Tissue chamber

Abstract

The invention relates to systems and methods for tissue treatment and analysis. The tissue chamber is configured to allow single container chemical processing, imaging, and wax embedding of a tissue sample in a single container without manipulating between steps. Tissue chambers having features for supporting the tissue sample and allowing fluid flow between the tissue sample and a surface of the tissue chamber are disclosed. These features may be refractive index matched to the sample structure of interest or may be dissolved in a transparentizing solution to allow for intra-chamber imaging with minimal distortion.

Description

Technical Field

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

[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 histological analysis are a century or more old and require trained medical professionals.

[0003] For standard histology methods, current procedures involve placing a tissue sample in a plastic cassette with perforated walls that allow fluid entry, and exposing the cassette to a fluid environment that changes composition over time to provide chemical fixation of the tissue.

[0004] The tissue is finally dehydrated and infiltrated with paraffin, and the embedded sample is then removed from the cassette by melting the paraffin. After paraffin infiltration and remelting, the sample is repositioned in molten paraffin and allowed to cool again to fix the sample in a certain orientation, which allows the sample to be cut into slices in the selected plane, thereby optimizing for clinical interpretation. The slices are then placed on slides, stained, fixed on coverslips, and then viewed directly by a pathologist or presented to an imager for digitization.

[0005] The above methods require manipulation of tissue during processing, resulting in labor costs and significant processing time. In addition, small samples, including small skin biopsies and biopsies from the gastrointestinal tract or long core biopsies (where orientation is critical for interpretive review of layers or complete cross-sections), can rotate and bend freely when the cartridge is submerged in fluid and exposed to agitation or flow. Therefore, these samples may require significant manipulation and processing time for optimal histological analysis.

[0006] Furthermore, the entire set of processing steps must be completed before a pathologist can begin substantive analysis of the sample, thereby incurring labor costs and delaying diagnosis.

[0007] When considering how to alleviate these technical and physical challenges of tissue preparation, improvements that allow tissue to be imaged directly without physical sectioning are possible and highly desirable. If a low-cost single tissue container / box can be used for both processing and imaging, the greatest advantage of this method for minimizing the time, labor, and cost of processing is achieved. However, there are several key considerations. One is that multiple fluids that must be exchanged in chronological order are involved during processing. Another is that imaging usually requires the sample to be placed flat on a sealed surface, which reduces the entry of chemicals during incubation. The third problem is the air or bubbles that may be present in the processing imaging box, which is particularly important for imaging samples in the imaging fluid. Minimizing bubbles during incubation is crucial, especially because some reagents (e.g., dyes) are expensive. Continuously adding reagents may avoid the problems caused by bubbles, but doing so is not advisable, and bubbles reduce the contact area for reagent diffusion, and bubbles can also cause severe distortion of imaging. In this way, filling the tissue chamber with these reagents requires special efforts to avoid or disperse bubbles. These and other practical considerations have brought considerable design challenges. Summary of the Invention

[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 benefits such as earlier access to diagnostic information and potentially avoiding the costly steps of manual cutting, staining, and slide distribution, where the need for such activities can be eliminated based on initial imaging results.

[0009] The single chamber solution provided herein allows the user to initially orient the sample in the chamber in the desired location for imaging and / or sectioning, and then perform all sample processing, including fixation and / or dehydration, initial staining, imaging, and optionally paraffin embedding, without having to touch or otherwise manually transfer the sample again, thereby reducing errors, lowering manufacturing costs, and reducing processing labor requirements. Therefore, the tissue chamber of the present invention allows the use of various processing devices described herein for automatic sample processing. Container aspects, such as specific geometric shapes, materials, and other configurations, allow for successful processing (e.g., dehydration, fixation, staining, transparency, and optionally embedding in a solid medium) of the sample without the need for direct manipulation of the sample. In certain embodiments, features on the container surface can facilitate fluid entry, exchange, and / or flow to all sides of the sample. Wherein there are sealable ports of needle and needleless types, such as "self-sealing" and "self-repairing" syringe injection ports, which allow fluid exposure, movement, and exchange while preventing possible bubble formation and entrapment that may affect imaging. A key aspect is that the resulting chamber is airtight in order to prevent rapid evaporation of highly volatile agents (e.g., alcohols) used as part of the process during incubation, as this would uncontrollably alter the dye concentration in the residual fluid and risk completely drying out the sample, leading to irreversible morphological changes. It is also of practical importance to design the chamber with a durable seal that enables long-term storage of samples in liquids, waxes (e.g., paraffin), or polymer embedding materials.

[0010] Initial attempts to address the bubble problem focused on ensuring that air was not introduced into the chamber during fluid filling. This approach works well in environments where the chamber size is small enough so that capillary action maintains fluid attachment to all cross-sectional chamber channel sides and there is no risk of air being trapped. In horizontally aligned tubular chambers, these conditions are related to the degree of intermolecular attraction of the fluid relative to gravity, as described by a dimensionless ratio known as the Bond number. The relevant parameters are the density of the fluid, the surface tension of the fluid (and tissue), and the size of the fluid chamber. For water in a glass tube, the fluid will remain attached with a characteristic size of approximately 2 mm. In addition, this increases the risk that, when air is in the chamber, the fluid will separate from the chamber wall and a portion may reach the outlet, after which the fluid continues to flow around the trapped bubbles and the bubbles become difficult to discharge. Crucially, it has been observed that incubating tissue with a methanol mixture at a temperature within the range of 40-50 degrees Celsius produces bubbles, which similarly affect fluid exchange in dehydration, staining, and organic solvent treatment steps, and brings additional challenges in reducing the harmful effects of bubbles.

[0011] Port location and chamber orientation are relevant to reducing the bubble effect. By positioning the outlet port higher than the inlet port during filling, and at or near the highest point within the chamber, gas can escape during the filling process. Any air / gas within the chamber will rise toward the elevated outlet port and be swept away as the fluid fills the chamber. One method that has been demonstrated to increase dye access to large imaging surfaces is to use index-matched or solvent-sensitive features between the imaging window and the sample to allow fluid exposure at the imaging surface during processing. These features can be transparent during imaging or dissolved prior to imaging, thereby substantially reducing or eliminating optical distortion during imaging. It has also been shown that including 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, helping to ensure that positioning is maintained, preventing artifacts of tissue compression, allowing fluid to flow around the non-imaging side of the sample, which can accelerate overall fluid exchange, and providing a surface for improved 'wetting.' When optically transmitted, it can also allow imaging through more than one surface, which is of particular value for using a multiphoton modality called second harmonic generation, which can be detected in transmission or reflection but is best detected in transmission.

[0012] Alternative approaches for fixing the positioning of small specimens during tissue processing, such that repositioning at the wax embedding step is unnecessary, have been explored, such as the techniques described in US 8796038 and US 20080227144, incorporated herein by reference. However, none of these existing methods allow for imaging directly after the clearing step—these methods are all designed for final removal by manual cutting and staining prior to visual interpretation or digital scanning. Additionally, these will generally not be suitable for orienting many common types of biopsies, including gastrointestinal or skin biopsy specimens, which are best imaged in very specific orientations and have irregular geometries.

