particularly for processes incorporating fluorescent staining, where dye cost can be a major or significant cost component.

[0016] The single-chamber technique described herein also reduces processing time over existing methods, where it is more economical to wait until sufficient sample is received and "totaled" (placed in a cassette) before loading into a tissue processor. The single-chamber approach can be combined with specialized tissue processors operable to accept the disclosed tissue chambers, for example, through interfacing with the chamber's fluid inlet / outlet. Once the chamber is loaded with a sample, the sample can be immediately plugged and processed, reducing the time the sample must idle waiting for additional samples, as in multiplex processing. The same applies to steps subsequent to embedding, including slide matching, slide staining, and organizing and scanning slides for digitization. As noted above, the single-chamber system described herein allows for varying geometries to more closely correspond to individual sample geometries, reducing chamber fluid volume and reagent consumption. Chambers within tissue cassettes can be designed to fit the smallest size container that will accommodate the sample. For example, a long core biopsy can be placed in a long, thin channel.

[0017] The container can be coded (e.g., with a machine-readable symbol such as a matrix barcode (QR) or UPC code, or any symbol of a recognizable shape, color, or reflective pattern). This can be a universally unique identifier (UUID), which uniquely identifies the cassette prior to sample addition and allows the tissue processor to automatically recognize the geometry of the tissue chamber being used and adjust the input volume accordingly, thereby further minimizing wasted reagent. In various embodiments, the container itself may be color-coded and indicate a geometry that can be recognized by the processing machine to determine the appropriate fluid volume and incubation time for a specific sample, minimizing average processing time while resulting in considerable savings in reagent costs, reduction in waste, and consistent processing results. In some embodiments, the identifier includes information that can be useful for tracking production or user details, such as client, date, and location. The unique identifier can be linked to the sample by the user through a laboratory and / or hospital information system (LIS / HIS), thereby creating a means by which data can be associated with the source subject. In some embodiments, the cassette incorporates an area to allow a second label to be printed or affixed if the facility desires to further identify the sample / cassette by visual means or any of a variety of known methods for machine reading of the label. For example, this would allow the user to read a local sample number or patient name and also satisfy requirements for identification established by laboratory accreditation bodies.

[0018] Similarly, microscope scanning and imaging times can also be reduced by using sample-specific sized chambers. Microscope slide scanners use various approaches to minimize slide scanning time, but they remain inefficient and manually dependent to varying degrees. Current systems typically capture low-power images and use image processing algorithms to estimate tissue location and size, but they are adversely affected by difficult-to-control artifacts such as mounting variability, dust, and dirt. As a result, manual oversight may be required to ensure that tissue is not missed and large empty spaces are not imaged. Operator adjustment of the scan area is a time-consuming component of slide scanning that can result in repetitive scans and high average slide scan times.

[0019] Coded sample-size-specific chambers as described herein can help maximize efficiency for image scanning while avoiding manual intervention. Because tissue is placed in the smallest chamber it will fit into and the imager can read the chamber coding to determine the size of the area to scan, the entire potential tissue location area can be imaged without wasting extra time imaging areas without tissue. The opportunity for error can be reduced and operator intervention should not be required, providing efficient imaging with reduced labor costs and errors.

[0020] The tissue holder of the present invention includes a trough area or cavity cutout sized to contain the sample and various processing fluids through which sample processing occurs. The trough is in fluid communication with one or more fluid inlets or outlets operable to provide processing fluids to the tissue-containing chamber. The tissue holder may include additional material surrounding the trough to allow for easy manipulation and / or orientation of the chamber within various processing equipment. In a preferred embodiment, the internal cavity is formed by two components. The first component is bounded on one side by an imaging window whose thickness and refractive index are optically matched to the requirements of the imaging microscope objective design. In a preferred embodiment, the refractive index is 1.5-1.7 and the thickness is less than 600 microns. In a more preferred embodiment, the refractive index is 1.5-1.6 and the thickness is at or less than 500 microns. In some embodiments, the thickness is at or less than 150 microns. In some optimized versions, the thickness is 100 micrometers or less. In some embodiments, the window has a refractive index equal to or similar to that of the glass commonly used in coverslips, i.e., about 1.515. In some embodiments, the window is made of coverslip glass. When discussing specific refractive index values, it is understood that there are standard wavelengths for measurement, and values ​​may vary with the wavelength used for measurement. In this context, the most relevant refractive index is at the wavelength of excitation at which the window contributes as little optical deviation as possible. In that regard, if the window is thin, it may deviate more in refractive index from that of the immersion fluid, within limits that depend on the lens used. In other preferred embodiments, the refractive index is matched to the microscope immersion fluid so that the quality of imaging by the microscope objective is largely independent of the window thickness. The window may be constructed in any manner that ensures a smooth and thin surface of optical quality. This may be accomplished through careful design of injection molding, machining, or by affixing a thin film to the structural component, which can be done by any of several methods known to those skilled in the art, such as adhesives, thermal bonding, or ultrasonic welding.

[0021] In some embodiments, the second cavity boundary component is fabricated at least in part from a solid, flexible material such as silicone, fluorosilicone, vulcanized rubber such as ethylene propylene diene monomer (EPDM) rubber or related derivatives / blends (e.g., Santoprene), other fluoroelastomer polymers (e.g., FKM, Viton), or other thermoplastic elastomers that can deform in response to pressure, which may be styrene (i.e., TPS, such as styrene block copolymers like Styrolux), polyolefin-based (e.g., cyclic olefin copolymer TPE), or copolyester-based (TPC). The cavity boundary component can incorporate one or more regions designed as self-sealing injectable ports for the introduction and egress of tissue treatment fluids, and optionally, long-term tissue fluids, which may be fluids designed to solidify upon polymerization upon cooling, exposure to a secondary chemical, or exposure to light. The flexible material may be structural in itself or made more structural by being adhered to a more rigid material by any of the means known to those skilled in the art, such as adhesives, heat welding, or overmolding. In some embodiments, the flexible material component incorporates an external open cavity designed to allow for the installation of a (third) solid component, which can compress the flexible material against the walls of the first, internal cavity-forming component, adding strength to the pressed seal and improving the airtight qualities of the internal cavity.

