Tissue chamber

Abstract

To provide systems and methods for tissue processing and analysis.SOLUTION: The present invention relates to systems and methods for tissue processing and analysis. Tissue chambers are configured to allow single-container chemical processing, imaging, and wax embedding of tissue samples in a single container without manipulation between steps. Tissue chambers with features to support the tissue sample and allow fluid flow between the tissue sample and the tissue chamber surface are disclosed. The features may be index matched to sample structures of interest or dissolvable in clearing solution to allow for in-chamber imaging with minimal distortion. Specialized tissue processing and wax removal apparatuses are also disclosed including for use with tissue chambers having frangible portions to permit ease of wax removal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Nonprovisional Application No. 16 / 164,343, filed October 18, 2018, which claims priority to and the benefit of Provisional Application No. 62 / 674,911, filed May 22, 2018, the contents of each of which are incorporated by reference.

[0002] FIELD OF THE INVENTION The present disclosure generally relates to histological systems and methods for simplified positioning, chemical processing and / or imaging of tissue samples, including imaging, staining, fixation, wax embedding, and wax removal in a single chamber. [Background technology]

[0003] (background) 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 over 100 years old, and histological analysis is primarily performed by trained medical professionals.

[0004] For standard histological methods, such as creating digital histological images, 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 whose composition changes over time to provide chemical fixation of the tissue. The tissue is finally dehydrated and infiltrated with wax before removing the wax-embedded sample from the cassette by melting the wax. At each processing step, the tissue sample generally rests on the floor and / or walls of the plastic cassette, providing poor fluid access to those portions of the sample without agitation.

[0005] After wax infiltration and removal, the sample is repositioned in molten wax and allowed to cool again, immobilizing the sample in an orientation that allows for sectioning slices of the sample in selected planes optimized for clinical interpretation. The slices are then mounted on slides, stained, and then either viewed directly by a pathologist or presented to an imager for digitization.

[0006] The above approaches require manipulation of the tissue during processing, resulting in labor costs and significant processing time. Furthermore, small specimens, including small skin biopsies and biopsies from the gastrointestinal tract where orientation is critical, can freely rotate and flex while the cassette is submerged in fluid and exposed to agitation or flow. These specimens can therefore require significant manipulation and processing time for histological analysis.

[0007] Additionally, 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. Summary of the Invention [Means for solving the problem]

[0008] (Abstract) The present invention provides a single-chamber solution for sample processing (e.g., dehydration, fixation, staining, and wax embedding), thereby reducing the cost, labor, and time required for histological analysis. Additionally, 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 the potential to avoid the costly steps of manual cutting, staining, and dispensing slides, 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 first orient the sample within the chamber in the desired position for imaging and / or sectioning, and then perform all sample processing, including fixation and / or dehydration, initial staining, imaging, and final wax embedding, without the need to touch or otherwise manipulate the sample again. Thus, the tissue chamber of the present invention enables automated sample processing using the various processing devices described herein. Container sides, such as features on the container surface to allow fluid access, exchange, and / or flow to all sides of the sample, allow the sample to be successfully processed (e.g., dehydrated, fixed, stained, cleared, and wax-embedded) without the need for sample agitation or manipulation. Sealable ports, such as "self-sealing" syringe injection ports, allow fluid exposure, movement, and exchange while preventing potential bubble formation and entrapment that could affect imaging. Refractive index-matching or solvent-sensitive features allow initial sample imaging through features without significant distortion. Additionally, the inclusion of a substantially non-fluorescent and non-reflective sponge for mounting and / or positioning helps ensure that position is maintained, prevents tissue compression artifacts, allows fluid flow around the sample, and provides a surface for improved "wetting." When properly index-matched and optically transparent, this can also enable imaging through more than one surface, which is particularly valuable for the use of a multiphoton modality known as second-harmonic generation.

[0010] Alternatives, such as the techniques described in U.S. Pat. No. 8,796,038 and U.S. Pat. No. 20080227144 (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 after the clearing step, typically do not work to orient all types of biopsies, including gastrointestinal or skin biopsy samples, and still require final removal by manual cutting and staining prior to visual interpretation or digital scanning.

[0011] The present invention provides significant advantages over existing techniques by allowing samples to be placed in a container for histological analysis prior to chemical treatment (e.g., fixation or exposure to a dehydration solution) and subjected to chemical treatment in an oriented position within a single container device that can be used for all steps of dehydration, staining, clearing, and imaging. Additionally, the tissue chamber of the present invention can then be used for wax embedding and automated or machine-assisted removal of the wax-embedded sample using a wax removal device configured to cooperate with the tissue chamber of the present invention.

