Methods And Apparatuses For Sectioning And Imaging Samples

Abstract

The present disclosure relates to methods and apparatuses for sectioning and imaging tissue or other samples, which are then automatically captured to enable subsequent analysis. The apparatus acts as a slice capture mechanism for serial sectioning microscopy in a fashion which enables subsequent interfacing with secondary microscopic interrogations or for processing with molecular diagnostic tools. The slices are spatially indexed to allow specific slices to be recalled from a library via automated handling techniques described herein.

Claims

1. A method of capturing and extracting sequential slices of an object from a microtome, the method comprising: extracting the sequential slices of the object as each sequential slice is sliced from the object by a blade of the microtome by directing each sequential slice away from the microtome using a mechanical conveyance that adheres the sequential slice to a conveyor; indexing the sequential slices by adhering each sequential slice to the conveyor and advancing the conveyor a known distance using a computer-controlled motor as each sequential slice is adhered to the conveyor.

2. The method of claim 1, wherein the conveyor is any of: a mesh, a tape, or a film.

3. The method of claim 1, wherein the conveyor is any of: a mesh, a tape, or a film, and wherein each sequential slice is adhered to the conveyor before or after slicing.

4. The method of claim 1, wherein the conveyor applies direct manipulation of each sequential slice by mechanical motion.

5. The method of claim 1, wherein the conveyor applies direct manipulation of each sequential slice by mechanical motion, the mechanical motion is generated by a computer-controlled motor able to provide a reliable and constant amount of tension or movement as each sequential slice is sliced from the object.

6. The method of claim 1, wherein each sequential slice is extracted by a constant tension applied to the conveyor which removes the sequential slice after slicing.

7. The method of claim 1, wherein each indexed sequential slice is stored for later retrieval.

8. The method of claim 1, further comprising: storing each indexed sequential slice; and retrieving one or more sequential sections for one or more secondary interrogations; wherein the one or more secondary interrogations include any of: secondary staining, molecular analysis, sequencing, electron microscopy, or visual microscopy.

9. The method of claim 1, wherein the indexing the sequential slices further comprises: electrostatically charging the object; electrostatically charging an extractor opposite to the object; and wherein each sequential slice is attracted to the extractor as the sequential slice is being sliced from the object and adhered to the conveyor.

10. The method of claim 1, further comprising: imaging one or more of the sequential slices; and generating a model from the images of the one or more sequential slices.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0035] FIG. 1A is a section view of a portion of an exemplary Slice Capture System during a slice capture step employing a fluid and a fluid pump system to capture a sample as it is sliced by the KESM, according to many embodiments.

[0036] FIG. 1B is a section view of a portion of an exemplary Slice Capture System during a slice capture step employing a series of mechanized roller or a tape to capture a sample as it sliced by the KESM, according to many embodiments.

[0037] FIG. 1C is a section view of a portion of an exemplary Slice Capture System during a slice capture step employing an electrostatic force to capture a sample as it is sliced by the KESM, according to many embodiments.

[0038] FIG. 2A is a section view of a portion of an exemplary Slice Capture System during a slice manipulation step employing a thermal cycling force to remove deformations from a sample, according to many embodiments.

[0039] FIG. 2B is section view of a portion of an exemplary Slice Capture System during a slice manipulation step employing a mechanical roller to remove deformations from a sample, according to many embodiments.

[0040] FIG. 2C is a section view of a portion of an exemplary Slice Capture System during a slice manipulation step employing an electrostatic force to remove deformations from a sample, according to many embodiments.

[0041] FIG. 3A is a schematic illustrating an exemplary Slice Capture System during a slice indexing step employing a linear tape to index the location of a sample, according to many embodiments.

[0042] FIG. 3B is a schematic illustrating an exemplary Slice Capture System during a slice indexing step employing a filter array to index the location of a sample, according to many embodiments.