[0022] In other embodiments, the second cavity-bounding component is fabricated from a solid plastic material with a specific set of properties: low manufacturing cost, sufficient flexibility to form an airtight seal with the first cavity-forming component, long-term resistance to various tissue processing reagents, including organic solvents such as acidified methanol and benzyl alcohol / benzyl benzoate combinations, and, optionally, direct printability (to produce indelible identification marks). The plastic should also not interact with fluorescent dyes involved in processing, such as Hoechst dyes, DAPI, eosin, rhodamines (e.g., rhodamine-6G, rhodamine 123, etc.), sulforhodamines, acridine orange, thiazole orange, TO-PRO, SYTOX® (e.g., SYTOX Green), AlexA® dyes (e.g., Alexa 647-NHS ester, Alexa 594-NHS ester, and chemical equivalents), and others. The plastic with this unique set of properties is polypropylene. A higher cost alternative is an acetal copolymer or homopolymer known alternatively by the trademark Delrin®. In some embodiments, this second component includes one or more ports made of a flexible, self-sealing material, such as any of the elastomeric materials listed above. In other embodiments, the ports self-close via any of a range of other self-closing mechanisms commonly known to those skilled in the art, such as spring-loaded ball valves or flexible port valves.

[0023] The tissue holder may also include one or more positioning members, preferably external to the tissue-containing chamber, such as posts, configured to fit into corresponding recesses in various processing devices, thereby positioning the tissue chamber relative to fluid inlets / outlets, imaging objectives, embedding removal tools, or other items. In various embodiments, the positioning members may be on the processing device, and the tissue chamber may include corresponding recesses for receiving the members.

[0024] In some embodiments, for ease of use, the tissue container may have overall external dimensions approximately those of a typical microscope slide, with an added thickness to accommodate coarsely cut samples, as opposed to thinly (10 μm or less) sliced ​​samples, i.e., approximately 75 mm × 25 mm × 5 mm. In some preferred embodiments, also for ease of use and with an aim to minimize manufacturing, material, and storage costs, the tissue container has overall external dimensions approximately those of a typical standard tissue cassette, typically approximately 40 mm × 30 mm × 6 mm, ranging from approximately 24-40 mm × 24-30 mm × 4-7 mm. The external dimensions of the container are also designed to accommodate internal cavity dimensions within which the majority of sample sizes commonly employed in standard histological analysis can be adequately fitted and processed, reducing modifications to common practice and facilitating the adoption of novel microscopy techniques.

[0025] Aspects of the present invention include a container for holding a tissue sample, the container comprising a cavity or trough for receiving the tissue sample and a wall having inner and outer surfaces adjacent to the cavity. In some embodiments, the chamber is loaded from the imaging side, and an optically transparent imaging cover or sheet is affixed after tissue loading in a manner that prevents the tissue from easily moving in any direction while leaving the chamber substantially sealed against liquid and air. In other embodiments, the tissue is loaded directly onto the imaging window portion of the first component, which forms the boundary (one surface and sidewall) of the cavity, while the second component is used to effectively enclose the sample within the sealed cavity, with the sidewalls of the first and second components forming an airtight seal. Alternatively, the tissue can be first loaded into the cavity on a surface facing the imaging surface and adjacent to the underside of the imaging surface. In either process, tissue is loaded onto the surface, optionally in a specific orientation, and then the members are brought together to form an internal closed cavity, completely enclosing the tissue and rendering it inaccessible to manual access within the chamber, and substantially sealing the chamber against liquid or air. In some embodiments, one component includes an optical window, which may have multiple features on its inner surface configured to contact the tissue sample and allow fluid flow between the tissue sample and the window. In some embodiments, the surface opposite the optical window may also be optically transparent or transmissive to a specific wavelength and used as an imaging window. The transmission wavelength may correspond to exactly half the wavelength (double the frequency) of the excitation laser wavelength, as would be generated by second harmonic generation (second harmonic generation).

[0026] In some embodiments, the elements defining the cavity are joined together in two separate steps. In the first step, the imaging window portion and the seal base component are joined together so that they form a seal with a specific cavity depth greater than the thickness of the sample being processed, e.g., 2 mm, 3 mm, 4 mm, or 5 mm. Tissue staining and dehydration then occur. The cassette may be oriented so that the tissue is held by gravity against the surface opposite the imaging surface during processing. This allows the staining fluid to reach the tissue surface that will be closest to the imaging window. Note that this may be accomplished in a manner such that the inlet port remains lower than the outlet port so that complete filling can occur without the formation of large air pockets. Note that the positioning of the outlet port refers to the path the fluid follows relative to the chamber, which may be in a different location than the outlet connection port. For example, the chamber may have an outlet channel extending from the main cavity to another portion of the cassette, with channel dimensions that allow capillary action to effectively draw the outlet fluid or gas from a specific portion of the chamber while positioning a convenient connector for easy insertion into a processing machine. The initial relative positioning of the two chamber-forming components can be reliably achieved by having features that provide some resistance to manual compression, such as by incorporating ridges, bumps, or overhangs that interfere with further compression of the two components but are surmountable with additional manual pressure. This second step, bringing the imaging surface and the base of the sealing component closer together, can occur either before or after the clearing step. Generally, the displacement of the dehydrating agent with the clearing fluid will proceed more rapidly if additional compression of the chamber occurs after the sample has been incubated with the clearing fluid for a period of time, given the greater surface area available for diffusional exchange. However, from a practical standpoint, it may be preferable to compress the sample at the moment it is filled with clearing fluid, so as to minimize the amount of time the sample needs to spend in a processing slot in the processor, allowing for improvements in the overall throughput of a given device.

[0027] As noted above, the optical window can have a refractive index approximately equal to the refractive index of a fluid (e.g., a clearing solution) in which the tissue sample is immersed prior to imaging. The refractive index of the clearing solution or other fluid to be used in processing the tissue sample can be approximately equal to the refractive index of the structure of the tissue sample to be analyzed. The optical window can have a refractive index of about 1.5 to about 1.7.