[0012] Single-chamber processing as described herein can provide the added benefit of minimizing reagent usage. 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, wax, and / or other processing fluids. The chamber walls and features can be optically clear and / or index-matched to the clearing solution and / or structures of the sample to be examined (e.g., organelles or proteins). In certain embodiments, the features may comprise a material that dissolves in the presence of certain solutions used in sample processing (e.g., clearing solutions) so that the features space the sample from the container 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 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 solution and / or structures of the sample to be examined.

[0013] Processing samples in a chamber at a fixed location for imaging allows for strict control over the volume of the chamber and reagents used in processing. Instead of placing a tissue-containing cassette open to a fluid environment that may be hundreds of times the volume of the tissue, as in conventional techniques, a single sample processing container as described herein has minimal dead volume and can reduce reagent expenditures. Reagent savings are particularly important for controlling dye costs, which may otherwise be prohibitive, especially for fluorescent markers. Thus, the sample preparation reagent container is particularly suitable for staining unembedded samples, especially for processes incorporating fluorescent stains, where dye costs may be the largest cost component.

[0014] The single-chamber technique described herein also reduces processing time over existing methods, where it is more economical to wait until enough samples are received and "totaled" (placed in a cassette) before loading into a tissue processor. The single-chamber approach can be combined with specialized tissue processors, for example, operable to receive the tissue chambers of the present disclosure through interfacial contact 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 sit idle waiting for additional samples, as in multiplex processing. The same applies to steps following embedding, including slide contrast, slide staining, and organizing and scanning slides for digitization.

[0015] As described above, the single-chamber system described herein allows for varying geometry to more closely match individual sample geometries, reducing chamber dead volume and reagent consumption. The chamber can be designed so that the sample fits into the smallest size container that will accommodate the specimen. For example, a long core biopsy can be placed in a long, thin channel. In certain embodiments, the chamber has a geometry similar to that of a thick, typical microscope slide, with dimensions of approximately 2.5 mm x 75 mm x 10 mm.

[0016] The container can be coded (e.g., with a machine-readable symbol (e.g., a matrix barcode (QR) or UPC code), or any symbol of a recognizable shape, color, or reflective pattern) to provide sample and patient identification and enable 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 to indicate the geometry. Similarly, microscope scanning and imaging time can also be reduced by using sample-specific sized chambers. Microscope slide scanning devices use various approaches to minimize slide scanning time, but they remain inefficient and manually dependent to various extents. Current systems typically take low-power images and use image processing algorithms to estimate tissue location and size, but they are adversely affected by artifacts (e.g., mounting variability, dust, and dirt) that are difficult to control. As a result, manual oversight is 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.

[0017] Coded sample-size-specific chambers as described herein can help avoid manual intervention while maximizing efficiency for image scanning. The tissue is placed in the smallest chamber that it will fit into, and the imager can read the chamber coding to determine the size of the area to scan, so the entire potential tissue location area is imaged. The opportunity for error can be reduced, and operator intervention should not be required, providing efficient imaging with reduced labor costs and errors.

[0018] The tissue chamber may include a trough area or cavity cutout sized to contain the sample and various processing fluids through which processing of the sample occurs. The trough may be in fluid communication with one or more fluid inlets or outlets operable to provide processing fluids to the trough. The tissue chamber may include additional material surrounding the trough to allow for easy manipulation and / or orientation of the chamber within various processing devices. The tissue chamber may include one or more positioning members (e.g., posts) configured to fit within corresponding recesses within various processing devices, thereby positioning the tissue chamber relative to fluid inlets / outlets, imaging objectives, wax 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.

[0019] The tissue chamber may comprise a frangible area in the floor or wall of the trough to provide for controlled separation of the floor or wall from the remainder of the tissue chamber upon application of sufficient force thereto. Such frangible area may comprise an area of ​​thinner or weaker material and can enable efficient mechanical separation of the oriented wax-embedded sample from the tissue chamber.

[0020] The tissue chamber can include a wax retention member (eg, a barb) within the trough configured to stabilize the wax-embedded sample within the trough during processing.