[0043] FIG. 4 is a flow chart illustrating an exemplary method of processing slices of a sample using Slice Capture Systems, according to many embodiments.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Example embodiments of the present disclosure and their advantages are best understood by referring now to the drawings herein, in which like numerals and letters refer to like parts.

[0045] In the following description of the various embodiments, reference is made to the accompanying drawings, which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. It should be understood that various alternatives to the embodiments of the systems and methods described herein may be employed in practicing the embodiments described herein. It is intended that the following claims define the scope of disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Definitions

[0046] Section or slice refers to a single strip of contiguous material that was removed from the block face of a sample by way of a relative motion between the sample and the knife or other cutting mechanism.

[0047] Serial Section Microscopy refers to the practice of taking serial sections with a microtome and imaging them, such as traditionally by mounting the slices to glass and staining.

[0048] Knife Edge Scanning Microscope or KESM refers to a microscope that performs Serial Section Microscopy in an automated fashion (such as that described in U.S. Pat. No. 6,744,572, the contents of which are fully incorporated herein by reference)

[0049] Microtome refers to a device in which a block of material is precisely cut such that a very thin layer of material is removed, or sectioned, from the surface of the block.

[0050] Imagery shall include any technique designed to measure an image, a spatial map of an optical or electronic response. The techniques include optical/electron microscopy techniques, to name a few.

[0051] Imaging generally refers to data collection in order to generate a visualization of a given area.

[0052] Stain refers to a chemical treatment, which aims to change the photonic response of all or parts of a medium by methods including but not limited to attaching a pigment, a genetically expressed fluorophore, or chemistry designed to modify the target structure to be imaged.

[0053] Molecular Diagnostic refers to a form of chemical test or assay, which takes a sample of tissue and identifies biological markers to make a diagnostic.

[0054] Multiplex refers to a method of selecting one location within a matrix by having two selective addressing systems on both sides of the matrix, thus needing only 2N selectors to address N{circumflex over ()}2 locations.

[0055] Tubes and connections refers to a system of NPT threaded PVC pipes, tubes, and connectors that allows water to flow through the KESM system.

[0056] Relay refers to an electrically operated switch, which uses a coil to isolate separate currents from interacting with each other.

[0057] Transistor refers to a semiconductor device used to amplify and switch electronic signals and electrical power. Transistors are composed of semiconductor material with at least three terminals for connection to an external circuit.

[0058] Switch refers to a switch is an electrical component that can break an electrical circuit, interrupting the current or diverting it from one conductor to another.

[0059] Ball Valve refers to a valve with a spherical disc, the part of the valve, which controls the flow through it. The sphere has a hole, or port, through the middle so that when the port is in line with both ends of the valve, flow will occur. When the valve is closed, the hole is perpendicular to the ends of the valve, and flow is blocked.

[0060] Solenoid valve refers to an electro-mechanically operated two-port valve. The valve is controlled by an electric current through a solenoid, which controls the flow of water through the valve. The valve remains closed until a current, such as a 12 VDC, is applied to the two terminals on the valve, the valve opens and water can flow through.

[0061] Deformations refer to the changes in shape that take place in a slice incurred by the sectioning process which result in varying warping, curling, elongation, tearing, crinkling, waving in a generated section.

[0062] Slice Capture System

[0063] Embodiments of the Slice Capture System may provide systems and methods for capturing and storing slices of a sample in the water flow coming off the KESM system