[0028] The chamber size accommodates tissue samples for microscopic analysis, but in preferred embodiments may be any size bounded by currently common sizes of tissue and existing tissue cassettes. Typical samples that are manually (coarsely) cut for processing may have thicknesses ranging from less than 0.5 mm to up to 4 mm thick, and other dimensions that can reach lengths of over 30 mm. A particular value feature for specific aspects of the invention described herein is that the internal cavity size and dimensions can be determined by varying only the second cavity-forming component. That is, to simplify the use and manufacture of components to accommodate differently sized tissues, the element with the windowed imaging surface can remain the same, and a second element may be configured to form a variable internal geometry while forming a seal with that first element, which can serve to achieve the following: 1. orient the elongated element in a restricted elongated orientation to improve imaging, 2. reduce the volume of expensive dye reagent for smaller samples by filling a portion of the chamber with a solid material, and 3. still allow the use of the same imaging window component for larger and / or thicker samples. Additionally, this second element can incorporate a self-sealing injectable port with a position that is constant with respect to various internal cavity geometries, so that a closed sample container can be utilized with a single type of processing instrumentation, including needles, and fluids to be injected and exchanged.

[0029] In various embodiments, the external planar dimensions of the chamber are those of a typical tissue cassette, i.e., generally about 40 mm x 30 mm x 6 mm. The imaging portion of the chamber may be substantially smaller in some embodiments. For example, the imaging portion of the chamber for needle core biopsies may be about 1.5 mm x 15 mm to about 3 mm x 40 mm, thereby fitting within a chamber with the external dimensions of a standard tissue cassette. In some embodiments, the planar dimensions may be larger to accommodate specific sample types having dimensions larger than the core biopsies mentioned above. For example, for imaging enucleated eye samples, the chamber may have dimensions of about 25 mm x 25 mm to about 30 mm x 40 mm. In other embodiments, the imaging chamber dimensions may be in the range of about 65 mm x 50 mm in planar dimensions, which may be large enough to accommodate large-format histological samples or samples typically referred to as "whole tissue specimens." Similarly, the imaging chamber height may be any height required to accommodate a specific sample type. For example, the height may be anywhere from about 200 μm to about 15 mm. In some embodiments, the imaging chamber may be about 200 μm to 500 μm high, which is optimal for cytology samples and very small biopsies. In other embodiments, the chamber height may be about 500 μm to 1.5 mm, which is typically optimal for small core biopsies. In other embodiments, the chamber height may be 1 to 2 mm, which is most suitable for samples that are manually thinly sectioned.

[0030] Incorporation of a sponge support, as discussed above, may require a taller chamber height to accommodate both the sponge and the sample. For example, a chamber incorporating a sponge support may have a height of about 1 mm to about 3 mm for small core biopsies. In other embodiments, the chamber height may be about 3 mm to about 6 mm, which may be optimal for regular tissue sections. In yet other embodiments, the chamber height may be about 6 mm to 15 mm, a dimension that may accommodate large-format histological specimens as well as typical medium- to large-sized unsectioned samples.

[0031] The external dimensions of the chambered container may vary depending on the dimensions of the enclosed tissue chamber and will be large enough to allow for any required fluid channels or external port plugs as described herein. In some embodiments, the external dimensions of the container may include a height of about 1 mm to 10 mm. In other embodiments, the container height may be about 10 mm to 20 mm.

[0032] The features may comprise a material having a refractive index approximately equal to the refractive index of a fluid (e.g., a clearing fluid) in which the tissue sample is immersed prior to imaging. In certain embodiments, the refractive index of the clearing fluid or other fluid to be used in processing the tissue sample may be approximately equal to the refractive index of the structure of the tissue sample to be analyzed.

[0033] The plurality of features can comprise a material having a refractive index of about 1.5 to about 1.7. The plurality of features can comprise a material having a refractive index of about 1.51 to about 1.57. The plurality of features can comprise a material that dissolves in the presence of an organic solvent. The organic solvent can be a clearing liquid such as benzyl alcohol and benzyl benzoate (BABB).

[0034] In various embodiments, the container may comprise a porous compressible material configured to contact the tissue sample on the side opposite the optically clear window. In some embodiments, the porous compressible material is a plastic sponge. The sponge cell size may be any size that allows for adequate tissue support with little compression, and may be anywhere between 10 μm and 5 mm. The sponge cell size may be within a range that facilitates wetting both the tissue and the optical surface without entrapment of air bubbles. In preferred embodiments, the sponge cell size is between 50 and 500 μm when dry. In other preferred embodiments, the sponge cell size is between 50 and 200 μm. The sponge may be open-cell or closed-cell. In preferred embodiments, the sponge is open-cell. In preferred embodiments, the sponge is substantially non-fluorescent. In some embodiments, the sponge is fabricated from a material having a refractive index of about 1.45 to about 1.7. In some embodiments, the sponge has a refractive index of about 1.50 to 1.57. The sponge material may be selected to approximately match the refractive index of the cleared tissue sample to be imaged.

[0035] The container may include one or more fluid ports that fluidly communicate with the cavity for receiving the tissue sample and the space outside the chamber. In certain embodiments, the chamber contains two ports. In other preferred embodiments, the chamber contains three ports. The use of separate ports—one for the infusion of dehydration / staining fluid, one for the clearing fluid (e.g., BABB), and a third for fluid egress / ejection—has the advantage of eliminating the risk of contamination of the dehydration / staining agent and the clearing fluid. This can be particularly important during processing, where we have found that small amounts of clearing fluid can interfere with proper staining of the sample. Therefore, preserving reagent purity through port isolation is a useful strategy. Two or three connection ports may be located on the same cassette surface to facilitate connection to a fluid exchange system or processor. In preferred embodiments, the fluid ports are self-sealing, such as with vulcanized or silicone plugs or surfaces that allow for needle introduction but seal upon needle removal. In some embodiments, the self-sealing port is a needleless connector (such as one that includes a self-closing valve that opens when a tubing connector is attached).