[0021] Aspects of the 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 an inner and outer surface adjacent to the cavity. In some embodiments, the chamber is loaded on the imaging side, and an optically transparent imaging cover or sheet may be 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 another embodiment, the tissue can be loaded into the cavity on a surface adjacent to the underside of the imaging surface, facing the imaging surface. A cover can be placed over the cavity after the tissue is loaded to prevent the tissue from substantially moving within the chamber and substantially seal the chamber against liquid or air. The wall may include an optical window, the window comprising multiple features on the inner surface, the multiple features 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 (e.g., may also be optically transparent or transparent to specific wavelengths 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 generated by second harmonic generation (second harmonic generation).

[0022] The optical window can have a refractive index approximately equal to the refractive index of a fluid (e.g., a clear liquid) in which the tissue sample is immersed prior to imaging. The refractive index of the clear liquid, 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.

[0023] The size of the chamber may be any size that accommodates a tissue sample for microscopic analysis. In various embodiments, the exterior planar dimensions of the chamber are approximately those of a microscope slide (generally, about 2.5 mm x 75 mm or 1 inch x 3 inches). The imaging portion of the chamber may, in some embodiments, be substantially smaller. For example, the imaging portion of the chamber for a prostate core biopsy may be about 1 mm x 15 mm to about 3 mm x 40 mm, thereby fitting within a chamber with the exterior dimensions of a standard microscope slide. 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 an enucleation sample, the chamber may have dimensions of about 25 mm x 25 mm to about 50 mm x 50 mm. In other embodiments, the imaging chamber dimensions may be large enough to accommodate large-format histological samples, or samples referred to as "whole tissue specimens," which typically have planar dimensions in the range of about 65 mm x 50 mm. 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 in height, 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.

[0024] 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 size that can accommodate large-format histological specimens as well as typical medium- to large-sized unsectioned samples.

[0025] The exterior dimensions of the chambered container can vary depending on the dimensions of the enclosed tissue chamber and are large enough to allow for any required fluid channels or external port plugs as described herein. In some embodiments, the exterior dimensions of the container can include a height of about 1 mm to 10 mm. In other embodiments, the container height can be about 500 μm to 1 mm. In other embodiments, the container height can be about 10 mm to 20 mm.

[0026] The features may comprise a material having a refractive index approximately equal to the refractive index of a fluid (e.g., a clear liquid) in which the tissue sample is immersed prior to imaging. In certain embodiments, the refractive index of the clear liquid, 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.

[0027] The features can comprise a material having a refractive index of about 1.5 to about 1.7. The features can comprise a material having a refractive index of about 1.53 to about 1.60. The features can comprise a material that dissolves in the presence of an organic solvent. The organic solvent can be a clear liquid (e.g., benzyl alcohol and benzyl benzoate (BABB)).

[0028] 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 1.53 to 1.60. The sponge material may be selected to approximately match the refractive index of the cleared tissue sample to be imaged.

[0029] 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 a preferred embodiment, the chamber contains two ports. The two ports may be on the same surface, facilitating connection to a fluid exchange system or processor. In a preferred embodiment, the fluid port is self-sealing, such as with a rubberized or silicone plug or surface that allows the introduction of a needle but seals upon removal of the needle. 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.

[0030] The container can comprise a material that is resistant to acids, organic solvents such as BABB, alcohol, and / or temperatures up to about 75 degrees Celsius. The sponge can comprise a material that is resistant to acids, organic solvents such as BABB, alcohol, and / or temperatures up to about 75 degrees Celsius.

[0031] In some embodiments, the cavity for receiving the tissue sample may comprise a frangible area. The frangible area may be located at the periphery of the wall of the cavity. The frangible area may comprise an area of ​​material that is thinned relative to the rest of the wall of the cavity.

[0032] The container may include one or more wax retaining members extending from an inner surface of the wall and / or one or more positioning members extending from an outer surface of the wall.

[0033] Aspects of the invention may include a method for analyzing a tissue sample, the method including the steps of 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 clear solution) in which the tissue sample is immersed prior to imaging, imaging the tissue sample at the desired position within the tissue chamber, and / or wax-embedding the tissue sample at the desired position within the tissue chamber.