[0013] The present invention provides significant advantages over the prior art by allowing samples to be placed in containers for histological analysis before full chemical processing (i.e., before or after exposure to formalin fixative), and allowing the sample to undergo chemical processing up to the clearing stage in a directional position in a single container device that can be used for all steps of dehydration, staining, clearing, and imaging. In addition, the tissue chamber of the present invention can then optionally be used for paraffin embedding or polymer embedding, and optionally for subsequent automatic or machine-assisted retrieval of the embedded sample using a retrieval device configured to work with the tissue chamber of the present invention.

[0014] As mentioned above, the tissue chamber can include features, and these features are operable to minimize the contact area between the base plate and / or wall of the chamber and the sample, thereby allowing good fluid paths in all areas of the sample to be provided to fixative, staining agent, dehydration solution, molten embedding material and / or other treatment fluids. The wall of the chamber and feature can be optically transparent and / or matched with the refractive index of the structure (for example, organelle, film or protein) of the clarification solution and / or the sample to be inspected. Feature can comprise the irregular surface that effectively serves as microchannel, wherein size is in the range of tens of microns to 500 microns. In certain embodiments, the surface irregularity forms a channel that is highly 100 microns. In other embodiments, the depth of channel is 200 microns. In other embodiments, the depth of channel is 50 microns. In still other embodiments, the surface feature is a projection of similar size. In some embodiments, the features may comprise materials that dissolve in the presence of certain solutions used in sample processing (e.g., clearing solutions) such that the features space the sample from the vessel walls and provide good fluid contact to all portions of the sample for processing, but dissolve prior to any initial imaging of the sample within the chamber and thus do not disrupt the imaging process. In preferred embodiments, the walls of the tissue chamber itself (or the imaging window portion therein) are substantially optically transparent and / or match the refractive index of the clearing solution and / or the structure of the sample to be examined. In some embodiments, the refractive index is substantially close to or approaches that of a common coverslip, typically made of glass having a refractive index of approximately 1.515 at specific visible wavelengths.

[0015] Single chamber processing as described herein can provide the other advantage that reagent is used minimized.The processing of the sample in the chamber allows the volume of the strict control chamber and the reagent used when processing.Replace the box containing tissue that is open to fluid environment (its volume can be hundreds of times the volume of tissue) as placed in the prior art, the volume of single sample processing vessel as described herein can be minimized with respect to the target size of tissue, thereby reducing the expenditure in terms of reagent.The optimal ratio of fluid to tissue can change according to the expectation compromise between the speed of processing and the depth of imaging.As a general rule, the reagent volume should be at least 11 times of the tissue volume, but the depth of imaging to hundreds of microns needs to be smaller than this ratio.Reagent preservation is particularly important for controlling dye cost, otherwise this dye cost may be too high, particularly for some fluorescent markers.Therefore, the reagent vessel of sample customization is particularly suitable for being incorporated into the processing of the dyeing of the sample without embedding, and especially fluorescent staining, and wherein the cost of dye can be main or significant cost composition.

[0016] Compared to existing methods, the single-chamber technology described herein also reduces processing time. In the case of current methods, it is more economical to wait until enough samples have been received and "roughly processed" (placed in a box) before loading the tissue processor. The single-chamber method can be combined with a dedicated tissue processor that is operable to receive the tissue chamber of the present disclosure by, for example, interfacing with the fluid inlet / outlet of the chamber. Once the chamber is loaded with a sample, the sample can be inserted and processed immediately; reducing the time that the sample must wait for another sample and is idle in multiplex processing. The same applies to the subsequent steps of embedding, including slide arrangement, slide staining, and slide organization and scanning for digitization. As described above, the single-chamber system described herein allows for varying geometries to more closely correspond to individual sample geometries, thereby reducing chamber fluid volume and reagent consumption. The chamber within the tissue cassette can be designed so that the sample matches the smallest size vessel that can accommodate the sample. For example, a long core biopsy can be placed in an elongated channel.

[0017] The vessel can be coded (for example, using 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 box before adding the sample, to allow the tissue processor to automatically identify the geometry of the tissue chamber being used and adjust the input volume accordingly, thereby further minimizing the reagent wasted. In various embodiments, the vessel itself can be color-coded to indicate the geometry, which can be identified by the processing machine to determine the sufficient fluid volume and incubation time for a specific sample, thereby significantly saving reagent costs, reducing waste and producing consistent processing results, while minimizing the average processing time. In certain embodiments, the identifier includes information that can help track manufacturing or user details (such as customer, 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 tool that can associate data with a source subject. In certain embodiments, when the institution expects to further identify the sample / box by visual means or any various known methods for machine-readable labels, the box is incorporated into the area that allows printing or pasting a second label. This would allow the user to read a local sample number or patient name, for example, and also meet the requirements for identification established by laboratory accreditation bodies.

[0018] Similarly, with chambers of set sizes specific to the sample, microscope scanning and imaging times can also be reduced. Although microscope slide scanners use various methods to minimize slide scanning times, they remain inefficient and rely on manual labor to varying degrees. Current systems typically acquire low-power images and use image processing algorithms to estimate the location and size of tissue, but are adversely affected by artifacts that are difficult to control, such as mounting variability, dirt, and smears. Therefore, human supervision may be required to ensure that tissue is not missed and large blank spaces are not imaged. Operator adjustment of the scan area is a time-consuming component of slide scanning, which can lead to repeated scans and long average slide scan times.

[0019] The coded sample-size-specific chambers described herein can help avoid manual intervention while maximizing the efficiency of image scanning. Because the tissue is placed in the smallest chamber it fits into, and the imager is able to read the chamber code to determine the size of the area to be scanned, the entire area of ​​possible tissue locations can be imaged without wasting excessive time imaging areas without tissue. The chance of error can be reduced, and no operator intervention is required, thereby providing efficient imaging and reducing labor costs and errors.

[0020] The tissue container of the present invention includes a trough area or cavity cutout sized to accommodate a sample and various processing fluids, wherein sample processing is performed. The trough is in fluid communication with one or more fluid inlets or outlets operable to provide processing fluids to the chamber containing the tissue. The tissue container may include additional material surrounding the trough to allow for easy manipulation and / or chamber orientation within various processing devices. In a preferred embodiment, the internal cavity is formed by two components. The first component is bounded on one side by an imaging window, which is optically matched in thickness and refractive index to the design requirements of the imaging microscope objective. In a preferred embodiment, this refractive index is between 1.5 and 1.7 and the thickness is less than 600 microns. In another preferred embodiment, this refractive index is between 1.5 and 1.6 and the thickness is 500 microns or less. In certain embodiments, the thickness is 150 microns or less. In some optimized versions, the thickness is 100 microns or less. In some embodiments, the refractive index of the window is equal to or similar to the refractive index of glass commonly used in cover glasses, or is approximately 1.515. In some embodiments, the window is made of cover glass. When discussing specific refractive index values, it should be understood that there are standard wavelengths for measurement, and these values ​​can vary depending on the wavelength used for measurement. In this context, the most directly relevant refractive index is the refractive index at the excitation wavelength, so that the window's contribution to light deviations is as small as possible. In this regard, if the window is thin, the refractive index can deviate more from that of the immersion fluid, within limits dependent on the lens used. In other preferred embodiments, the refractive index is matched to that of the microscope immersion fluid, so that the quality of image formation by the microscope objective is largely independent of the window thickness. The window can be constructed in any manner that ensures a smooth and thin surface of optical quality. This can be accomplished by injection molding, elaborate machining, or by securing a thin film to a structural component, which can be performed by any of several methods known to those skilled in the art, such as adhesives, heat bonding, or ultrasonic welding.