[0036] The container can comprise a material that is acid resistant and inert, resistant to organic solvents such as BABB, resistant to alcohol, and / or resistant to temperatures up to about 75 degrees Celsius. The sponge can comprise a material that is acid resistant, resistant to organic solvents such as BABB, resistant to alcohol, and / or resistant to temperatures up to about 75 degrees Celsius.

[0037] Aspects of the invention may include a method for analyzing a tissue sample, including orienting a tissue sample in a desired position within a tissue chamber, exposing the tissue sample to a first solution for chemical treatment at the desired position within the tissue chamber, exposing the tissue at the desired position within the tissue chamber to a fluid (e.g., a clearing solution) in which the tissue sample is immersed prior to imaging, imaging the tissue sample at the desired position within the tissue chamber, and optionally embedding the tissue sample at the desired position within the tissue chamber.

[0038] The clearing agent may be BABB. The first solution may comprise a dehydrating agent, a fixative, a dye, and / or some combination thereof (including when the fixative may be a dehydrating agent). In certain embodiments, the dye may be a fluorescent dye, and the imaging step may comprise fluorescent imaging. The desired location may be a desired location for sectioning the wax-embedded tissue sample and / or imaging the tissue sample. The tissue chamber may comprise a plurality of features disposed on an interior surface of the tissue chamber and configured to contact the tissue sample and allow fluid flow between the tissue sample and the interior surface. [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 shows a tissue chamber having a trough and a fluid inlet and outlet. [Figure 2] FIG. 2 shows a top view of the tissue chamber. [Figure 3] FIG. 3 shows a cutaway view of the tissue chamber with the positioning member. [Figure 4] FIG. 4 shows a tissue chamber with multiple features for spacing the sample from the chamber walls. [Figure 5] FIG. 5 illustrates a container 1001 containing a support sponge 1013, according to one embodiment. [Figure 6] FIG. 6 illustrates some of the internal components of the container 1001 shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description The present invention provides devices, systems, and methods for chemically processing (e.g., fixing, dehydrating, staining, and clearing), and optionally visual histological analysis of tissue during embedding, while reducing manual intervention, human contact, and labor costs during processing. The systems and methods allow for the initial placement of a tissue sample in a single container in a preferred orientation for embedding and / or imaging prior to embedding. The tissue sample can then be chemically processed (optionally fixed, dehydrated, stained, and / or cleared) and optionally embedded in paraffin or other media within the single container without subsequent direct manual repositioning of the sample. Furthermore, the tissue sample can be stained, cleared, and imaged intact to provide an initial pathological analysis that potentially eliminates the need for ongoing, expensive, and labor-intensive processing, embedding, sectioning, staining, and analysis. The systems and methods of the present invention allow for the positioning of the sample in a specific orientation for automated processing to reduce the likelihood of gas bubble interference.

[0041] FIG. 1 shows a tissue cassette 101 having a trough 107 for receiving and processing a tissue sample. The sample may be obtained, for example, during surgery, biopsy, fine needle aspiration, culture, or autopsy, and is preferably obtained for histological analysis. The tissue cassette 101 and / or the trough 107 therein may be provided in various sizes and may include (human- and / or machine-readable) markings 109 that correspond to the trough 107 or chamber 101 size and / or provide information regarding the subject (from which the sample is to be obtained), the type of sample, and / or the type of analysis to be performed. Once read by a machine or human, the markings 109 may be used to adjust tissue processing (e.g., reagent selection, reagent volumes, or processing equipment selection and configuration) and / or label imaging data. Additional information derived from laboratory or hospital information systems may be used to further adjust the details of the tissue processing protocol.

[0042] The tissue cassette 101 may include a remaining area surrounding the trough 107 to increase overall size and allow for easier manipulation. This remaining area may incorporate channels for directing fluids between specific locations relative to the chamber cavity and connection ports. A cutout 103 or opening in the cassette 101 can reduce the material required for production, production time, and their associated costs, as well as reduce the mass of the cassette 101. One or more fluid inlets / outlets 105 fluidly communicate with the chamber (created by the trough 107 and portions of the cassette) and the exterior surface of the cassette 101. The fluid inlets / outlets 107 interface with corresponding fluid inlets / outlets in various processing equipment to provide and remove processing fluids such as fixatives, dehydration fluids, stains / dyes, clearing solutions, or wax for embedding. Features such as rails, grooves, or recesses may be incorporated into the exterior side of the cassette to allow precise positioning relative to the viewing window.

[0043] The walls of the tissue chamber, or relevant portions thereof (e.g., imaging window), may be optically clear and / or index-matched to the clearing fluid and / or sample structure to be measured. Tissue cassette 101 is operable to contain the tissue sample for all processing steps for histological analysis, thereby enabling periodic imaging of the intact and wax-free sample, including, for example, fluorescent dye-based imaging techniques. After staining, fixation, dehydration, and / or any other processing steps have been performed, an embedding medium, such as paraffin, other wax, polymer, or the like, can be introduced into the chamber formed by trough 107 and cassette 101 via fluid inlet / outlet 105 to provide a wax-embedded sample within a block of wax ready for sectioning and subsequent analysis.

[0044] Thus, the tissue sample can be first oriented in the trough 107 or on the imaging portion of the cassette 101 in the desired position for both initial imaging and subsequent sectioning, and then left untouched throughout the remaining processing, imaging, and embedding steps.

[0045] The tissue chamber may be constructed from materials such as metal, plastic, cyclic olefin polymer, copolymer, styrene butadiene copolymer, or glass. Preferably, the chamber material does not react with either the tissue sample or the processing solutions that its surfaces contact. The chamber, in some embodiments, can be constructed from multiple materials. For example, the imaging window of the cassette may be constructed from a non-reactive, index-matched material, while the remainder of the cassette may be constructed from a different, less expensive material to reduce costs.