[0034] The clearing agent may be BABB. The first solution may include 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 include 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 include a plurality of features disposed on an inner surface of the tissue chamber and configured to contact the tissue sample and allow fluid flow between the tissue sample and the inner surface. The present invention provides, for example, the following items. (Item 1) A container for holding a tissue sample, the container comprising a surface having a plurality of features, the plurality of features configured to contact the tissue sample and allow fluid flow between the tissue sample and the surface. (Item 2) Item 10. The container of item 1, wherein the plurality of features comprises a material having a refractive index approximately equal to the refractive index of a fluid to be used in processing the tissue sample. (Item 3) 3. The container of claim 2, 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. (Item 4) Item 4. The container according to item 3, wherein the plurality of features comprises a material having a refractive index of about 1.5 to about 1.7. (Item 5) Item 5. The container according to item 4, wherein the plurality of features comprises a material having a refractive index of about 1.53 to about 1.60. (Item 6) Item 7. The container of item 1, wherein the plurality of features comprises a material that dissolves in the presence of an organic solvent. 7. The container according to item 6, wherein the organic solvent is a clear liquid. (Item 8) 8. The container of claim 7, wherein the clarifying solution is benzyl alcohol and benzyl benzoate (BABB). (Item 9) Item 10. The container of item 1, comprising a porous compressible material configured to contact the tissue sample on a side of the tissue sample opposite the surface. (Item 10) 10. The container of claim 9, wherein the porous compressible material has a refractive index approximately equal to the refractive index of the tissue sample to be analyzed. (Item 11) Item 10. The container of item 1, wherein at least a portion of the surface has a refractive index approximately equal to the refractive index of a fluid to be used in processing the tissue sample. (Item 12) Item 12. The container of item 11, 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. (Item 13) Item 12. The container according to item 11, wherein at least a portion of the surface has a refractive index of about 1.5 to about 1.7. (Item 14) Item 1. The container according to item 1, comprising a material that is resistant to acid. (Item 15) Item 1. The container according to item 1, comprising a material that is resistant to organic solvents. (Item 16) Item 1. The container according to item 1, comprising a material that is resistant to alcohol. (Item 17) Item 1. The container of item 1, comprising a material that can withstand temperatures up to about 75 degrees Celsius. (Item 18) Item 10. The container of item 1, wherein the surface comprises a frangible area. (Item 19) Item 19. The container of item 18, wherein the frangible area defines a perimeter. (Item 20) 20. The container of claim 19, wherein the frangible area comprises an area of ​​material that is thinned relative to an area inside the perimeter. (Item 21) Item 10. The container of item 1, further comprising one or more wax retention members extending from the surface on the same side as the plurality of features. (Item 22) Item 10. The container of item 1, further comprising one or more positioning members extending from a side of the surface opposite the plurality of features. (Item 23) Item 10. The container of item 1, wherein the surface at least partially defines a cavity for receiving the tissue sample. (Item 24) 24. The container according to item 23, comprising one or more fluid ports in fluid communication with the cavity for receiving the tissue sample and a space outside the cavity. (Item 25) 24. The container of claim 23, wherein the cavity for receiving the tissue sample comprises a frangible area. (Item 26) 26. The container of claim 25, wherein the frangible area is located on a periphery of the surface where the surface intersects with one or more walls further defining the cavity. (Item 27) 1. A method for analyzing a tissue sample, the method comprising: orienting the tissue sample in the tissue chamber to a desired position; exposing the tissue sample to a first solution for chemical treatment at the desired location within the tissue chamber; immersing the chemically treated tissue sample in a fluid at the desired location within the tissue chamber; imaging the immersed tissue sample at the desired location within the tissue chamber without repositioning the tissue sample after orientation; A method comprising: (Item 28) 28. The method of claim 27, further comprising wax-embedding the tissue sample at the desired location within the tissue chamber. (Item 29) 28. The method of claim 27, further comprising sealing the tissue sample in the tissue chamber after orientation, wherein the first solution and the fluid are introduced into the tissue chamber through a sealable port. (Item 30) 28. The method of claim 27, wherein the fluid comprises a clarifier. (Item 31) 31. The method of claim 30, wherein the fining agent is BABB. (Item 32) 28. The method of claim 27, wherein the first solution comprises a dehydrating agent. (Item 33) 28. The method of claim 27, wherein the first solution comprises a fixative. (Item 34) 34. The method of claim 33, wherein the fixative is a dehydrating agent. (Item 35) 28. The method of claim 27, wherein the first solution comprises a dye. (Item 36) 36. The method of claim 35, wherein the dye is a fluorescent dye and the imaging comprises fluorescent imaging. (Item 37) 29. The method of claim 28, wherein the desired location is a desired location for sectioning a wax-embedded tissue sample. (Item 38) 28. The method of claim 27, wherein the desired location is a desired location for imaging of the tissue sample. (Item 39) 28. The method of claim 27, wherein the tissue chamber comprises a plurality of features disposed on an interior surface of the tissue chamber, the plurality of features configured to contact the tissue sample and to allow fluid flow between the tissue sample and the interior surface. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 shows a tissue chamber having a trough and fluid inlets and outlets.