[0021] In some embodiments, the second cavity-defining component is at least partially made of 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, which can be styrenic (i.e., TPS, such as styrenic block copolymers such as Styrolux), polyolefin-based (e.g., cyclic olefin copolymer TPE), or copolyester-based (TPC), which can deform in response to pressure. The cavity-defining component can incorporate one or more areas designed as self-sealing injectable ports for introducing and allowing exit of tissue treatment fluids and optionally long-term tissue fluids, the latter of which can be fluids designed to solidify upon cooling, exposure to secondary chemicals, or polymerization based on light exposure. The flexible material can itself be structural, or made more structural by adhering to a harder material by any means known to those of ordinary skill in the art, such as adhesives, heat welding, or overmolding. In some embodiments, a flexible material component is incorporated into an external open cavity designed to accommodate a (third) solid component, which can press the flexible material against the walls of the first internal cavity forming component to, for example, increase the strength of the pressed seal, which enhances the airtight quality of the internal cavity.

[0022] In other embodiments, the second cavity-defining component is made of a solid plastic material having the following specific properties: low manufacturing cost, flexibility sufficient to form an airtight seal with the first cavity-forming component, long-term resistance to various tissue processing reagents including acidified methanol and organic solvents (such as a benzyl alcohol / benzyl benzoate combination), and optionally direct printability (for producing an indelible identification mark). The plastic should also not interact with fluorescent dyes involved in the processing, such as Hoechst dye, DAPI, eosin, rhodamine (e.g., rhodamine-6G, rhodamine 123, etc.), sulforhodamine, acridine orange, thiazole orange, TO-PRO, (e.g., SYTOX Green), dyes (e.g., Alexa 647-NHS ester, Alexa 594-NHS ester, and chemical equivalents), etc. The plastic with this unique set of properties is polypropylene. A more expensive alternative is the alternative trade name In some embodiments, the second component comprises one or more ports comprised of a flexible, self-sealing material, such as any of the elastomeric materials listed above. In other embodiments, the ports are self-closing by any of a variety of other self-closing mechanisms generally known to those skilled in the art, such as a spring-loaded ball valve or a flexible port valve.

[0023] The tissue container may also include one or more positioning members (e.g., posts) preferably located outside the chamber containing the tissue, the one or more positioning members being configured to fit within corresponding recesses in various processing devices to thereby position the tissue chamber relative to fluid inlets / outlets, imaging objectives, embedding retrieval tools, or other items. In various embodiments, the positioning members may be located on the processing device, and the tissue chamber may include corresponding recesses to receive the members.

[0024] In some embodiments, for ease of use, the overall outer dimensions of the tissue container can be approximately that of a common microscope slide, but with increased thickness to accommodate specimens that are roughly cut rather than thinly sliced ​​(10 μm or thinner), or approximately 75 mm × 25 mm × 5 mm. In some preferred embodiments, also for ease of use and to minimize manufacturing, material, and storage costs, the overall outer dimensions of the tissue container are approximately that of a common standard tissue cassette, typically approximately 40 mm × 30 mm × 6 mm, and ranging from approximately 24-40 mm × 24-30 mm × 4-7 mm. The outer dimensions of the container are also designed to accommodate internal cavity dimensions within which most sample sizes commonly employed in standard histological analyses can be adequately fitted and processed, thereby reducing changes to routine practice and facilitating the adoption of new microscopy techniques.

[0025] Aspects of the present invention include containers for accommodating tissue samples, wherein the container includes a cavity or groove for receiving the tissue sample and a wall with an inner surface and an outer surface adjacent to the cavity. In some embodiments, the chamber is loaded from the imaging side, and after the tissue is loaded, the optically transparent imaging cover or sheet is fixed in a manner that the tissue is not easily movable in any direction, while the chamber is substantially sealed to liquid and air. In other embodiments, the tissue is directly loaded onto the imaging window portion of the first assembly, which forms the boundary (a surface and sidewall) of the cavity, and the second assembly is used to effectively enclose the sample in the sealed cavity, wherein the sidewalls of the first assembly and the second assembly form an airtight seal. Alternatively, the tissue can first be loaded into a cavity on a surface opposite to the imaging surface and adjacent to the lower side of the imaging surface. In either process, the tissue is optionally loaded onto the surface in a configuration with a specific orientation, and then the components are put together to form an internal closed cavity, and the tissue is fully contained in the chamber and cannot be manually entered, and so that the chamber is substantially sealed to liquid or air. In some embodiments, a component includes an optical window, wherein the window includes a plurality of features on an inner surface that are configured to contact a tissue sample and permit fluid flow between the tissue sample and the window. In some embodiments, the surface opposite the optical window is also optically transparent or transmissive for a particular wavelength and can serve as an imaging window. The transmission wavelength can be a transmission wavelength corresponding to exactly half the wavelength (twice 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 put together in two separate steps. In the first step, the imaging window portion and the sealing base assembly are put together so that they form a seal, with a specific cavity depth greater than the thickness of the sample being processed (such as 2, 3, 4 or 5 mm). Tissue staining and dehydration are then performed. The box can be oriented during processing so that the tissue is held against the surface opposite to the imaging surface by gravity. This allows the dyeing fluid to reach the tissue surface that will be closest to the imaging window. Note that this can be done in such a way that the inlet port remains lower than the outlet port so that complete filling can occur without forming a large air pocket. It should be noted that the outlet port positioning refers to the path taken by the fluid relative to the chamber, and this can be a position different from the position of the outlet connection port. For example, the chamber can have an outlet channel extending from a large cavity to another part of the box, wherein the channel size is such that capillary action allows the outlet fluid or gas to be effectively extracted from the specific components of the chamber, while making the connector positioning convenient so as to be easily inserted into the processing machine. The initial relative positioning of the two chamber-forming assemblies can be reliably achieved by having features that provide some resistance to manual compression, such as by incorporating ridges, bumps, or protrusions that interfere with further compression of the two assemblies but can be overcome by additional manual pressure. This second step of bringing the imaging surface and the base of the sealing assembly closer together can be performed before or after the clearing step. Generally, replacing the dehydrating agent with the clearing fluid will proceed faster if additional compression of the chamber occurs after incubating the sample with the clearing fluid for a period of time, given the greater surface area available for diffusion exchange. However, from a practical point of view, it may be preferable to compress the sample as it is filled with the clearing fluid so as to minimize the amount of time the sample needs to spend in the processing tank within the processor, thereby allowing the overall throughput of a given device to be increased.

