Abstract:
Methods, apparatus and systems for collecting thin tissue samples for imaging. Thin tissue sections may be cut from tissue samples using a microtome-quality knife. In one example, tissue samples are mounted to a substrate that is rotated such that thin tissue sections are acquired via lathing. Collection of thin tissue sections may be facilitated by a conveyor belt. Thin tissue sections may be mounted to a thin substrate (e.g., by adhering thin tissue sections to a thin substrate via a roller mechanism) that may be imaged, for example, by an electron beam (e.g., in an electron microscope). Thin tissue sections may be strengthened before cutting via a blockface thinfilm deposition technique and/or a blockface taping technique. An automated reel-to-reel imaging technique may be employed for collected/mounted tissue sections to facilitate random-access imaging of tissue sections and maintaining a comprehensive library including a large volume of samples.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a divisional of U.S. application Ser. No. 13/734,121, entitled “METHODS, APPARATUS AND SYSTEMS FOR PRODUCTION, COLLECTION, HANDLING, AND IMAGING OF TISSUE SECTIONS,” filed Jan. 4, 2013, which is a continuation of U.S. application Ser. No. 12/695,297, entitled “METHODS, APPARATUS AND SYSTEMS FOR PRODUCTION, COLLECTION, HANDLING, AND IMAGING OF TISSUE SECTIONS,” filed Jan. 28, 2010, now U.S. Pat. No. 8,366,857, which is a continuation of U.S. application Ser. No. 10/886,799, entitled “METHODS AND APPARATUSES FOR THE AUTOMATED PRODUCTION, COLLECTION, HANDLING, AND IMAGING OF LARGE NUMBERS OF SERIAL TISSUE SECTIONS,” filed on Jul. 8, 2004, now U.S. Pat. No. 7,677,289, each of which is hereby incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to automating the process of producing thousands of serial tissue sections from an embedded tissue block in such a fashion as to allow said tissue sections to be reliably collected, handled, stored, and digitally imaged (via automated retrieval) using both light and transmission electron microscopes in order to produce 3D reconstructions of the original tissue&#39;s structure. 
       BACKGROUND OF THE INVENTION 
       [0003]    Today neuroscientists are routinely carrying out evermore-advanced physiological experiments and cognitive scientists are proposing and testing evermore-comprehensive models of brain function. Unfortunately, these experiments and models involve brain systems where incomplete information regarding the system&#39;s underlying neural circuitry presents one of the largest barriers to research success. It is widely accepted within the neuroscience community that what is needed is a comprehensive and reliable wiring diagram of the brain that will provide a neuroanatomical scaffolding (and a set of foundational constraints) for the rest of experimental and theoretical work in the neuro- and cognitive sciences. Unfortunately, the current approach of attempting to integrate the deluge of thousands of individual in vivo tracing experiments into a coherent whole is proving to be a virtually impossible task. 
         [0004]    There is an alternative approach that avoids the problem of stitching together the results of thousands of in vivo tracer injection experiments. The imaging of a single post-mortem brain at a sufficiently high resolution to resolve individual neuronal processes and synapses, while maintaining registration across size-scales, would allow direct tracing of a brain&#39;s connectivity. Researchers using the raw data in such a synapse-resolution brain connectivity atlas would be able to map all the regions, axonal pathways, and synaptic circuits of the brain; and unlike separate specialized experiments, the results would immediately and easily be integrated because they are all performed on the same physical brain. 
         [0005]    Today, the creation of such a synapse-resolution atlas has only been achieved for tiny invertebrate animals such as  C. Elegans  (a round worm measuring 1 mm in length and less than 100 μm in diameter). This is because the fundamental technology used, that of serial section electron reconstruction, currently requires the painstaking manual production of thousands of extremely thin (&lt;1 μm) tissue slices using a standard ultramicrotome in which newly sliced tissue sections are floated away from the cutting knife on water and manually placed on slotted TEM specimen grids a few sections at a time. 