[0036] [Figure 2] FIG. 2 shows a top view of a tissue chamber with a wax retention member.

[0037] [Figure 3]FIG. 3 shows a cutaway view of the tissue chamber with the positioning member and wax retaining member.

[0038] [Figure 4] FIG. 4 shows a tissue chamber with several features for spacing the sample from the chamber walls.

[0039] [Figure 5] FIG. 5 shows the wax removal device in the open position.

[0040] [Figure 6] FIG. 6 shows the wax removal device in the open position with a tissue chamber loaded therein.

[0041] [Figure 7] FIG. 7 shows the wax removal device in the wax cutting position.

[0042] [Figure 8] FIG. 8 shows the wax removal device in the wax removal position.

[0043] [Figure 9A] FIG. 9A shows a cutaway view of the wax removal device in the open position with a tissue chamber loaded therein.

[0044] [Figure 9B] FIG. 9B shows a cutaway view of the wax removal device in the wax cutting position.

[0045] [Figure 9C] FIG. 9C shows a cutaway view of the wax removal device in the wax removal position.

[0046] [Figure 10] FIG. 10 illustrates a container 1001 containing a support sponge 1013, according to one embodiment.

[0047] [Figure 11] FIG. 11 illustrates some of the internal components of the container 1001 shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention provides devices, systems, and methods for visual histological analysis of tissue during chemical processing (e.g., fixation, dehydration, drying, and staining) and wax 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 wax embedding and sectioning and / or imaging. The tissue sample can then be chemically processed (fixed, dehydrated, and / or dried) and wax-embedded in a single container without subsequent repositioning. Furthermore, the tissue sample can be stained, cleared, and imaged intact to provide an initial pathological analysis that potentially negates the need for subsequent expensive processing, embedding, sectioning, staining, and analysis. The systems and methods of the present invention allow for simple mechanical separation of wax-packaged samples prepared for sectioning in a microtome.

[0049] FIG. 1 shows a tissue chamber 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 dissection, and is preferably obtained for histological analysis. The tissue chamber 101 and / or the trough 107 therein may be provided in various sizes and may include markings 109 (human- and / or machine-readable) that correspond to the trough 107 or chamber 101 size and / or provide information regarding the subject from which the sample was obtained, the type of sample, and / or the type of analysis to be performed. When read by a machine or human, the markings 109 may be used to adjust tissue processing (e.g., reagent selection, reagent volume, or processing equipment selection and configuration) and / or to label imaging data.

[0050] The tissue chamber 101 may include a remaining area surrounding the trough 107 to increase overall size and allow for easier manipulation. A notch 103 or opening in the chamber 101 can reduce the material required for production, production time, and their associated costs, as well as reduce the mass of the chamber 101. One or more fluid inlets / outlets 105 are in fluid communication with the trough 107 and the exterior of the chamber 101. The fluid inlets / outlets 107 interface with corresponding fluid inlets / outlets in various processing equipment to provide and remove processing fluids (e.g., fixatives, dehydration fluids, stains / dyes, clearing solutions, or wax for embedding).

[0051] 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. The tissue chamber 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, wax can be introduced into the trough 107 via the fluid inlet / outlet 105 to provide a wax-embedded sample in a block of wax ready for sectioning and subsequent analysis.

[0052] Thus, the tissue sample can be first oriented within the trough 107 in the desired position for both initial imaging and subsequent sectioning, and then left untouched throughout the remaining processing, imaging, wax embedding, and removal steps.

[0053] The tissue chamber may be constructed from materials such as metal, plastic, cyclic olefin polymer, or glass. Preferably, the chamber material does not react with the tissue sample or any of the processing solutions that its surface comes into contact with. The chamber can, in some embodiments, be constructed from multiple materials. For example, the trough may be constructed from a non-reactive, index-matching material, while the remainder of the chamber may be constructed from a different, less expensive material to reduce costs.