[0027] As described above, the refractive index of the optical window can be approximately equal to the refractive index of the fluid (e.g., 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 when 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 comprise a refractive index of about 1.5 to about 1.7.

[0028] The size of the chamber can be any size for accommodating tissue samples for microscopic analysis, but in a preferred embodiment, this size is defined by the currently familiar size of tissue and existing tissue cassettes. The thickness of the common sample of manual (rough) cutting to be processed can be in the thickness range of less than 0.5mm to 4mm, and can reach other sizes that length exceeds 30mm. For the particular aspects of the present invention described herein, the feature with special value is that the internal cavity size and size can be determined by only changing the second cavity forming assembly. That is, in order to simplify the use and manufacture of the assembly for accommodating tissues of different sizes, the element with the window imaging surface can remain the same, and the second element can be configured to form a seal against the first element, while forming a variable internal geometry that can help realize the following: 1. orienting the elongated element with limited elongated orientation to improve imaging, 2. reducing the volume of the expensive dye reagent for smaller samples by filling the parts of the chamber with solid material, and 3. still being able to use the same imaging window assembly for larger and / or thicker samples. Additionally, this second element may incorporate self-sealing injection ports whose positioning is constant for various internal cavity geometries, such that the closed sample container may be used with a single type of processing instrument containing the needle and the fluids to be injected and exchanged.

[0029] In various embodiments, the outer planar dimensions of the chamber are those of a common tissue cassette, typically approximately 40 mm × 30 mm × 6 mm. In some embodiments, the imaging portion of the chamber can be significantly smaller. For example, the imaging portion of a chamber for a core needle biopsy can be between approximately 1.5 mm × 15 mm and approximately 3 mm × 40 mm, thereby fitting within a chamber with the outer dimensions of a standard tissue cassette. In some embodiments, the planar dimensions can be larger to accommodate specific sample types that are larger than those described above for core biopsies. For example, for imaging ocular enucleation specimens, the chamber dimensions can be between approximately 25 mm × 25 mm and approximately 30 mm × 40 mm. In other embodiments, the imaging chamber dimensions can be large enough to accommodate what are commonly referred to as large-format histology specimens or "whole specimens," which have planar dimensions in the range of approximately 65 mm × 50 mm. Similarly, the imaging chamber height can be any height required to accommodate a specific sample type. For example, the height can be anywhere between approximately 200 μm and approximately 15 mm. In some embodiments, the imaging chamber height can be between about 200 μm and 500 μm, which is best suited for cytology samples and very small biopsies. In other embodiments, the chamber height can be between about 500 μm and 1.5 mm, which is generally best suited for small biopsies and core biopsies. In other embodiments, the chamber height can be between 1 mm and 2 mm, which is best suited for hand-thinned samples.

[0030] Incorporating the sponge support as discussed above may require a higher chamber height to accommodate both sponge and sample. For example, the height of the chamber incorporating the sponge support can be between approximately 1mm and approximately 3mm, for small biopsies and core biopsies. In other embodiments, the chamber height can be between approximately 3mm and approximately 6mm, which can be most suitable for conventional tissue sections. In still other embodiments, the chamber height can be between approximately 6mm and 15mm, which is the size that can accommodate large format histological specimens and general medium to large unsectioned samples.

[0031] The outer dimensions of the container containing the chamber can vary according to the size of the enclosed tissue chamber and will be large enough to allow any desired fluid passages or external port plugs as described herein. In some embodiments, the height of the outer dimensions of the container can be between about 1 mm and 10 mm. In other embodiments, the container height can be between about 10 mm and 20 mm.

[0032] The plurality of features may comprise a material having a refractive index approximately equal to the refractive index of the fluid (e.g., a clearing solution) in which the tissue sample is immersed prior to imaging. In certain embodiments, the refractive index of the clearing solution or other fluid to be used when 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 may comprise a material having a refractive index of about 1.5 to about 1.7. The plurality of features may comprise a material having a refractive index between about 1.51 and about 1.57. The plurality of features may comprise a material that dissolves in the presence of an organic solvent. The organic solvent may be a clearing solution 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 transparent window. In some embodiments, the porous, compressible material is a plastic sponge. The sponge cell size can be any size that can adequately support the tissue under minimal compression and can be any size in the range of 10 μm to 5 mm. The sponge cell size can be within a range that facilitates wetting both the tissue and the optical surface without creating bubbles. In a preferred embodiment, the sponge cell size is between 50 μm and 500 μm when dry. In other preferred embodiments, the sponge cell size is between 50 μm and 200 μm. The sponge can be open cell or closed cell. In a preferred embodiment, the sponge is open cell. In a preferred embodiment, the sponge is substantially non-fluorescent. In some embodiments, the sponge is made of a material with a refractive index between approximately 1.45 and approximately 1.7. In some embodiments, the sponge has a refractive index between approximately 1.50 and approximately 1.57. The sponge material can be selected to approximately match the refractive index of the cleared tissue sample to be imaged.

[0035] Container can comprise one or more fluid ports and the space outside chamber that are communicated with the cavity fluid for receiving tissue sample.In certain embodiments, chamber contains two ports.In other preferred embodiments, chamber contains three ports.The advantage of using the independent port for injecting dehydration / staining fluid and for clarification fluid (for example, BABB) plus the 3rd port for fluid to leave / discharge is to eliminate the risk of contaminating dehydration / staining agent with clarification fluid.This can be particularly important during processing, because it has been found that a small amount of clarification fluid interferes with the sufficient dyeing of sample.So, keeping reagent purity by port separation is a useful strategy.Two or three connection ports can be located on the same box surface, thereby promote to be connected to fluid exchange system or processor.In a preferred embodiment, fluid port is self-sealing, as with permission to introduce needle but can seal when needle is taken out rubberized or silicone plug or surface.In certain embodiments, self-sealing port is needleless connector, as included in the needleless connector of the self-closing valve that opens when tube connector is attached.

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

[0037] Aspects of the present invention may include a method for analyzing a tissue sample comprising the steps of: orienting the tissue sample in a desired position in a tissue chamber; exposing the tissue sample to a first solution for chemical treatment in the desired position in the tissue chamber; exposing the tissue in the desired position in 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 in the desired position in the tissue chamber; and optionally securely embedding the tissue sample in the desired position in the tissue chamber.

[0038] The transparentizing agent can be BABB. The first solution can include a dehydrating agent, a fixing agent, a dye and / or some combination thereof, including wherein the fixing agent can be a dehydrating agent. In certain embodiments, the dye can be a fluorescent dye, and the imaging step can include fluorescence imaging. The desired positioning can be a desired positioning for slicing a wax-embedded tissue sample and / or imaging the tissue sample. The tissue chamber can include a plurality of features that are placed on the inner surface of the tissue chamber and are configured to contact the tissue sample and allow fluid flow between the tissue sample and the inner surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A tissue chamber is shown with a trough and fluid inlet and outlet ports.