[0046] FIG. 3 shows a cutaway view of tissue chamber 301, with fluid inlet / outlet 305 shown providing fluid access from the exterior of chamber 301 to trough 307. The bottom surface of trough 307 can include positioning members 313, such as posts or tabs (or corresponding recesses for receiving such members). Positioning members 313 may correspond to complementary positioning recesses on the surfaces of various processing and imaging devices. While described herein with respect to members present on chamber 301 and corresponding recesses present on the device, it will be readily apparent that the reverse arrangement would also provide the same function. When positioned within their corresponding recesses, positioning members 313 may serve to position chamber 301 and trough 307 within the device relative to, for example, fluid coupling for fluid inlet / outlet 305, wax cutting blades, plungers for separating the floor of trough 307 along frangible area 311, imaging objectives, light sources, or various other processing tools.

[0047] The tissue chamber may be a reusable or single use item.

[0048] FIG. 4 shows a tissue chamber 401 with multiple features 403 for spacing the sample from the chamber wall 405. Spacing the sample away from the otherwise flat surface of the chamber wall 405 allows processing solutions, such as dehydration, fixation, clearing, and stain solutions, to access all sides of the sample. In the absence of such features 403, the sample would rest against the flat surface of the chamber wall 405, sealing it away from fluids, increasing processing time, reducing processing efficiency (and subsequent analytical quality), and / or requiring manipulation or agitation to reorient the sample and expose the blocked surface to fluids. The features may be any shape, including cones, pyramids, needles, cylinders, spheres, cubes, ridges, spikes, or other three-dimensional shapes. The features may also include porous structures or recesses within the material surface to allow fluid penetration or access. The features 403 should be shaped and spaced so that they still support the sample above the surface of the wall 405, while providing little surface area for contact with the sample and sufficient weight distribution so as not to puncture or otherwise penetrate the sample.

[0049] The features should have a height or depth sufficient to allow fluid flow between the supported sample and the surface of the chamber wall. In various embodiments, the features may have a height or depth of about 1 μm to about 5 mm. In preferred embodiments, the features have a height or depth of about 100 μm to about 500 μm.

[0050] An obvious drawback of such features 403 would be their detrimental effect on imaging quality. Thus, in various embodiments, the features may be constructed from a similar or the same material as the walls 405 of the chamber 401 and index-matched to the clearing liquid and / or the sample structure to be inspected. Since the imaging window is also fabricated from a material that is index-matched to the clearing liquid, the features may be etched or otherwise molded into the imaging window itself. The features 403 would thereby provide little distortion during imaging. In other embodiments, the features 403 may be constructed from a different material than the walls 405 of the chamber 401, and that material may be configured to dissolve in the presence of one or more of the processing solutions (e.g., the clearing liquid). The material ideally has a combination of features that will not impair imaging by dissolving in the clearing liquid, while making it most suitable for achieving the goal of proper fluid exposure during dehydration and staining. With this in mind, an ideal material would remain essentially unaffected over the dehydration incubation period (ranging from a few minutes to 12 hours or more), yet be sufficiently resistant to alcohols such as methanol and acidified alcohols such as alcohol with acetic acid, so as to dissolve rapidly (within seconds to minutes) upon exposure to organic solvents such as benzyl alcohol or benzyl benzoate, or a mixture of the two. An example of such a material is polyvinyl chloride. Another preferred example of such a material is methyl methacrylate butadiene styrene copolymer (MBS), such as that sold under the name Zylar 670. This latter material has the added advantage of having a refractive index closely matched to that of the clearing fluid known as BABB (benzyl alcohol / benzyl benzoate). This is because it does not significantly perturb the optical properties of the dissolving fluid upon dissolution, meaning that ideal imaging conditions can be maintained even if some residual material remains in the imaging path. The clearing liquid is generally applied before imaging, so that if the feature 403 dissolves in the presence of the clearing liquid, the feature 403 will not be present to distort subsequent imaging.The clearing liquid may comprise benzyl alcohol and benzyl benzoate (BABB), and thus feature 403 may comprise a material known to dissolve in BABB. In an ideal embodiment, the material that dissolves rapidly in BABB is also chemically resistant to acidified methanol. In a more preferred embodiment, the material that dissolves rapidly in BABB is resistant to acidified methanol and does not react with typical common protein and nuclear fluorescent dyes. As explained, the role of this material is to act as a dissolvable spacer between the tissue and the imaging window. This spacer can be configured in many shapes to achieve the spacing goal. One such configuration is as a sheet with multiple holes, gaps, or strips devoid of material. If the sheet incorporates strips of removed material, the strips effectively act as channels to allow fluid exchange for most of the sample surface (which would otherwise be isolated from fluid access due to its proximity to the final imaging window), while providing sufficient rigidity to the sheet to prevent complete deformation.

[0051] In certain embodiments, the bottom surface of the chamber may include features for receiving or positioning certain tissue sample shapes or types. For example, a V-shaped or semi-cylindrical notch may be formed in the bottom surface to receive and position an elongated sample, such as a core biopsy sample. Such an embodiment is depicted in FIG. 13 , where a core biopsy sample is placed within the notch on the bottom surface of the chamber. Such features can restrict the orientation of the tissue sample to a generally straight line while allowing fluid exchange on all sides, which is beneficial for high-speed imaging by reducing the number of imaging scans required to capture the entire sample.

[0052] The treatment device of the present invention may include a receptacle for the cassette, which orients the cassette in such a way that the inlet port is at or near the lowest position relative to the ground surface and the outlet port is at or near the highest position relative to the ground surface. Introducing fluid through the inlet port in this way ensures that air or gas will first escape through the outlet port, reducing the likelihood of bubbles being trapped adjacent to the tissue, which would have the effect of slowing fluid exchange or staining. As bubbles may form during the treatment incubation period, the treatment device may also incorporate a mechanism for periodically or continuously withdrawing and refilling fluid. Given the orientation and geometry of the fluid ports, this results in effective mixing of the fluid, increasing the concentration gradient at the tissue surface, which will improve diffusive exchange and aid in the dispersion and evacuation of gas bubbles.