[0054] 2 shows a top view of tissue chamber 201 with fluid inlet / outlet 205, notch 203, and trough 207. There may be wax retention members 209 (e.g., barbed posts) within trough 207 configured to position and retain hardened wax and the sample held therein on the floor of trough 207. Trough 207 may include a frangible area 211 on its floor, e.g., comprised of an area of ​​thinner or weaker material, such that in response to the application of a shear force between the floor of trough 207 and the remainder of chamber 201, the floor of trough 207 separates from the remainder of chamber 201 along a line defined by frangible area 211. The frangible area 211 can be sized and positioned to define the floor of the trough 207 and wax retention members 209 such that, upon separation along the frangible area 211, a wax-packaged sample bound to the floor of the trough 211 can be removed from the remainder of the chamber 201 for further processing (e.g., sectioning in a microtome). The wax retention members 209 can be spaced apart in a manner such that an associated sample can be fitted between them within the trough 207, if desired.

[0055] 3 shows a cross-sectional view of tissue chamber 301, with fluid inlet / outlet 305 shown providing fluid access from the exterior of chamber 301 to trough 307. Trough 307 includes wax retention members 309 and frangible area 311, as described above. The bottom surface of trough 307 can include positioning members 313 (e.g., 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 being present on chamber 301 and corresponding recesses being present on the device, it will be readily apparent that the reverse arrangement also provides the same functionality. When positioned within their corresponding recesses, the positioning members 313 may serve to position the chamber 301 and trough 307 within the apparatus relative to, for example, a fluid coupling for the fluid inlet / outlet 305, a wax cutting blade, a plunger for separating the floor of the trough 307 along the frangible area 311, an imaging objective, a light source, or various other processing tools.

[0056] In one embodiment, the positioning member 313 is attached to the floor of the trough 307 and remains in that state after separation of the frangible area 311, which is now secured to the floor of the separated trough 307 by the wax retaining member 309, causing the wax-embedded sample in the wax block positionable by the positioning member 313 to protrude from the surface of the trough floor 307 opposite the surface retaining the wax-embedded sample. The positioning member 313 can thus be used to position the wax block containing the sample for subsequent processing, for example, in a microtome for sectioning. The tissue chamber may be a reusable or single-use item. For example, a frangible tissue chamber is generally considered a single-use item.

[0057] 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 surfaces of the chamber wall 405 allows processing solutions (e.g., dehydration, fixation, clarification, and dye solutions) to access all sides of the sample. In the absence of such features 403, the sample would rest against the flat surfaces 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. Because features 403 are designed to reduce surface area contact between the sample and the vessel or chamber wall 405, shapes such as cones or pyramids that provide a large base area for contact with the chamber wall 405 with few contact points at the top that support the sample are preferred. Features 403 should be shaped and spaced so that they provide little surface area for contact with the sample and provide sufficient weight distribution so as not to puncture or otherwise penetrate the sample, while still supporting the sample above the surface of wall 405.

[0058] The features should have a height or depth sufficient to allow fluid to 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.

[0059] An obvious drawback of such features 403 is their detrimental effect on imaging quality. Thus, in various embodiments, the features may be constructed from a material similar to the walls 405 of the chamber 401 and index-matched to the clearing fluid and / or the sample structure to be inspected. The features 403 thereby provide little distortion during imaging. In other embodiments, the features 403 may be constructed from a material different from the walls 405 of the chamber 401, which material may be configured to dissolve in the presence of one or more of the processing solutions (e.g., the clearing fluid). The clearing fluid is generally applied before imaging, so that if the features 403 dissolve in the presence of the clearing fluid, they are not present to distort subsequent imaging. The clearing fluid may include benzyl alcohol and benzyl benzoate (BABB), and thus the features 403 may include materials known to dissolve in BABB.

[0060] The processing device of the present invention may include a wax removal device operable to manipulate the tissue chamber described herein. Such a device may include a base with a spring-loaded platform on which the tissue chamber may be placed. A hole may be present in the middle of the platform to accommodate a plateau shaped to match the base of a sample trough that includes locating holes that match the locating posts on the bottom of the tissue chamber. When pressed from above, the tissue chamber can be lowered against the spring-loaded platform, causing the central plateau to push up against the bottom of the sample trough, breaking it along its thinned perimeter and thus releasing the sample from the chamber.

[0061] The wax removal device can also include a central piston that holds two knives that point downward from above toward the end of the sample trough. When lowered, the knives cut into the wax, separating the portion of wax within the sample trough from the wax that extends to the fluid inlet and outlet.