[0040] Figure 2 A top view of the tissue chamber is shown.

[0041] Figure 3 A cross-sectional view of a tissue chamber with a positioning member is shown.

[0042] Figure 4 A tissue chamber is shown having multiple features for isolating the sample from the chamber walls.

[0043] Figure 5 A container 1001 including a support sponge 1013 is shown according to some embodiments.

[0044] Figure 6 Shown Figure 5 Some internal configurations of the container 1001 are shown. DETAILED DESCRIPTION

[0045] The present invention provides chemical treatment (for example, fixing, dehydration, dyeing and transparency) and optionally embedding during tissue is carried out visual histological analysis equipment, system and method, reduce manual intervention, people's contact and labor cost during treatment simultaneously.System and method allow tissue sample to be initially placed in single container for embedding and / or before embedding, carry out imaging with preferred orientation.Then tissue sample can be chemically treated (optionally fixing, dehydration, dyeing and transparency) and optionally in single container, this tissue sample is embedded in paraffin or other medium, without the need to directly manually reposition sample subsequently.In addition, tissue sample dyeing, transparency and complete imaging can be to provide initial pathological analysis, thereby eliminate potentially the needs to lasting expensive and labor-intensive processing, embedding, sectioning, dyeing and analysis.System and method of the present invention allow sample to be positioned in specific orientation to carry out automated processing, to reduce the possibility of bubble interference.

[0046] Figure 1 Shown is a tissue cassette 101 with the groove 107 for taking in and processing tissue sample.Sample can for example obtain during surgical operation, biopsy, fine needle aspiration, cultivation or autopsy, and preferably obtains for histological analysis.Tissue cassette 101 and / or groove 107 therein can be provided with various sizes, and can comprise mark 109 (people and / or machine readable), this mark can correspond to the size of groove 107 or chamber 101 and / or provide the information about the type of experimenter, sample and / or the type of analysis to be performed therefrom obtaining sample.Once read by machine or people, mark 109 can be used for customized tissue processing (for example, reagent selection, reagent volume or treatment equipment selection and configuration) and / or for marking imaging data.The other information that obtains from laboratory or hospital information system can be used for the details of further customized tissue processing scheme.

[0047] Tissue cassette 101 can comprise the remainder area around groove 107, to increase overall size and allow easy manipulation.This remainder area can be incorporated into the passage for guiding fluid between the specific position and the connection port relative to the chamber cavity.The incision 103 in the box 101 or opening can reduce the quality of box 101, reduce the time of material required in the production, production and its associated cost simultaneously.One or more fluid inlets / outlets 105 are communicated with the outer surface fluid of chamber (produced by the part of groove 107 and box) and box 101.Fluid inlet / outlet 107 can be interfaced with the corresponding fluid inlet / outlet in various treatment equipment, to provide and remove treatment fluid, as fixative, dehydrating fluid, stain / dye, clarification solution or the wax for embedding.Features such as track, groove or recess can be incorporated into the outside aspect of box, so that it is possible to realize accurate positioning with respect to observation window.

[0048] The wall of the tissue chamber or its relevant part (for example, imaging window) can be optically transparent and / or with the refractive index matching of the clearing solution and / or the sample structure to be measured. Tissue cassette 101 is thus operable to hold tissue sample to carry out all processing steps for histological analysis, while allowing for example complete and periodic imaging without wax sample, including imaging techniques based on fluorescent dyes. After performing fixation, dyeing, dehydration and / or any other processing step, embedding medium such as paraffin, other waxes or polymers can be introduced into the chamber formed by groove 107 and cassette 101 via fluid inlet / outlet 105, so that the wax-embedded sample in the wax block can be provided, ready to be sectioned and subsequently analyzed.

[0049] Thus, a tissue sample can be initially oriented within well 107 or on the imaging portion of cassette 101 in a desired location for both initial imaging and later sectioning, and then held stationary for the remainder of the processing, imaging, and embedding steps.

[0050] The tissue chamber can be constructed from materials such as metal, plastic, cyclic olefin copolymer, styrene butadiene copolymer, or glass. Preferably, the chamber material is non-reactive with the tissue sample or any processing solutions that come into contact with its surface. In certain embodiments, the chamber can be constructed from a variety of materials. For example, the imaging window of the cartridge can be constructed from a non-reactive and refractive index-matched material, while the remainder of the cartridge can be constructed from a different, less expensive material to reduce costs.

[0051] Figure 3 A cross-sectional view of a tissue chamber 301 is shown, wherein a fluid inlet / outlet 305 is shown providing a fluid pathway from the outer surface of the chamber 301 to the groove 307. The bottom surface of the groove 307 may include a positioning member 313, such as a post or tab (or a corresponding recess for receiving such a member). The positioning member 313 may correspond to a complementary positioning recess on the surface of various processing and imaging devices. It will be apparent that although this document describes a member present on the chamber 301 and a corresponding recess present on the device, the reverse arrangement will also provide the same functionality. The positioning member 313, when positioned in its corresponding recess, can be used to position the chamber 301 and the groove 307 within the device relative to, for example: a fluid coupling for the fluid inlet / outlet 305, a wax cutting blade, a plunger for separating the bottom plate of the groove 307 along the fragile region 311, an imaging objective, a light source, or various other processing tools.

[0052] The tissue chamber may be a reusable or a disposable item.

[0053] Figure 4Shown is a tissue chamber 401 having a plurality of features 403 for spacing a sample from a chamber wall 405. Spacing the sample from other flat surfaces of the chamber wall 405 allows processing solutions (such as dehydration, fixation, clearing, and dye solutions) to enter all sides of the sample. In the absence of such features 403, the sample will be against the flat surface of the chamber wall 405, thereby isolating the sample from the fluid and increasing processing time, reducing processing efficiency (and subsequent analysis quality), and / or requiring manipulation or stirring to redirect the sample and expose the obstructed surface to the fluid. Features can be any shape, including cones, pyramids, needles, cylinders, spheres, cubes, ridges, spikes, or other 3D shapes. Features can include porous structures or recesses in the material surface that allow fluid to penetrate or enter. Features 403 are shaped and spaced so that these features provide a minimum contact surface area with the sample while still supporting the sample above the surface of the wall 405 and providing sufficient weight distribution so as not to pierce or otherwise penetrate the sample.

[0054] The height or depth of the features should be sufficient to allow fluid to flow between the supported sample and the surface of the chamber wall. In various embodiments, the height or depth of the features can be about 1 μm to about 5 mm. In preferred embodiments, the height or depth of the features is about 100 μm to about 500 μm.