         [0006]    Because of the manual nature of this current process, this technique is totally impractical to apply to larger brain structures and so it is currently unable to address the needs of the larger community of neuroscientists who require a map of the brain connectivity of rodent and primate brains. The key challenge in extending these imaging technologies to map structures that are 1×10 5  (mouse brain) and 1×10 8  (human brain) times as large as  C. Elegans  is the invention of a reliable automated process for producing these thin serial tissue sections. The invention described herein is targeted at this automation challenge. 
       BACKGROUND OF THE INVENTION 
     Prior Art 
       [0007]    We are unaware of any current microtome design (either in production or disclosed in the open literature) that adequately addresses this need for automating the production, collection, handling, and imaging of large numbers of thin tissue sections suitable for use in light and transmission electron microscopic 3D reconstruction work. Although there is a vast number of patents pertaining to microtomes and their automation, these designs are targeted toward automating the slicing process only, and do not address the tissue collection and handling processes. Today the term “automated microtome” has become synonymous with a manual microtome merely having motorized knife advance. Thus, current “automated microtome” designs still require manual slice retrieval and manual slide or grid mounting for imaging. Such manual slice retrieval necessitates skilled, delicate, and incredibly time-consuming work be expended on each tissue slice (or small series of slices) as it involves “fishing” each tissue slice out of a water boat attached to the knife of the ultramicrotome instrument and onto a TEM grid. 
         [0008]    One published microtome design that does somewhat address the automation of tissue collection is U.S. Utility Pat. No. 5,746,855 by Bolles. In that design, the standard manual method of blockface taping (whose advantages for slice collection and handling are described more fully in U.S. Utility Pat. No. 4,545,831 by Ornstein) is proposed to be automated by a pressure roller pressing a reel of transparent adhesive tape against the blockface just before the microtome blade cuts the next slice of tissue off the block. Thus the slice is adhered to the tape and can be carried away automatically by simply advancing the tape reel. (The advantages of tape as a collection, storage, and imaging medium for tissue sections is described in U.S. Utility Pat. No. 3,939,019 by Pickett.) 
         [0009]    The automatic taping lathe-microtome invention described herein is most similar to this taping designed proposed by Bolles; however, our design improves and extends the application of that design significantly. For example, a key disadvantage of Bolles&#39; design is that it makes no modification to the current standard microtome design which involves a discontinuous ratcheting motion of the flat block across the knife. Thus the Bolles design requires the tape to be freshly applied to the block after each slice also in a discontinuous fashion. This seems difficult to automate reliably especially for very thin tissue slices as would be required for most neural reconstruction work. 
         [0010]    Another proposed method for automating the collection of tissues from a microtome disclosed in U.S. Utility Pat. No. 6,387,653 by Voneiff and Gibson, proposed the use of a series of rollers to collect the tissue from the blade of a microtome and move it directly to a glass slide. That design also makes no modification to the current standard microtome design, and thus also suffers from the discontinuous ratcheting action. The Voneiff and Gibson design, however, uses neither blockface taping nor tape as a collection medium. 
         [0011]    It should be noted that neither the Bolles&#39; design nor the Voneiff and Gibson design target the collection of tissue slices for electron microscopic (ultrastructure) imaging. Imaging by an electron beam requires that the tissue slice is unobstructed by any holding substrate thicker than a few nanometers. The tape in Bolles&#39; design and the glass slide in the Voneiff and Gibson design are much too thick for this. The design disclosed herein directly targets collection of slices for light and transmission electron microscopic imaging, and makes modifications to the tape collection medium in order to accommodate this. 
         [0012]    The automatic taping lathe-microtome invention described herein completely redesigns the basic cutting motion of the microtome, replacing the standard discontinuous ratcheting motion with the continuous rotary motion of a lathe. As will be described below, this continuous lathe cutting design makes possible continuous taping and slice collection. The result is a mechanically more stable, more reliable, faster, and more easily constructed design which should finally make possible the fully automated production, collection, handling, imaging, and storage of thousands of semithin and ultrathin tissue sections for use in light and transmission electron microscopic serial 3D reconstruction of neural (and other) tissue. 