[0053] FIG. 5 illustrates a container 1001 including a support sponge 1013 according to an embodiment. The container 1001 includes a sample chamber 1005 for receiving a tissue sample and two fluid ports 1007 for introducing and removing fluid from the sample chamber 1005. The container 1001 includes a cover 1003 that encloses the sample chamber 1005 after a tissue sample is placed therein. The fluid port 1007 may be sealable or self-sealing, particularly where the cover 1003 is operable to form a fluid and air tight seal with the top of the container 1001 to create a sealed environment within the sample chamber 1005. As noted above, the self-sealing fluid port 1007 may include a vulcanized or silicone plug or surface that allows the introduction of a needle but seals upon needle removal. In some embodiments, the self-sealing port is a needleless connector, such as one that includes a self-closing valve that opens when a tubing connector is attached. In other embodiments, the port is sealable, such as by heating or by the introduction of an external device such as a plug. The container 1001 may include a bottom cover 1015 with a sponge support 1013 or other support as discussed herein. The sponge support 1013 and / or bottom cover 1015 may form the bottom of the sample chamber 1005 and may comprise an optically transmissive, transparent, or index-matched material (e.g., having approximately the same index of refraction as the cleared tissue sample to be imaged), such as an optically transmissive window 1011 in the bottom cover 1015.

[0054] The sponge or other porous compressible material is configured to contact and hold the tissue sample in place after placement within the tissue chamber for chemical processing, clearing, and / or imaging. In some embodiments, the porous compressible material is a plastic sponge. The sponge cell size may be any size that allows for adequate tissue support with little or no compression, and may be anywhere between 10 μm and 5 mm. The sponge cell size may be within a range that facilitates wetting both the tissue and the optical surface without entrapment of air bubbles. In preferred embodiments, the sponge cell size is between 50 and 500 μm when dry. In other preferred embodiments, the sponge cell size is between 50 and 200 μm. The sponge may be open-cell or closed-cell. In preferred embodiments, the sponge is open-cell. In preferred embodiments, the sponge is substantially non-fluorescent. In some embodiments, the sponge is fabricated from a material having a refractive index of about 1.45 to about 1.7. In some embodiments, the sponge has a refractive index of about 1.50 to 1.57. The sponge material may be selected to approximately match the refractive index of the cleared tissue sample to be imaged. The sponge may comprise a material that is resistant to acids, organic solvents such as BABB, alcohols, and / or temperatures up to about 75 degrees Celsius.

[0055] 6 illustrates some of the internal configuration of the container 1001 shown in FIG. 10, including the internal fluid passages 1017 leading from the fluid ports 1007 to the sample chamber 1005. The fluid ports 1007 are optionally positioned at a planar level offset from the level of the sample chamber 1005 so that they can be oriented higher than the sample chamber 1005 during fluid exchange. Due to the lower density of air and other gases relative to the processing fluids, such an orientation aids in the removal of gas from the sample chamber 1005 during fluid exchange, ensuring optimal surface contact for dyes and processing chemicals, and preventing imaging distortion due to entrapped gas.

[0056] In certain embodiments, the chamber may be large enough that careful avoidance of air introduction and capillary action with the chamber walls may not be sufficient to prevent gas bubble formation. Thus, not only may inlet / outlet and / or fluid ports be positioned above the sample chamber to allow air purging, but in certain embodiments, fluid filling and exchange can be performed with the fluid ports elevated and the chamber itself oriented to allow gas to escape. For example, for a fluid chamber with a cross-sectional dimension perpendicular to the fluid flow direction that is at least 2.5 mm in at least one dimension, keeping the chamber flat on a surface (with the imaging surface parallel to the floor) during loading with an aqueous or alcohol-based solution may result in a high probability that not all of the gas will be able to escape from the chamber before the fluid reaches the outlet. Attractive forces between the fluid and the surface affect the specific dimensions at which this risk is high. Thus, the dimensions at which capillary forces prevent bubble formation during fluid loading in a horizontal configuration may depend on the details of the fluid and the surrounding solid material.

[0057] In some embodiments, therefore, the chamber may be oriented vertically during filling so that the outlet is at or near the highest point of the internal chamber, and in certain preferred embodiments, higher than the inlet port, allowing the gas to completely escape prior to the chamber being filled. The chamber can be filled in a vertical orientation. As fluid enters the chamber, gas is displaced and exits out an elevated outlet port at the top of the chamber. Approximately 4 mm 2For chamber cross-sectional areas of approximately 100 μL / s, using a low fill rate of less than about 200 μL / s, preferably less than 100 μL / s, can also significantly reduce the risk of bubble formation along the tissue or within the chamber. Larger cross-sectional areas can accommodate faster fill rates, assuming capillary action is less likely to play a significant role in gas entrapment. This may be particularly true for higher viscosity liquids, such as those used for clearing (benzyl alcohol / benzyl benzoate), but can also apply to low viscosity liquids, such as alcohol-based reagents. In various embodiments, the outlet height may be only slightly higher than the inlet height, such as those designed to allow gravity to hold the sample away from the imaging window during exposure to the staining chemical. A greater height difference further reduces the risk of bubble formation, but larger samples are more likely to require imaging surface access during staining and are located in larger cavities, which are less susceptible to bubble entrapment. Ideally, the outlet may be located at or near the highest point within the chamber when oriented for filling. Surface tension of the fluid can also help ensure that air is completely removed during filling. The location of the inlet port is not critical, as long as it is lower than the outlet; however, the higher it is, the greater the risk of air being trapped below the inlet level; therefore, a lower point for the inlet is preferred. In some embodiments, the chamber may be tilted away from a fully vertical position (perpendicular to the floor) during filling without significantly affecting the ability to completely remove air; this can help reduce the difference between the highest point of the chamber and the outlet port. From a practical standpoint, the angle of the chamber relative to the horizontal during fluid filling may be about 5 degrees to about 90 degrees. Optimally, for small-diameter chambers, it may be about 45 degrees to about 90 degrees, and for larger chambers with a lower risk of bubble entrapment, it may be about 15 degrees to 30 degrees. Such an orientation can also eliminate the need to completely remove gas bubbles from the inlet tubing. By using a slow injection, such bubbles, even if they enter the chamber, will quickly rise above the fluid level, thereby quickly exiting the chamber and maintaining a gas-free environment surrounding the tissue during processing and subsequent imaging.As discussed above, the inlet and outlet ports may be valved or otherwise sealable, which allows the chamber to be air-free or substantially air-free after the clearing step, meaning that the chamber can be reoriented to a horizontal position for imaging after the inlet and outlet connectors are removed.