[0055] The obvious disadvantage of this type of feature 403 is its deleterious effect on imaging quality. Therefore, in various embodiments, the feature can be constructed of a material similar or identical to the wall 405 of chamber 401, and matched with the refractive index of the clearing solution and / or the sample structure to be inspected. Since the imaging window is also made of a material that matches the refractive index of the clearing solution, the feature can be etched or otherwise molded into the imaging window itself. Feature 403 will thus provide minimal distortion during imaging. In other embodiments, feature 403 can be constructed of a material that is different from the wall 405 of chamber 401, and the material can be configured to dissolve when there is one or more treatment solutions in the treatment solution (e.g., clearing solution). The material ideally has a combination of features, which makes the material most suitable for achieving the goal of sufficient fluid exposure during dehydration and staining without damaging imaging due to being dissolved in the clearing solution. In view of this, the ideal material has sufficient resistance to alcohols such as methanol and acidified alcohols such as methanol plus acetic acid so as to remain essentially unaffected during the dehydration incubation period (ranging from several minutes to 12 hours or longer), but dissolves rapidly (several seconds to several minutes) when exposed to organic solvents such as benzyl alcohol or benzyl benzoate or mixtures 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 those sold under the name Zylar 670. This latter material has the additional advantage that its refractive index closely matches that of the clearing fluid known as BABB (benzyl alcohol / benzyl benzoate), because this means that once dissolved, the material does not significantly interfere with the optical properties of the dissolving fluid and is able to maintain ideal imaging conditions even when some residual material remains in the imaging path. Because the clearing solution is typically applied before imaging, if features 403 dissolve in the presence of the clearing solution, these features will not appear and distort subsequent imaging. The clearing solution can include benzyl alcohol and benzyl benzoate (BABB), and therefore, feature 403 can include materials known to dissolve in BABB. In an ideal embodiment, the material that dissolves quickly in BABB is also chemically resistant to acidified methanol. In another preferred embodiment, the material that dissolves quickly in BABB is resistant to acidified methanol and does not react with typical general proteins and nuclear fluorescent dyes. As described above, the role of this material is to serve as a soluble spacer between the tissue and the imaging window. This spacer can be configured to be in many shapes to achieve the spacing target. One such configuration is a sheet with multiple holes, gaps or strips without material. If the sheet is incorporated with strips of removed material, the strips effectively act as channels that allow fluid exchange to a large part of the sample surface, otherwise the sample will be isolated from the fluid path due to its proximity to the final imaging window, while providing enough rigidity to prevent complete deformation to the sheet.

[0056] In certain embodiments, the bottom surface of the chamber can include features for accommodating or positioning certain tissue sample shapes or types. For example, a V-shaped or semi-cylindrical recess can be formed in the bottom surface for accommodating and positioning long, narrow samples, such as core biopsy samples. Such an embodiment is depicted in FIG13 , in which the core biopsy sample rests in a recess on the bottom surface of the chamber. Such features can allow fluid exchange on all sides while constraining the orientation of the tissue sample to a fairly straight line, which is beneficial for rapid imaging by reducing the number of imaging scans required to capture the entire sample.

[0057] The treatment device of the present invention can include a housing for the box, which is oriented in such a way that the inlet port is located at or near the lowest positioning, and the outlet port is located at or near the highest positioning relative to the ground. In this way, introducing fluid via the inlet port ensures that air or gas can first escape through the outlet port, thereby reducing the possibility of bubbles being trapped near the tissue, which will have the effect of slowing down fluid exchange or dyeing. Since bubbles can be formed during the incubation period of treatment, the treatment device can also be incorporated into a mechanism for periodically or continuously extracting fluid and refilling fluid. Taking into account the orientation and geometry of a given fluid port, this will cause effective mixing of the fluid, thereby increasing the concentration gradient at the tissue surface, which improves diffusion exchange, and will also contribute to the dispersion and emptying of bubbles.

[0058] Figure 5A container 1001 including a support sponge 1013 is shown according to certain embodiments. 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 lid 1003 that closes the sample chamber 1005 after the tissue sample has been placed therein. The fluid ports 1007 can be sealable or self-sealing, particularly where the lid 1003 is operable to form a fluid and airtight seal with the top of the container 1001 to create a sealed environment within the sample chamber 1005. As described above, the self-sealing fluid port 1007 can include a rubberized or silicone plug or surface that allows the introduction of a needle but seals when the needle is removed. In some embodiments, the self-sealing port is a needleless connector, such as a needleless connector including a self-closing valve that opens when a tube connector is attached. In other embodiments, the port is sealable, such as by heating or by introducing an external device such as a plug. The container 1001 can include a bottom cover 1015 having a sponge support 1013 or other supports as discussed herein. The sponge support 1013 and / or the bottom cover 1015 can form the bottom of the sample chamber 1005 and can contain an optically transmissive, transparent, or index-matched material (e.g., having a refractive index approximately the same as that of the cleared tissue sample to be imaged), such as an optically transmissive window 1011 in the bottom cover 1015.

[0059] A sponge or other porous, compressible material is configured to contact a tissue sample and hold it in place after it is positioned within a tissue chamber for chemical processing, clearing, and / or imaging. In some embodiments, the porous, compressible material is a plastic sponge. The sponge cell size can be any size that adequately supports the tissue under minimal compression and can be anywhere in the range of 10 μm to 5 mm. The sponge cell size can be within a range that facilitates wetting both the tissue and the optical surface without creating bubbles. In a preferred embodiment, the sponge cell size is between 50 μm and 500 μm when dry. In other preferred embodiments, the sponge cell size is between 50 μm and 200 μm. The sponge can be open cell or closed cell. In a preferred embodiment, the sponge is open cell. In a preferred embodiment, the sponge is substantially non-fluorescent. In some embodiments, the sponge is made of a material with a refractive index between approximately 1.45 and approximately 1.7. In some embodiments, the sponge has a refractive index between approximately 1.50 and approximately 1.57. The sponge material can be selected to approximately match the refractive index of the cleared tissue sample to be imaged. The sponge may comprise an acid resistant material, an organic solvent resistant material, such as BABB, an alcohol resistant material, and / or a temperature resistant material up to about 75 degrees Celsius.

[0060] Figure 610 shows some internal configurations of the container 1001, including internal fluid channels 1017 from the fluid ports 1007 to the sample chamber 1005. The fluid ports 1007 are optionally positioned horizontally in a plane that is offset relative to the level of the sample chamber 1005, so that the fluid ports can be oriented higher than the sample chamber 1005 during fluid exchange. Because the density of air and other gases is lower relative to the density of the processing fluid, this orientation facilitates the removal of gases from the sample chamber 1005 during fluid exchange, thereby ensuring optimal surface contact of the dye and processing chemicals and preventing imaging distortion due to trapped gases.