       SUMMARY OF THE INVENTION 
       [0013]    The central invention described herein, the “automatic taping lathe-microtome”, produces a continuous ribbon of tissue by lathing an extremely thin strip off the surface of a cylindrical block containing a multitude of embedded tissue samples. The automatic taping lathe-microtome includes mechanisms for sandwiching and adhering this thin and fragile ribbon of tissue between a pair of supporting top and bottom base tapes. One of these base tapes is adhered to the ribbon of tissue before the ribbon is lathed from the block (using a blockface taping method) and the other base tape is adhered after the ribbon is lathed off. If the top and bottom base tapes are composed of transparent material, the resulting composite tape-sandwich allows for light microscopic imaging of the tissue sections while providing complete environmental protection of the fragile tissue sections by hermetically sealing them between the top and bottom base tapes. If, on the other hand, special holes are cut in the top and bottom base tapes above and below each tissue slice (holes cut either before or after the lathing action) then the tissue sections will be framed by the supporting base tapes but the view of these tissue sections will remain unobstructed by the base tapes. Such hole-cut base tapes can then act as TEM slot grids and allow for direct viewing of each tissue slice in a standard transmission electron microscope (TEM). 
         [0014]    Such TEM-ready composite tape-sandwiches are designed to be mounted on reels within an “electron tomography tape cassette”, a related invention disclosed herein. This tape cassette is hermetically sealed and is designed to allow direct coupling to a standard TEM&#39;s specimen port sharing its vacuum. Any tissue section of the composite tape-sandwich can thus be reeled into the electron beam of the TEM much like the film in a movie projector. This allows for random-access imaging of any section on the tape (each tape perhaps containing tens of thousands of serial sections representing an equivalent of several cubic millimeters of brain volume) all without requiring the vacuum seal on the TEM to be broken. Cassettes include tape drive motors, positioning clamps and motors, and a precision tomographic tilt motor for 3D electron tomography. This combined system of the automatic taping lathe-microtome, the TEM-ready composite tape-sandwich, and the electron tomography tape cassette is intended to make possible the efficient creation of synapse-resolution brain connectivity atlases for the neuroscience research community. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]    In all drawings like reference numbers represent corresponding parts throughout: 
           [0016]      FIG. 1A  is a perspective view of the bottom silicone rubber mold. 
           [0017]      FIG. 1B  is a perspective view of the toothed silicone rubber mold. 
           [0018]      FIGS. 1C, 1D, and 1E  are a series of perspective views showing the first three sequential steps in the tissue embedding process. 
           [0019]      FIG. 2A  is a close-up view of the tooth-indentation cavities. 
           [0020]      FIG. 2B  is a close-up view of tissue cubes being placed in the tooth-indentation cavities. 
           [0021]      FIG. 2C  is a perspective view of the top silicone rubber mold. 
           [0022]      FIGS. 2D, 2E, and 2F  are a series of perspective views showing the fourth, fifth, and sixth steps in the tissue embedding process. 
           [0023]      FIGS. 3A and 3B  are perspective views showing the last two steps in the tissue embedding process. 
           [0024]      FIG. 3C  is an enlarged view of the final cured axle-mounted cylindrical tissue block. 
           [0025]      FIG. 3D  is a view showing where the axle-mounted tissue block gets inserted in the automatic taping lathe-microtome. 
           [0026]      FIG. 4A  is a perspective view showing the preferred embodiment of the automatic taping lathe-microtome. The supporting housing of the taping mechanism is removed in this and following figures to better display the mechanism. 
           [0027]      FIG. 4B  is a close-up view of where the knife cuts the tissue block in the automatic taping lathe-microtome. 
           [0028]      FIG. 5A  is a perspective view detailing the tape-web mechanism and cylindrical tissue block (with lathe body removed). 
           [0029]      FIG. 5B  is a close-up view from the back of the tissue block. This view details the blockface applicator mechanisms. 