[0064] Slice Capture

[0065] Aspects of the present disclosure provide methods of capturing sequential slices of an object from a microtome. FIG. 1A shows an exemplary method 100a of capture and extraction comprising a step of moving each slice 140 away from the knife edge 120 of the KESM with a constant flow of fluid 162 over the surface of the microtome blade 110. The fluid 162 may be suctioned by a fluid pump system connected by tubes and connections to a suction channel 170 located directly over the microtome edge 120. The force provided by fluidic drag on the slice 140 may keep the section taut and may pull it free of the knife 110 and block 130, thereby extracting the section 140. The pump may provide suction 172, moving the fluid 162 from an immersion bath 160 which the sample 130 and knife 110 are immersed in, while also moving the captured section 150 through the suction channel 170 and conveying it away from the microtome edge 120 along the fluid path 164. The captured slice 150 may be suctioned through a series of tubes and connections to either a capture or a bypass system. The bypass system may have a filter for non-captured slice disposal. The fluid 162 may be returned to the pump and finally back to the immersion bath 160. The mechanism for the power and the wiring of the capture and bypass system may be configured to be controlled manually or by computerized control. The activation of a valve to the bypass system may direct the flow of water carrying a slice through a series of tubes to be flushed from the system.

[0066] Further methods of capturing sequential slices of an object from a microtome are also provided. FIG. 1B shows an exemplary method 100b of capture and extraction comprising a step of moving each slice 140 away from the knife edge 120 of the KESM by a mechanical conveyance 180. The slice 140 may be conveyed onto a mesh, a tape, or a film 182, herein referred to as a conveyor, which may adhere to the slice 140 before or after cutting. By applying a conveyor 182 to the sample 130 before cutting with the knife 110, the section 140 may be extracted by a constant tension applied to the conveyor 182 which removes the section 140 after slicing. Similarly, the section 140 may be gathered by the conveyor 182 as it is being sectioned, allowing no interruption of cutting dynamics. In either case, the conveyor 182 may allow direct manipulation of the captured section 150 by mechanical motion. This motion may be generated by a small controlled motor able to provide a reliable and constant amount of tension or movement as each section 140 is generated.

[0067] FIG. 1C shows an exemplary method 100c of capture and extraction comprising a step of moving each slice 140 away from the knife edge 120 of the KESM by an electrostatic force. This electrostatic charge may be built up in an extractor 190, allowing the section 140 to be pulled away from the knife block 110 by the force of the electrostatic charge on the section 140. For example, the sample block 130 may be positively charged 192 such that the slice 140 may be attracted to a negatively charged 194 extractor 190. Alternatively, the charges may be the reverse, with the sample 130 being negatively charged 194 and the extractor 190 being positively charged 192. This may allow the section 140 to be rapidly extracted. Thin sections 140 have large susceptibility to electric charge, having enormous surface area to volume ratios. An electrode may induce a large charge in a captured section 150, repelling it away from the knife edge 120 after sectioning.

[0068] Slice Manipulation

[0069] Aspects of the present disclosure provide methods of capturing sequential slices of an object from a microtome and manipulating the slice to prepare it for storage. FIG. 2A shows an exemplary method 200a of manipulation comprising a step of applying a thermal cycling force 220 to prepare the section 140 for a storage step. Thermal cycling 220 may remove deformations 242 after sectioning to allow the section 140 to more easily be stored flat 244 on a flat surface 210. Thermal cycling 220 may occur through any variety of heating methods, including but not limited to radiation, fluidic heat transfer in air or water or other media, mechanical vibration, or pressure. Combined with the other methods presented, the thermal cycling 220 may absorb extra variance in sections 140 when captured at large scales.

[0070] Further methods of capturing sequential slices of an object from a microtome and manipulating the slice to prepare it for a storage step are also provided. FIG. 2B shows an exemplary method 200b of manipulation comprising a step of manipulating a slice 140 with a mechanical roller 230. The roller 230 may be comprised of plastic or other materials. This roller 230 may correct any curling or other slice deformations 242 that occur as a result of sectioning, and may manipulate the slice 140 into a flat storage state 244. Two oppositely rotating rollers 230 may pick up the leading edge of a curled section 140. As the section 140 is pulled through the rollers 230, a compressive force may then be applied, extruding the section 140 on the opposite side and relieving built up stress in the section. This may create a flattened section 242 necessary for the downstream indexing process.