[0062] The wax removal device may be operable (mechanically or manually via a handle) through three positions. An open position, in which the arm is raised, may lift both the central piston and the larger chamber piston sufficiently above the base to allow placement of the tissue chamber on the base. A wax-cutting position may be where the piston is lowered in tandem by the arm to the point where the knife in the central piston cuts through the wax. A stop on the guide rail of the base may prevent the central piston from descending further into the tissue chamber base. A wax-removing position, in which the arm is lowered further so that the larger chamber piston presses down on the tissue chamber, may push the sample trough base out of the tissue chamber. A spring mechanism on the insert connecting the handle to the central piston may allow movement to the wax-removing position while the central piston remains stationary and pressed against the stop.

[0063] After moving to the wax removal position and breaking the sample trough, the arm is raised back to the open position and the sample / wax / trough base may be removed. The sample is then ready to be placed into the microtome for sectioning.

[0064] 5-8 show a wax removal device configured to cooperate with the tissue chamber described herein, according to an embodiment of the present invention. The wax removal device is useful for removing wax-embedded samples from the tissue chamber after wax embedding. FIG. 5 illustrates the wax removal device 501 in an open position with the handle 503 in an elevated position, providing access to a spring-loaded platform 507 for placement of a tissue chamber 509 thereon. The spring-loaded platform 507 comprises an outer portion, the size and shape of which generally conform to the dimensions of the tissue chamber 509 to be placed thereon and configured to support the tissue chamber 509 from below. The spring-loaded platform 507 also includes an inner portion, the size and shape of which generally conform to the dimensions of a frangible trough portion of the tissue chamber 509 containing the wax-embedded sample 511.

[0065] The inner portion is firmly supported from below, while the outer portion may be supported from below by a spring or may otherwise be capable of being pressed below the level of the central portion in response to a downward force on the spring-loaded platform 507.

[0066] The handle 503 is operatively associated with a central portion 513 having a wax-cutting blade 517 at its end proximal to a spring-loaded platform 507. The handle 503 is operable to apply a downward force to the central portion 513, which in response moves the wax-cutting blade 517 toward the spring-loaded platform 507 and the tissue chamber 509 mounted thereon. The tissue chamber 509, spring-loaded platform 507, and wax-cutting blade 517 are positioned relative to one another such that, when the handle 503 is operated, the wax-cutting blade 517 is forced into and through the wax in the tissue chamber 509, severing the wax-embedded sample 511 from the surrounding wax in the tissue chamber 509. The wax removal device 501 includes a stop 505 operable to limit the downward movement of the central portion 513 and the associated depth reached by the wax-cutting blade 517 so that the wax-cutting blade 517 cuts only through the wax inside the tissue chamber 509, but not through the floor of the tissue chamber 509.

[0067] The handle 503 is also operatively associated with an outer portion 515 having a plunger 523 at its end proximal to the spring-loaded platform 507. A stop 505 limits the downward movement of the central portion 513, but the outer portion 515 is able to continue its downward movement in response to further movement of the handle 503. The plunger 523 generally conforms to the dimensions of the outer portion of the spring-loaded platform 507 and includes an opening that generally conforms in size and shape to the dimensions of the central portion of the spring-loaded platform 507. Thus, when urged downward into contact with the tissue chamber 509 on the spring-loaded platform 507, the plunger 523 applies downward pressure only to the outer portion of the spring-loaded platform, which in turn is depressed below the level of the rigidly supported inner portion. The inner portion thereby applies an upward force to the frangible trough portion of the tissue chamber 509 containing the wax-embedded sample 511, while the plunger applies a downward force to the remainder 525 of the tissue chamber 509 surrounding the trough portion. These opposing forces create a shear force in the thinned or otherwise frangible area such that the frangible area breaks, releasing the severed wax-embedded sample 511 from the tissue chamber 509.

[0068] Thus, full movement of the handle 503 is operable to move both the central portion 513 and the outer portion 515 downwardly towards the spring-loaded platform 507. The wax-cutting blade 517 cuts through the wax surrounding the wax-embedded sample 511 and is stopped, while the outer portion 515 and associated plunger 523 continues downwardly, breaking the remainder 525 of the tissue chamber away from the severed wax-embedded sample 511 and depressing the remainder 525 and the outer portion of the spring-loaded platform 507 below the level of the now separated wax-embedded sample 523, which can then be removed from the wax removal device 501 for further processing.