           [0030]      FIG. 5C  is a close-up view of the side of the tissue block during operation of the automatic taping lathe-microtome. 
           [0031]      FIG. 6A  is a perspective view of the final composite tape sandwich, where each layer in the composite sandwich is peeled away and labeled. 
           [0032]      FIG. 6B  is a view of the final composite tape sandwich from the underside. 
           [0033]      FIG. 7A  is a perspective view showing the electron tomography tape cassette with side panels removed to reveal the tissue tape reels within. 
           [0034]      FIG. 7B  is a close-up view of the specimen stage tip of the electron tomography tape cassette. 
           [0035]      FIG. 7C  is a close-up view of the specimen stage tip of the electron tomography tape cassette where the sides of the tip have been removed to reveal the tape path and clamping mechanism within. 
           [0036]      FIG. 8A  is a perspective view of the electron tomography tape cassette with arrows drawn to display the main degrees of freedom of movement allowed by the mechanism. 
           [0037]      FIG. 8B  depicts a stylized transmission electron microscope (TEM) with the electron tomography tape cassette inserted into its specimen port. 
           [0038]      FIGS. 8C, 8D, and 8E  are three close-up views of the electron tomography tape cassette detailing how the entire cassette mechanism can rotate relative to the TEM in order to perform a tomographic tilt-series on the tissue sample at the tip. 
           [0039]      FIG. 9A  is a schematic side view of alternative embodiment #1. 
           [0040]      FIG. 9B  is a schematic side view of alternative embodiment #2. 
           [0041]      FIG. 9C  is a schematic side view of alternative embodiment #3. 
           [0042]      FIG. 9D  is a schematic side view of alternative embodiment #4. 
           [0043]      FIG. 9E  is a schematic side view of the preferred embodiment. 
       
    
    
     DRAWINGS 
     Reference Numerals 
       [0000]    
       
         
           
               100 —Axle 
               102 —Central hole in axle 
               104 —Center-drilled ends of axle 
               106 —Silicone rubber bottom embedding mold 
               108 —Mold cavity (of part  106 ) 
               110 —Axle seal hole (of part  106 ) 
               112 —Liquid embedding resin 
               120 —Silicone rubber toothed embedding mold 
               122 —Axle seal hole (of part  120 ) 
               124 —Mold teeth (count of 30) 
               126 —Schematic of curing oven 
               130 —Partially cured embedding resin 
               132 —Tooth-indentation cavities (count of 30) 
               140 —1 mm 3  tissue cube (count of 27) 
               150 —Silicone rubber top embedding mold 
               152 —Axle seal hole (of part  150 ) 
               154 —Resin fill-ports (of part  150 ) 
               160 —Axle-mounted cylindrical tissue block 
               162 —Part of tissue block without tissue cube (count of 3) 
               200 —Automatic taping lathe-microtome 
               201 —Lathe body 
               202 —Lathe headstock spindle bearing 
               204 —Lathe tailstock with live center 
               206 —Lathe spindle and belt drive 
               208 —Precision DC gearhead motor 
               210 —Precision linear translation stage 
               212 —Precision linear motorized actuator 
               214 —Microtome knife 
               216 —Lathe dog 
               218 —Film and adhesive blockface-application mechanism 
               300 —Tape-web assembly 
               301 —Tape (full web) 
               302 —Final composite tissue tape-sandwich take-up reel 
               304 —Top base tape feed roll 
               305 —Top base tape (unprocessed) 
               306 —Tape hole puncher mechanism 
               308 —Tape drive rollers 
               309 —Web slack region before blockface taping 
               310 —Freshly cut surface of cylindrical tissue block 
               312 —TEM support film applicator head 
               314 —TEM support film smoothing and drying roller mechanism 
               316 —Block surface with TEM support film 
               318 —Adhesive strip applicator heads (count of 2) 
               320 —Adhesive strips smoothing and drying roller mechanism 
               322 —Block surface with TEM support film and adhesive strips 
               330 —Blockface taping pressure roller 
               332 —Section of top base tape adhered to blockface 
               334 —Freshly microtomed ribbon of tissue adhered to top base tape 
               336 —Bottom base tape applicator (print head) and blowout hole mechanism 
               338 —Final TEM-ready composite tissue tape-sandwich (abbreviated: tissue tape) 