[0058] In this vertical configuration, a technique we have determined to further reduce gas bubble persistence is to periodically or continuously withdraw fluid from the chamber and refill it. Fluid withdrawal does not necessarily have to be complete; fluid may remain within the chamber during fluid withdrawal. Fluid movement produces shear forces that help separate bubbles adjacent to the tissue or chamber wall. Due to their lower density, bubbles may rise against fluid movement. However, bubbles may also break away and be expelled from the chamber during a subsequent refill action. Thus, the geometry and orientation of the chamber can be coupled with fluid movement to minimize the effects of undesired bubbles.

[0059] In some embodiments, a two- or three-component chamber may be used. One component may be fabricated from index-matched plastic and incorporate an imaging window of a specified thickness. In some embodiments, an imaging window thickness of approximately 500 μm maximizes access to deep imaging while providing sufficient structural integrity, reducing the risk of breakage and allowing for injection-molded construction. In other embodiments, a thinner window, between 100 and 200 μm, is preferred because it can reduce the aberration effects of imperfect refractive index matching. In preferred embodiments, the window is 100 μm or less to minimize the aberration effects that any refractive index mismatch may have. Surrounding plastic around the imaging window can facilitate human manipulation while providing additional structural integrity against bending and other distortions. The first component with the imaging window may include a cavity, which is sized and shaped to receive the second component and form a sealed chamber. The second component may be constructed entirely from a silicone or similar flexible material, such as a thermoplastic elastomer or vulcanized vulcanizing compound, to facilitate sealing, or in certain embodiments, at least one of the first and second components may incorporate a flange, gasket, or other portion made from such a material to facilitate sealing.

[0060] In certain embodiments, flexible portions, such as parts made from or featuring silicone or rubber, may be thin enough to allow the part to flex. If such portions form a chamber wall or part of a chamber wall, flexibility therein can allow the chamber to accommodate variations in tissue thickness. In various embodiments, the flexible portion may be a wall facing the imaging window, applying resistance and compressing tissue against the imaging window. Such flexibility can also provide processing benefits by optionally expanding during fluid filling, exposing areas of tissue, such as the region against the imaging window of part 1 (which may otherwise be inaccessible to fluid), and / or being drawn inward in response to removal of excess fluid, compressing the sample against the window for imaging. Thus, the pressure differential between the fluid in the chamber and external pressure (e.g., ambient pressure) can be manipulated to achieve desired effects. Such negative pressure to cause sample compression may be achieved, for example, by the BABB fluid being drawn down the outlet tube by gravity (siphon effect), or by actively drawing the fluid, or by increasing the external pressure on the chamber.

[0061] In some embodiments, the flexible component incorporates an external cavity that serves as a receptacle for a third solid component designed to reinforce the cavity's airtight seal and potentially also as a medium for affixing identifying information, such as a label, or for directly printing such information. The sealing effect of this solid component is provided by generating a pressure radially against the flexible component that counteracts the pressure generated radially inward against the peripheral wall of the flexible component. The radial direction of compression by the imaging window-containing component and that of the external third solid component may be reversed, such that the flexible component forms the lower wall of the cavity by surrounding the cavity wall formed by the upper (imaging window) component, and the third solid component forms an outer ring around the flexible component, inducing the same counter-compression effect. Additionally, this third solid component may be designed with perforations that facilitate movement of the flexible side of the cavity wall but reduce pressure buildup between the outer wall of the flexible side of the cavity and this sealing component.

[0062] Despite silicone's reported incompatibility with benzyl alcohol and benzyl benzoate, the present system and method recognize that BABB's silicone absorption rate is slow and does not interfere with the material's use in the techniques described herein. Similar behavior applies to other thermoplastic elastomers (TPVs), such as those derived from vulcanizates. Furthermore, silicones and TPVs do not significantly lose structural integrity over a period of at least one year upon exposure to any of the chemicals used in the processing methods described herein. In some embodiments, fluorosilicones may be used to further reduce adverse reactions with chemicals used in processing. As mentioned, in other preferred embodiments, vulcanized rubber or ethylene propylene diene monomer (EPDM) rubber or compounds derived from blending EPDM and polypropylene may be used in place of silicone, providing a low-cost alternative with long-term resistance to organic solvents.

[0063] The methods of the present invention may involve chemical treatment, imaging, and embedding in a single chamber such that the tissue sample can be initially positioned within the chamber in a desired orientation for sectioning and / or imaging without further manipulation until optional removal of the embedded sample for sectioning.

[0064] Chemical processing may include fixation, dehydration, clearing, staining, and other steps known in the art and useful for both intact tissue imaging (e.g., fluorescent staining or fluorescent antibody staining and imaging) and histological analysis (e.g., wax embedding and microtome sectioning). In certain embodiments, a tissue sample may be exposed to one or more stains, fixatives, dehydrating agents, and / or clearing agents in a single tissue chamber as described herein. In some embodiments, fixation occurs prior to positioning the sample in the cassette chamber. In some cases, one or more of the above stains, fixatives, dehydrating agents, and / or clearing agents may be combined in a single solution. Suitable examples of chemical processing solutions and techniques are described in U.S. Publication Nos. 2016 / 0003716 and 20160003715, the contents of each of which are incorporated herein by reference.

[0065] Incorporation by Reference References and citations are made throughout this disclosure to other documents, such as patents, patent applications, patent publications, magazines, books, newspapers, web content, etc. All such documents are incorporated herein by reference in their entirety for all purposes.