[0061] In certain embodiments, the chamber can be large enough to carefully avoid the introduction of air and the capillary action with the chamber wall may not be enough to prevent bubble formation. Therefore, not only can the inlet / outlet and / or fluid port be positioned on the sample chamber to allow air purging, but also in certain embodiments, the chamber itself can be oriented so that the fluid port is high to allow gas to escape when performing fluid filling and exchange. For example, for a fluid chamber having a cross-sectional dimension of at least 2.5 mm in at least one dimension perpendicular to the direction of fluid flow, keeping the chamber flat on the surface (wherein the imaging surface is parallel to the base plate) during the loading of an aqueous or alcohol-based solution is likely to cause the gas to not completely escape the chamber before the fluid reaches the outlet. The attraction between the fluid and the surface affects the specific size at which this risk is higher. Therefore, the size at which capillary force prevents bubble formation during the loading of the fluid in a horizontal configuration can depend on the properties of the fluid and the surrounding solid material.

[0062] Thus, in some embodiments, the chamber can be oriented vertically during filling so that the outlet is located at or near the highest point of the interior chamber when filling, and in some preferred embodiments, above the inlet port, thereby allowing the gas to escape completely before the chamber is filled. The chamber can be filled in a vertical orientation. As fluid enters the chamber, the gas is displaced and exhausted from the elevated outlet port at the top of the chamber. For approximately 4 mm 2In some embodiments, the cross-sectional area of ​​the chamber is less than about 200uL / s and preferably less than 100uL / s. The risk of forming bubbles beside or in the chamber can be significantly reduced by using a slow filling speed that is less than about 200uL / s and preferably less than 100uL / s. Larger cross-sectional area can adapt to faster filling speeds because the possibility that capillary action plays an important role when trapping gas is lower. This is especially true for higher viscosity liquids, such as the liquid (benzyl alcohol / benzyl benzoate) used for transparentization, but can also be applied to low viscosity liquids such as alcohol-based reagents. In various embodiments, the outlet height can be only slightly higher than the inlet height, such as being designed to allow gravity to hold the sample away from the height of the imaging window portion during exposure to dyeing chemicals. Larger height difference further reduces the risk of bubble formation, but larger samples are more likely to need imaging surface access during dyeing and are located in a larger cavity that is not easy to be trapped by bubbles. Ideally, when directed to be used for filling, the outlet can be positioned at or near the highest point in the chamber. The surface tension of the fluid can still help ensure that air is fully discharged during filling. The positioning of the inlet port is not too important, as long as it is lower than the outlet, but the higher the inlet port, the greater the risk of air being trapped below the inlet level, so the lowest point of the inlet is preferred. In some embodiments, the chamber can be tilted away from a completely vertical positioning (perpendicular to the base) without significantly affecting the ability to completely exhaust air during filling, and can help reduce the difference between the highest point of the chamber and the outlet port. In practice, the angle of the chamber relative to the horizontal surface during fluid filling can be between about 5 degrees and about 90 degrees. Optimally, for small diameter chambers, the angle can be between about 45 degrees and about 90 degrees, and for large chambers with a lower risk of bubble entrapment, the angle can be between about 15 degrees and 30 degrees. Such an orientation can also eliminate the need to completely eliminate bubbles from the inlet tubing. In the case of slow injection, even if such bubbles enter the chamber, they will rise faster than the liquid level and thus quickly leave the chamber, thereby maintaining a gas-free environment around the tissue during processing and subsequent imaging. As discussed above, the inlet and outlet ports can 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 into a horizontal position for imaging after removal of the inlet and outlet connectors.

[0063] In this vertical configuration, a technique that has been determined to further reduce the persistence of bubbles is to periodically or continuously withdraw fluid from the chamber and refill it. The fluid does not necessarily need to be completely withdrawn—it can remain in the chamber during withdrawal. The fluid movement creates shear forces that help separate bubbles from adjacent tissue or chamber walls. Due to their lower density, bubbles can rise, which is opposite to the movement of the fluid. Moreover, these bubbles can be removed and expelled from the chamber during a subsequent refilling action. Therefore, the geometry and orientation of the chamber can be combined with the fluid movement to minimize the undesirable effects of bubbles.

[0064] In some embodiments, a two-part or three-part chamber may be used. One part may be made of index-matched plastic and incorporate an imaging window of a specified thickness. In some embodiments, an image window thickness of approximately 500 μm may maximize the opportunity to obtain deep imaging while providing sufficient structural integrity to reduce the risk of breakage while also allowing injection molded construction. In other embodiments, a thinner window between 100 μm and 200 μm is preferred as this can reduce the aberration effects of incomplete refractive index matching. In a preferred embodiment, the window is 100 μm or less, thereby minimizing the aberration effects that any refractive index mismatch may have. The surrounding plastic surrounding the imaging window can provide additional structural integrity to resist bending and other deformation while facilitating human manipulation. The first part with the imaging window may include a cavity that is sized and shaped to receive the second part to form a sealed chamber. The second component can be constructed entirely of silicone or a similar flexible material (such as a thermoplastic elastomer or a vulcanized rubber compound) to facilitate sealing, or in some embodiments, at least one of the first component and the second component can incorporate a flange, gasket or other portion made of such a material to facilitate sealing.

[0065] In certain embodiments, the flexible portion (e.g., a component made of or featuring silicone or rubber) can be thin enough to allow the component to bend. If such a portion forms a wall or part of a chamber, the flexibility therein can allow the chamber to adapt to variations in tissue thickness. In various embodiments, the flexible portion can be a wall opposite the imaging window and can apply resistance to press the tissue against the imaging window. Such flexibility can also provide processing advantages by optionally expanding during fluid filling to expose tissue areas that the fluid might otherwise not be able to enter, such as areas of the imaging window against component 1, and / or being pulled inward to press the sample against the window for imaging when excess fluid is removed. Thus, the pressure difference between the fluid in the chamber and the external pressure (e.g., ambient pressure) can be manipulated to achieve a desired effect. Such negative pressure that causes sample compression can be achieved, for example, by pulling the BABB fluid down the outlet tube by gravity (siphon effect), by actively withdrawing the fluid, or by increasing the external pressure on the chamber.

[0066] In certain embodiments, the flexible component is incorporated into an external cavity that serves as a receptacle for a third solid component designed to enhance the airtight seal of the cavity and may also serve as a medium for attaching identification information such as a label or for directly printing such information. The sealing effect of this solid component is provided by a pressure generated radially against the flexible component, which is opposite to the pressure generated radially inward against the peripheral wall of the flexible component. The radial direction of compression performed by the component containing the imaging window and the radial direction of compression performed by the external third solid component may be opposite, so 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, thereby inducing the same counter-compression effect. In addition, this third solid component may be designed with perforations that promote movement of the flexible aspect of the cavity wall but reduce the accumulation of pressure between the flexible outer wall of the cavity and this sealing component.

[0067] Although silicone is reported to be incompatible with benzyl alcohol and benzyl benzoate, the systems and methods of the present invention recognize that silicone absorption of BABB is slow and does not hinder the use of the material in the technology described herein. Similar behavior applies to other thermoplastic elastomers, such as thermoplastic elastomers derived from vulcanized rubber (TPV). In addition, silicone and TPV do not significantly lose structural integrity over a period of at least 1 year when exposed to any chemicals used in the treatment methods described herein. In certain embodiments, fluorosilicone can be used to further reduce adverse reactions with the chemicals used in the treatment. As mentioned above, in other preferred embodiments, vulcanized rubber or ethylene propylene diene monomer (EPDM) rubber or compounds derived from mixed EPDM and polypropylene can be used instead of silicone, thereby providing a low-cost alternative with lasting resistance to organic solvents.