               400 —Single microtomed tissue slice (1 mm 2 , 100 nm-1 μm thick) 
               402 —Ribbon of tissue slices in embedding medium 
               404 —TEM support film coating 
               406 —Adhesive strips 
               408 —Top base tape 
               409 —Viewing hole cut in top tape directly over tissue slice 
               410 —Bottom base tape 
               411 —Viewing hole cut in bottom tape directly over tissue slice 
               500 —Electron tomography tape cassette 
               502 —TEM mounting flange 
               504 —TEM specimen stage body (vacuum seals to TEM) 
               506 —Cassette reels and drive motors (count of 2) for tissue tape 
               508 —TEM sample stage tip mechanism 
               510 —Tissue slice clamped in position for TEM imaging 
               512 —Tomography tilt-series drive motor 
               520 —Tissue tape image-securing top clamps (count of 2) 
               521 —Tissue tape image-securing bottom clamps (count of 2) 
               522 —Blowout hole in wrap-around portion of tissue tape 
               524 —Blowout hole tape length adjust pulley 
               530 —Transmission electron microscope (TEM) 
               600 —Water boat for tissue collection 
               602 —Water 
               610 —Partially-submerged conveyor-belt 
               612 —Submerged pulley 
               614 —Bottom base tape hole puncher 
               616 —Pressure roller 
               618 —Tape-sandwich sealing mechanism 
           
         
       
     
       DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0121]    In the following description of the preferred embodiment, 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. 
       Overview 
       [0122]    The present invention discloses a device, an automated taping lathe-microtome, and a set of associated methods and apparatuses for fully automating the collection, handling, and imaging of large numbers of serial tissue sections. In order to most clearly describe these methods and apparatuses I will first briefly outline the current method of producing serial tissue sections for TEM (transmission electron microscopic) imaging. 
       Current State-of-the-Art 
       [0123]    Classical TEM tissue processing and imaging methods begin by embedding an approximately 1 mm 3  piece of biological tissue that has been fixed with mixed aldehydes, post-fixed with osmium tetroxide, and infiltrated with an embedding resin. This single tissue cube is then placed in a silicone rubber mold which is then filled with liquid embedding resin. The mold is placed in an oven in order to cure the resin and tissue into a hard tissue block. This tissue block is then clamped in the chuck of a microtome (for TEM use this is also called an ultramicrotome). 
         [0124]    This chuck is mechanically actuated to allow the tissue block to be driven down in a smooth trajectory across a very sharp knife thus liberating a slice of the embedded tissue suitably thinned to allow TEM imaging. In “sliding” and “rotary” microtome designs the chuck is moved along a linear trajectory across the blade. (The rotation in a “rotary” microtome is referring to a crank wheel that is rotated by the operator. That rotation is converted to a linear motion of the chuck.) In a “disc” microtome (see U.S. Utility Pat. No. 6,253,653) the trajectory of chuck movement is along a circular arc; however, the sliding, rotary, and disc microtomes&#39; motions are all inherently saltatory using a discontinuous ratcheting motion where the knife engages and disengages the tissue block every slice. Sequential tissue sections are produced by ratcheting the chuck forward toward the knife a small distance after each slice. 
         [0125]    The resulting thin tissue slices are typically less than 1 μm thick and are extremely fragile. In fact, they are so fragile that they would be destroyed if one attempted to remove them from the bare surface of the knife which produced them. For this reason, the knife has a boat of water attached to it in such a manner as to allow newly cut tissue to float on the water (supported by surface tension) immediately subsequent to its cutting from the tissue block. This technique protects the fragile tissue section from friction induced mechanical damage which would occur from extended contact with the knife&#39;s body. A histology technician then manually scoops the floating tissue section (or short series of sections) onto a slotted TEM specimen grid, one whose slot opening has been previously coated with an ultra-thin film of plastic TEM support film. This support film is strong enough to provide support for the tissue section bridging the slot&#39;s gap, but still thin enough to allow unobstructed TEM viewing. 