[0071] FIG. 2C shows an exemplary method 200c of manipulation comprising a step of manipulating the slice 140 with an electrostatic force 250. The electrostatic force 250 may cause the section 140 to conform to a flat surface 210 of charge to force the slice 140 into a flat storage state 244 from a deformed state 242. This may allow for an electrostatic charge on the microtome to greatly influence the dynamics of the section 140. The electrostatic charge on the knife 110 may be manipulated, and the induced charge on the section 140 may cause the section 140 repel from the opposing charge, flattening the section. The section 140 may for example be positively charged 192 while the flat surface of charge 210 may be negatively charged 194, or vice versa. The charge accumulated between other parts of the system, including but not limited to the fluid, conveyance, or final storage system, may also induce this self-repulsion and flattening 244 of the slice 140.

[0072] Aspects of the present disclosure also provide methods of capturing sequential slices of an object from a microtome and manipulating the slice to prepare it for a storage step. An exemplary method of manipulation may comprise a step of applying a fluidic force to prepare the section for indexing and storage. This fluid may heat, bend, float, flatten, and move the section with the purpose of removing deformations in order to reliably gather, index, and store of each section. The fluid may also move the section to a storage site, allowing other corrections to take place after storage. A flat reference plane may be presented by floating the section to the top of an open channel of fluid. Combined with heat, the section may adopt the shape of the flat fluid plane, creating a section ready for capture.

[0073] Slice Indexing

[0074] Aspects of the present disclosure provide methods of retrieving sections which have been indexed and stored. An exemplary method of storage and retrieval may be automated or manual. The storage process may be comprised of an index of sections in a variety of storage systems, which may allow consistent correlation between the index and the final storage location. The storage system may allow retrieval of slices from the storage system. The retrieval process may retrieve one section or it may retrieve multiple sections. The retrieval process may be guided by a computationally informed or guided decision or an automatic action.

[0075] Aspects of the present disclosure provide methods of capturing sequential slices of an object from a microtome and indexing the slice to prepare it for a storage step. FIG. 3A shows an exemplary method of indexing 300a comprising a step of indexing slices 140 of an object 130 on a linear tape 310 after they have been extracted and manipulated as described herein. As described herein, a particular section 140 may be correlated to a point on the linear index on the tape 312, which may be used to mark the known correlation between a particular section 140 and its final storage location. This linear tape 310 as described in this indexing step may be embodied as the mechanical conveyance 180 utilized in the first step. The linear tape 310 may maintain tension to present a flat surface which the sections 140 may adhere to. The indexing of the tape 310 may be carried out by a computer-controlled motor moving the tape, allowing known linear distances 312 on the tape 310 to correspond to particular sections 140 placed along the length of the tape 310.

[0076] Further methods of capturing sequential slices of an object from a microtome and indexing the slice to prepare it for a storage step are also provided. FIG. 3B shows an exemplary method 300b of indexing comprising a step of indexing a slice(s) 140 into a filter(s) 340. A filter 340 or an array of filters 320 may provide a known indexed storage place for one or more sections 140, which may allow a known slice(s) 140 to be correlated with a its final storage location. As described herein, if the method of a slice manipulation is with a fluid, then the filters 340 may be presented in a multiplexed fashion to the fluid flow via one or more connection tubes 350, allowing multiple storage sites for the same fluid flow to be alternately presented. As sections 140 accumulate in a filter 340, the filter 340 may be swapped out for another filter site 330 or the present filter site 330 may remain in the fluid flow to accumulate further sections 140. The swapping of filter sites 330 may be executed by a series of actuated solenoid valves, which may be computer-controlled. The solenoid valves may be arranged in any fashion, which may allow branching of the fluid flow into discrete filter sites 330, each uniquely indexed.