[0069] 6 shows the wax removal device 501 in the open position, with the tissue chamber 509 positioned on the spring-loaded platform 507. The handle 503 is still in the raised position.

[0070] FIG. 7 shows the wax removal device 501 in the wax-cutting position, in which the handle 503 is partially actuated so that the stop 505 is acting against the central portion 513 and the wax-cutting blade 517 is cutting the wax surrounding the wax-embedded sample 511, but the plunger 523 is not destroying the fragile area of ​​the tissue chamber 509.

[0071] FIG. 8 shows the wax removal device 501 in the wax removal position, in which the handle 503 is fully actuated, causing the outer portion 515 to force the plunger 523 downward, breaking the remainder of the tissue chamber 525 downward against the spring-loaded platform 507 and now away from the separate wax-embedded sample 511.

[0072] 9A-9C show cutaway views of a wax removal device according to an embodiment of the present invention. Shown are the central portion 513 and outer portion 515 of the wax removal device 501 and their respective association with the wax-cutting blade 517 and plunger 523. The cutaway views further show the inner and outer portions of the spring-loaded platform 507 and the ability of the wax removal device 501 to cut the wax-embedded sample 511 and separate it from the remainder of the tissue chamber 525 for further processing.

[0073] As shown in Figures 9A-9C, the tissue chamber 509 and / or spring-loaded platform 507 may include positioning posts, tabs, or other members 519 and complementary recesses for receiving the positioning tabs, posts, or other members. The positioning members 519 and corresponding recesses can serve to position the tissue chamber 509 on the spring-loaded platform 507 relative to its inner and outer portions, and relative to the wax-cutting blade 517 and plunger 523. A wax retention member 521 (e.g., a post or barb) is also shown in Figures 9A-9C as part of the tissue chamber 509. The wax retention member 521 is operable to hold and position the wax-embedded sample 511 within the tissue chamber 509 during operation of the wax removal device 501.

[0074] Figure 9A shows the wax removal device 501 in an open position, with the tissue chamber 509 loaded onto the spring-loaded platform 507. Figure 9B shows the wax removal device 501 in a wax-cutting position, with the stop 505 acting against the center portion 513 and the wax-cutting blade 517 cutting the wax surrounding the wax-embedded sample 511, but the plunger 523 not breaking the frangible area of ​​the tissue chamber 509. Figure 9C shows the wax removal device 501 in a wax-removing position, with the outer portion 515 forcing the plunger 523 downward, breaking the remainder of the tissue chamber 525 downward against the spring-loaded platform 507 and away from the now separate wax-embedded sample 511.

[0075] FIG. 10 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 fluids 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 self-sealing, particularly if 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 described above, the self-sealing fluid port 1007 may include a rubberized or silicone plug or surface that allows the introduction of a needle but seals upon removal of the needle. 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. 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.

[0076] The sponge or other porous compressible material is configured to contact and hold the tissue sample in place after positioning 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 1.53 to 1.60. The sponge material may be selected to approximately match the refractive index of the cleared tissue sample to be imaged. The sponge can comprise a material that is resistant to acids, organic solvents such as BABB, alcohols, and / or temperatures up to about 75 degrees Celsius.

[0077] 11 illustrates some of the internal configurations 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 relative to the processing fluid, such an orientation aids in the removal of air from the sample chamber 1005 during fluid exchange, ensuring optimal surface contact for the dyes and processing chemicals and preventing imaging distortion due to trapped air.

[0078] The methods of the present invention may involve chemical treatment, imaging, and wax embedding in a single chamber so that the tissue sample can be initially positioned within the chamber in a desired orientation for sectioning and / or imaging without further manipulation until removal of the wax-embedded sample for sectioning.

[0079] Chemical processing may include fixation, dehydration, clearing, drying, and other steps known in the art and useful for both intact tissue imaging (e.g., fluorescent staining and imaging) and histological analysis (e.g., wax embedding and microtome sectioning). In certain embodiments, tissue samples may be exposed to one or more staining agents, fixatives, dehydrating agents, and / or clearing agents in a single tissue chamber as described herein. In some cases, one or more of the above staining agents, 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.

[0080] Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc., have been made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes.

[0081] equivalent Various modifications of the invention and many additional embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire 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

[Claim 1] Devices, systems, methods, etc.

Images