         [0126]    To image this tissue section, the specimen grid is placed in a TEM specimen stage and manually inserted into the vacuum port of a TEM. Vacuum is pulled on the TEM, and the specimen is finally imaged via the TEM&#39;s electron beam. Reliably producing, collecting, and imaging thousands of serial sections from a single block is required to reconstruct even cubic millimetersized volumes of neural tissue, and this is virtually impossible to accomplish using these classical methods and microtome designs. 
       Key Innovations Disclosed Here 
       [0127]    The innovations disclosed here are designed to bring a high degree of automation to this entire process of tissue collection, handling, and imaging; thus allowing the mass production of serial sections for large-volume 3D reconstruction research. This automated mass production is accomplished by the following innovations to the classical methods and microtome designs:
       Innovation #1: Embed multiple tissue cubes in a single block and process them all at the same time.   Innovation #2: Use an axle-mounted cylindrical tissue block and continuous lathe slicing motion (instead of the traditional discontinuous ratcheting motion of current sliding, rotary, and disc microtome designs).   Innovation #3: Strengthen the tissue sections before cutting by use of blockface thinfilm deposition and blockface taping, thus making subsequent steps more reliable.   Innovation #4: Collect tissue sections by the thousands via a tape-sandwich that doubles as a durable handling and storage medium, as well as a TEM imaging specimen grid.   Innovation #5: Load entire tissue-tape (containing thousands of serial sections) into the electron microscope all at once, threading the tape through the electron beam in a fashion similar to film in a movie projector. This allows random-access imaging of any tissue section on the entire tape without forfeiting the time needed to crack the TEM&#39;s vacuum and re-pumping.       
 
         [0133]    The preferred embodiment of these innovations will now be described in the following logical order:
       1. Description of the axle-mounted cylindrical tissue block embedding method and mold tooling.   2. Description of the automatic taping lathe-microtome mechanical design and operation.   3. Description of the electron tomography tape cassette mechanical design and operation.       
 
       Axle-Mounted Cylindrical Tissue Block Embedding Method and Tooling 
       [0137]      FIG. 1A  shows a silicone rubber embedding mold  106 , having a cylindrical cavity  108 , and a central hole  110 .  FIG. 1C  shows how the mold  106  is slipped onto a metal axle  100  such that the hole  108  forms a liquid-tight seal against the axle  100 . The axle  100  has center-drilled ends  104  for eventual mounting on a lathe. In the first step of the embedding process depicted in  FIG. 1C , liquid embedding resin  112  is poured into mold  106  to fill it halfway. The axle  100  has a central hole  102  which is also filled by the resin  112 . 
         [0138]      FIG. 1B  shows a toothed silicone rubber mold  120 . This mold also has a central hole  122  which forms a seal with the axle  100 . The toothed silicone rubber mold  120  has a multitude of protruding teeth  124 .  FIG. 1D  depicts the second step of the embedding process. The toothed silicone rubber mold  120  is slipped onto the top of the axle  100  such that the teeth  124  become immersed a set distance into the liquid embedding resin  112 . A repeatable depth of immersion of the teeth  124  into the resin  112  is guaranteed when the toothed mold  120  is pressed against the bottom mold  106  such that they form a tight seal. 
         [0139]      FIG. 1E  depicts the third step of the embedding process. The axle  100  with filled molds  106  and  120  are placed in a curing oven schematically depicted by a rectangle and thermometer symbol  126 . The axle is left in the oven until the resin is partially cured. 