[0068] The methods of the present invention can include single chamber chemical processing, imaging, and embedding, such that a tissue sample can be initially positioned within the chamber in a desired orientation for sectioning and / or imaging, and no further manipulation is required until the embedded sample is optionally removed for sectioning.

[0069] Chemical treatment can include fixing, dehydrating, transparentizing, dyeing and other steps known in the art and can be used for complete tissue imaging (e.g., fluorescent staining or fluorescent antibody staining and imaging) and histological analysis (e.g., embedding and microtome sectioning). In certain embodiments, tissue sample can be exposed to one or more staining agents, fixatives, dehydrating agents and / or transparentizing agents in a single tissue chamber as described herein. In certain embodiments, fixation occurs before the sample is positioned in the box chamber. In some cases, one or more of the above-mentioned staining agents, fixatives, dehydrating agents and / or transparentizing agents can be combined in a single solution. Suitable examples of chemical treatment solutions and technology are described in U.S. Publication No. 2016 / 0003716; and U.S. Publication No. 20160003715, the contents of each U.S. disclosure in these U.S. disclosures are incorporated herein by reference.

[0070] Incorporated by Reference

[0071] Throughout this disclosure, other documents, such as patents, patent applications, patent publications, journals, books, articles, web page content, have been referenced and cited. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0072] Equivalent

[0073] Various modifications of the invention, in addition to those shown and described herein, as well as many additional embodiments thereof, will become apparent to those skilled in the art from the entire contents of this document, including references to scientific and patent literature cited herein. The subject matter herein contains important information, illustrations, and guidance that can be adapted to practice the invention in its various embodiments and its equivalents.

Claims

1. A container for containing a tissue sample, the container comprising: a first component made of a rigid material and comprising a cavity having an imaging window on one surface thereof; and A second component is at least partially made of a flexible material and is sized and shaped to create a hermetically sealed chamber when inserted into the cavity of the first component.

2. The container of claim 1, wherein the rigid material comprises optical grade plastic.

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

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

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

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

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

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

9. The container of claim 1, wherein one or more of the first component and the second component comprises one or more fluid ports.

10. The container of claim 1, wherein the second component comprises a deformable portion operable to press a tissue sample within the hermetically sealed chamber against the imaging window upon application of a negative pressure differential to the hermetically sealed chamber.

11. A container for processing a tissue sample, the container comprising: an airtight chamber for containing a tissue sample to be processed; a sealable inlet port providing fluid access to the airtight chamber; as well as A sealable outlet port provides fluid access to the airtight chamber and is located on the container such that when the container is positioned to be filled for tissue processing, the valved outlet port is at or near the highest point of the airtight chamber and above the valved inlet port.

12. The container of claim 10, further comprising a surface comprising a plurality of features configured to contact a tissue sample, reduce surface area contact between the tissue sample and the surface by spacing the tissue sample from the surface, and permit fluid flow between the tissue sample and the surface, the features comprising three-dimensional structures positioned on the surface.

13. The container of claim 11, wherein the plurality of features comprise a material having a refractive index approximately equal to a refractive index of a fluid to be used in processing the tissue sample.

14. The container of claim 12, wherein the refractive index of the fluid to be used in processing the tissue sample is approximately equal to the refractive index of the structure of the tissue sample to be analyzed.

15. The container of claim 13, wherein the plurality of features comprise a material having a refractive index of about 1.5 to about 1.

7.

16. The container of claim 14, wherein the plurality of features comprise a material having a refractive index between about 1.53 and about 1.

60.

17. The container of claim 11, wherein the plurality of features comprise a material that dissolves in the presence of an organic solvent.

18. The container of claim 16, wherein the organic solvent is a clearing solution.

19. The container of claim 11, comprising a porous compressible material configured to contact the tissue sample on a side of the tissue sample opposite the surface.

20. The container of claim 18, 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 of claim 11, wherein the refractive index of at least a portion of the surface is approximately equal to the refractive index of a fluid to be used in processing the tissue sample.

22. The container of claim 20, wherein the refractive index of the fluid to be used in processing the tissue sample is approximately equal to the refractive index of the structure of the tissue sample to be analyzed.

23. The container of claim 20, wherein the refractive index of the at least a portion of the surface is from about 1.5 to about 1.

7.

24. The container of claim 11, wherein the surface at least partially defines a cavity for receiving the tissue sample.

25. A method for analyzing a tissue sample, the method comprising: orienting a tissue sample in a desired position in a tissue chamber, the tissue chamber comprising: a valved inlet port providing fluid access to the tissue chamber; and a valved outlet port providing fluid access to the tissue chamber; positioning the tissue chamber in a treatment orientation such that the valved outlet port is positioned higher than the valved inlet port and at or near the highest point of the tissue chamber relative to the ground; exposing the tissue sample to the first solution for chemical treatment in the desired location within the tissue chamber in the processing orientation by introducing the first solution through the valved inlet port and allowing air to escape from the tissue chamber through the valved outlet port; immersing a chemically treated tissue sample in the desired position in the tissue chamber in the treatment orientation by introducing the fluid through the valved inlet port; as well as The immersed tissue sample is imaged in the desired position in the tissue chamber without repositioning the tissue sample after orienting.

26. The method of claim 29, further comprising embedding the tissue sample in the desired position in the tissue chamber in the processing orientation by introducing an embedding fluid through the valved inlet port.

27. The method of claim 30, wherein the desired position is a desired position for sectioning an embedded tissue sample.

28. The method of claim 29, wherein the fluid comprises a clearing agent.

29. The method of claim 32, wherein the clearing agent is BABB.

30. The method of claim 29, wherein the first solution comprises a dehydrating agent.

31. The method of claim 29, wherein the first solution comprises a fixative.

32. The method of claim 35, wherein the fixative is a dehydrating agent.

33. The method of claim 29, wherein the first solution comprises a dye.

34. The method of claim 37, wherein the dye is a fluorescent dye and the imaging step comprises fluorescence imaging.

35. The method of claim 29, wherein the desired position is a desired position for imaging the tissue sample.

36. The method of claim 29, wherein the tissue chamber comprises a plurality of features disposed on an interior surface of the tissue chamber and configured to contact the tissue sample and permit fluid flow between the tissue sample and the interior surface.

37. A container for holding a tissue sample, the container comprising a surface comprising features configured to receive a tissue sample and position the tissue sample in a desired position.

38. The container of claim 41, wherein the feature comprises a v-shaped notch and the tissue sample is a core biopsy sample.

39. The container of claim 3, wherein the window has a thickness between about 100 μm and about 500 μm.

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

Images