[0077] Aspects of the present disclosure provide methods of capturing sequential slices of an object from a microtome and indexing the slice to prepare it for a storage step. An exemplary method of indexing may comprise a step of indexing a slice(s) into a set of containers. The containers may be altered to work with other instrument which may necessitate a useful container structure. These containers may allow indexing of one or multiple sections, which may allow a known slice(s) to be correlated with its final storage location. Sections may be moved into a container by any of the slice manipulations described herein. Containers may be handed off in an automatic fashion to a second instrument, for example a DNA sequencer, mass spectroscopy machine, or any other secondary interrogation method.

[0078] Aspects of the present disclosure provide methods of capturing sequential slices of an object from a microtome. FIG. 4 shows an exemplary method 400 comprised of four steps. The method 400 may comprise one or more steps or sub-steps of the slice capture, manipulation, and indexing steps described above.

[0079] In a first step 410, a slice 140 may be captured and extracted from the knife edge of a KESM or microtome as the slice is made. The first step 410 may comprise one or more sub-steps including a step 412 of sectioning a slice 140 of an object and a step 414 of extracting the slice 140 away from the microtome for further manipulation. First step 410 may for example comprise any of the methods 100a, 100b, or 100c as previously described herein or similar.

[0080] In a second step 420, the section 140 may be manipulated by a variety of forces to prepare the section 140 for efficient downstream processing so that the section is laid flat. These manipulation forces may include one or more of a flattening 422, a movement 424, or a sensing 426. Alternatively, the section 140 may be directed to a bypass system 428 which may be used for slice 140 disposal as previously described herein. Second step 420 may for example comprise any of the methods 200a, 200b, or 200c as previously described herein or similar.

[0081] In a third step 430, a section 140 may be indexed into a storage medium to provide a unique reference point to the physical location of a section 140. The step 430 may comprise one or more sub-steps including the a sub-step 432 of creation of spatial separation between slices 140, a sub-step 434 of accounting of the slices 140, and a sub-step 436 of indexing the slices 140 into a storage medium for storage and retrieval of sections. The step 430 may, for example, comprise any of the method 300a or 300b as previously described herein or similar.

[0082] In a fourth step 440, the section 140 may be kept in a storage system 442 which may a sub-step 444 of storage of the sections 140 and a later sub-step 446 of automated or manual retrieval of the sections 140 for secondary interrogations.

[0083] In an exemplary embodiment of method 400 comprising the steps 100a, 100b, or 100c; 200a, 200b, or 200c; and 300a or 300b as described herein or similar, slices may come off the knife of the KESM and flow through a standard liquid or water channel. The apparatus may be configured such that slices may flow through a series of suction tubing before being multiplexed through a filter array. A particular filter well may be chosen from the filter where slices may be stored. The filter array may then be removed, maintaining the registered slices in the filters for further inspections.

[0084] As slices leave the knife-edge they may move at the average velocity of the liquid or water flow through a series of tubes and connections. The liquid or water flow may enter a manifold with a single input and multiple output channels, with multiple electronically controlled solenoids that open or close flow out of the manifold. This may present a series of rows over the top of the filter well matrix. The bottom of the filter well matrix may be comprised of a series of columns, which exit through an equivalent series of solenoids and a manifold. The orientation of the solenoids in the filter matrix described here can be arranged such that each slice moves through the water channels at the same average velocity, and the solenoids may be timed to place slices in specific locations within the filter well matrix. In this way, for example, 20 total solenoids10 on each side of the filter matrixcan address 100 locations within the matrix. This arrangement can reduce cost and complexity of the operating system. Multiple slices may be also kept in each filter assembly. Individual slices may be selected out of a collection by mounting all the slices in the collection and re-imaging in an automated slide scanner. The filter matrix embodiment can be more suited to storing a number of slices from a more general region, which can then be extracted for examination. Such a channel system is described in U.S. Provisional Application No. 62/140,093, filed Mar. 30, 2015, which application is incorporated herein by reference.

[0085] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.