         [0140]      FIG. 2A  (close-up) and  FIG. 2D  depict the fourth step of the embedding process where the toothed mold  120  is removed after curing to reveal a partially cured embedding resin  130  having a multitude (count of 30) of tooth-indentation cavities  132 . In  FIG. 2B  (close-up) and  FIG. 2E , the fifth step of the embedding process, a multitude of tissue cubes  140  are manually placed into some, but not all, of the tooth-indentation cavities  132 . A total of 27 tissue cubes are inserted into the tooth-indentation cavities leaving three cavities free of tissue. Each of these tissue cubes  140  have been previously been fixed with mixed-aldehydes, post-fixed with osmium tetroxide, en bloc stained with heavy metals, and infiltrated with resin as per standard TEM tissue processing procedures. The tooth-indentation cavities  132  provide a means to secure the tissue cubes  140  in a set orientation and position with respect to each other and to the axle  100  throughout the rest of the embedding process. 
         [0141]      FIG. 2C  shows a silicone rubber top mold  150 . This mold is also designed to slip onto axle  100  via a hole  152  in the center of the mold  150 . Two resin fill-ports  154  ensure that the hole  152  does not make a complete seal with the axle  100 , but instead leaves a path for resin filling. 
         [0142]      FIG. 2F  depicts the sixth step of the embedding process where the top mold  150  is slipped onto axle  100  and pressed into contact with bottom mold  106  such that the two form a liquid-tight seal against each other. Thus they together form a single tissue embedding mold cavity. 
         [0143]    In step seven of the embedding process, depicted in  FIG. 3A , new liquid resin is poured into one of the resin fill-ports  154  and air is allowed to escape through the other fill-port  154 . Enough resin is poured in to fill up the entire cavity of molds  106  and  150 . This assembly of axle  100  is then placed in curing oven  126  and allowed to completely cure. Removal of the top mold  150  and the bottom mold  106  is depicted in  FIG. 3B . This reveals a completely cured axle-mounted cylindrical tissue block  160 . If necessary, excess pieces of cured resin or inaccuracies in block shape can be removed or corrected by lathing down the sides of the axle-mounted cured cylindrical tissue block  160  at this step using a standard commercial lathe. 
         [0144]      FIG. 3C  show a more detailed view of the axle-mounted cylindrical tissue block  160 . The 27 embedded tissue cubes  140  are seen populating the circumference of the cylindrical tissue block. Three empty spots in this circumference  162  are also depicted (these empty positions along the circumference will be used later for “blowout hole” slots needed during the imaging processes). The automatic taping lathe-microtome described next will shave a thin spiral ribbon off the circumference of this block and mount that ribbon between protective base tapes. Every full rotation of the axle-mounted tissue block  160  within the automatic taping lathe-microtome will liberate a short stretch of ribbon containing 27 slices of the tissue cubes embedded within. A few thousands of revolutions will liberate a very long ribbon, and will have succeeded in reducing all 27 1 mm 3  tissue cubes into a tape containing thousands of thin tissue slices suitable for light or TEM imaging and 3D reconstruction work. 
         [0145]    In  FIG. 3D  shows the position where the axle-mounted cylindrical tissue block  160  is mounted within the automatic taping lathe microtome  200 . 
       Automatic Taping Lathe-Microtome Mechanical Design and Operation 
       [0146]      FIG. 4A  is a perspective drawing of the automatic taping lathe-microtome  200  depicting all of its main component parts except those associated with the tape-web assembly  300  which is detailed later in  FIG. 5A .  FIG. 4B  is a close-up view of the microtome knife  214  cutting the axle-mounted cylindrical tissue block  160 . The main body of the automatic taping lathe-microtome  200  is a small jeweler&#39;s lathe  201  having a standard headstock spindle bearing  202 , and tailstock with live center  204 . The axle-mounted cylindrical tissue block  160  is mounted between the headstock spindle bearing  202  and the tailstock  204  using the center-drilled ends  104  of axle  100 . A lathe dog  216  is attached to the axle  100  of the cylindrical tissue block  160  such that the lathe spindle and belt drive  206  can drive the axle-mounted cylindrical tissue block  160  in a rotary motion. The lathe spindle and belt drive  206  is driven by a precision DC gearhead motor  208  at a slow and steady rate setting the pace of the rest of the automatic taping lathe-microtome&#39;s mechanisms. 