[0066] equivalent Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the complete contents of this document, including the scientific and patent references cited herein. The subject matter of this specification contains important information, examples, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. A container for holding tissue samples, wherein the container is A first component made from a rigid material and having a cavity, the cavity having an imaging window on one of its surfaces, A second component, which is made of at least partially flexible material and is sized and molded to create an airtight, sealed chamber when inserted into the cavity of the first component, and A container equipped with [something].

2. The container according to claim 1, wherein the rigid material comprises optical-grade plastic.

3. The container according to claim 1, wherein the imaging window is refractive index matched to the refractive index of the fluid used when processing the tissue sample to be analyzed.

4. The container according to claim 1, wherein the imaging window is refractive index matched to the refractive index of the structure of the tissue sample to be analyzed.

5. The container according to claim 1, wherein the flexible material comprises silicone.

6. The container according to claim 1, wherein the flexible material comprises fluorosilicone.

7. The container according to claim 1, wherein the flexible material comprises ethylene propylene diene monomer rubber (EPDM).

8. The container according to claim 1, wherein the second component comprises a cavity for receiving a tissue sample.

9. The container according to claim 1, wherein one or more of the first and second components are provided with one or more fluid ports.

10. The container according to claim 1, wherein the second component comprises a deformable portion that is operable to compress a tissue sample in the airtight chamber relative to the imaging window in response to the application of a negative pressure difference to the airtight chamber.

11. A container for processing tissue samples, wherein the container is An airtight chamber for holding the tissue sample to be processed, A sealable inlet port providing fluid access to the airtight chamber, A sealable outlet port is provided which provides fluid access to the airtight chamber and when the container is positioned to be filled for tissue processing, the valved outlet port is positioned on the container at or near the highest point of the airtight chamber and higher than the valved inlet port A container equipped with [something].

12. The container according to claim 11, further comprising a surface, the surface comprising a plurality of feature portions, the plurality of feature portions being configured to contact a tissue sample and reduce surface area contact between the tissue sample and the surface by separating the tissue sample from the surface, thereby enabling fluid flow between the tissue sample and the surface, and the feature portions comprising a three-dimensional structure positioned on the surface.

13. The container according to claim 12, wherein the plurality of feature parts are made of a material having a refractive index approximately equal to the refractive index of the fluid to be used when processing the tissue sample.

14. The container according to claim 13, wherein the refractive index of the fluid to be used when processing the tissue sample is approximately equal to the refractive index of the structure of the tissue sample to be analyzed.

15. The container according to claim 14, wherein the plurality of feature parts are made of a material having a refractive index of about 1.5 to about 1.

7.

16. The container according to claim 15, wherein the plurality of feature parts are made of a material having a refractive index of about 1.53 to about 1.

60.

17. The container according to claim 12, wherein the plurality of feature parts are made of a material that dissolves in the presence of an organic solvent.

18. The container according to claim 17, wherein the organic solvent is a clearing solution.

19. The container according to claim 12, comprising a porous compressible material configured to contact the tissue sample on the side of the tissue sample facing the surface.

20. The container according to claim 19, wherein the porous compressible material has a refractive index approximately equal to the refractive index of the tissue sample to be analyzed.

21. The container according to claim 12, wherein at least a portion of the surface has a refractive index approximately equal to the refractive index of the fluid to be used when processing the tissue sample.

22. The container according to claim 21, wherein the refractive index of the fluid to be used when processing the tissue sample is approximately equal to the refractive index of the structure of the tissue sample to be analyzed.

23. The container according to claim 22, wherein at least a portion of the surface has a refractive index of about 1.5 to about 1.

7.

24. The container according to claim 12, wherein the surface defines, at least partially, a cavity for receiving the tissue sample.

25. A method for analyzing tissue samples, wherein the method is The method involves orienting a tissue sample within a tissue chamber to a desired position, wherein the tissue chamber is A valved inlet port providing fluid access to the tissue chamber, A valved outlet port that provides fluid access to the tissue chamber To be equipped with, Positioning the tissue chamber in a processing orientation such that the valved outlet port is higher than the valved inlet port and is located at or near the highest point of the tissue chamber with respect to the ground surface, The first solution is introduced through the valved inlet port, and air is allowed to escape the tissue chamber through the valved outlet port, thereby exposing the tissue sample to the first solution in the treatment orientation at the desired position within the tissue chamber for chemical treatment. By introducing the fluid through the valved inlet port, the chemically treated tissue sample is immersed in the fluid in the desired position within the tissue chamber in the treatment orientation. To image the immersed tissue sample at the desired position within the tissue chamber without repositioning the tissue sample after orientation. Methods that include...

26. The method according to claim 25, further comprising introducing an embedding fluid through the valved inlet port to embed the tissue sample in the processing orientation at the desired location within the tissue chamber.

27. The method according to claim 26, wherein the desired location is a desired location for sectioning the tissue sample to be embedded.

28. The method according to claim 25, wherein the fluid comprises a clearing agent.

29. The method according to claim 28, wherein the clarifying agent is BABB.

30. The method according to claim 25, wherein the first solution comprises a dehydrating agent.

31. The method according to claim 25, wherein the first solution comprises a fixative.

32. The method according to claim 31, wherein the fixing agent is a dehydrating agent.

33. The method according to claim 25, wherein the first solution comprises a dye.

34. The method according to claim 33, wherein the dye is a phosphor dye, and the imaging step includes phosphor imaging.

35. The method according to claim 25, wherein the desired position is a desired position for imaging the tissue sample.

36. The method according to claim 25, wherein the tissue chamber comprises a plurality of feature portions, the plurality of feature portions are arranged on the inner surface of the tissue chamber, and are configured to contact the tissue sample and enable fluid flow between the tissue sample and the inner surface.

37. A container for holding a tissue sample, wherein the container has a surface having a feature portion, and the feature portion is configured to receive and position the tissue sample at a desired location.

38. The container according to claim 37, wherein the feature portion comprises a V-shaped notch, and the tissue sample is a core biopsy sample.

39. The container according to claim 3, wherein the thickness of the window is approximately 100 to approximately 500 μm.

40. The container according to claim 4, wherein the imaging window is refractive index matched to approximately 1.515.

Images