         [0147]    On the side of the lathe body  201  the standard lathe cross-slide has been replaced by a precision linear translation stage  210 . This stage is driven by a precision linear motorized actuator  212  which is capable of providing the sub-micron movements necessary for TEM microtomy. Attached to the linear translation stage  210  is the microtome knife  214 . This assembly enables the knife  214  to be slowly pressed against the rotating cylindrical tissue block  160  in a lathe-like fashion thus liberating a ribbon of tissue into the tape-web assembly  300 . The tape-web assembly  300  holds the tape  301  inline with the rotating cylindrical tissue block  160  such that blockface taping can proceed at a continuous rate in synchrony with the continuous lathe-like cutting of the rotating cylindrical tissue block  160 . This continuous blockface taping process will be detailed below. Also shown in  FIG. 4A  is a thin-film and adhesive blockface-application mechanism  218 . This mechanism applies a thin-film coating onto the freshly cut surface of the cylindrical tissue block  160  and then applies two strips of adhesive to the tissue block  160 &#39;s surface as well. This adhesive is what will secure the tape to the blockface just prior to slicing at knife  214 . Final TEM-ready tissue tape is reeled up onto a take-up reel  302 . 
         [0148]      FIG. 5A  is a perspective view detailing the tape-web mechanism  300  and cylindrical tissue block  160  only. The lathe body and cross-slide components have been removed for clarity.  FIG. 5B  shows the same mechanism, but a close-up view from behind the tissue block detailing the blockface application mechanism.  FIG. 5C  is a close-up view of the side of the tissue block during operation. 
         [0149]    Starting at the top of the mechanism, a top base tape feed roll  304  supplies a continuous stream of plastic tape  305  into the mechanism. A tape hole puncher mechanism  306  punches square viewing holes into the plastic top base tape  305 . The tape is driven forward by tape drive rollers  308  which maintain a slack (no tension) region  309  in the web. This slack region assures that no tension forces from the tape disturb the motion of the cylindrical tissue block  160  or the blockface taping process. 
         [0150]    The slack, hole-cut tape  309  is adhered to the block  160 &#39;s surface at a blockface taping pressure roller  330 . The timing of the hole cutting performed by the tape hole puncher mechanism  306  is synchronized to the current angle of the cylindrical tissue block  160  such that each hole will be precisely aligned directly over an embedded tissue cube  140  when the tape  309  is adhered to the block  160 . A section  332  of top base tape is adhered for a quarter-turn of the block  160  before it is sliced off the block  160  at the knife  214  along with a thin ribbon  402  (detailed in  FIG. 6A ) of the tissue block  160 . The thickness of this ribbon of tissue is set by the relative rotary speed of the lathe spindle  206  and the linear speed of the knife  214 . Both speeds are constant and serve to cut off a continuous spiral ribbon of embedded tissue  402  which is already adhered to the tape  332  at the time of cutting producing a freshly microtomed ribbon of tissue adhered to top base tape  334 . 
         [0151]    The ribbon of tissue adhered to tape  334  is reeled up by a final composite tissue tape-sandwich take-up reel  302 , but before it gets there the tape  334  is driven past a bottom base tape applicator (and blowout hole mechanism)  336  that applies (prints) a covering bottom base tape  410  (detailed in  FIG. 6A ). The blowout hole function of  336  will be discussed later during the section on tape imaging. This produces a TEM-ready composite tape sandwich (abbreviated tissue-tape)  338  which is reeled up onto take-up reel  302 . 
         [0152]      FIGS. 5B and 5C  more clearly show the blockface preparation steps leading up to the production of the adhered section of tape  332 . The freshly cut surface of cylindrical tissue block  310  comes into contact with the TEM support film head  312  which lays down a thin-film on the entire surface of the block with the help of a smoothing and drying roller mechanism  314 . This produces a support film coated block surface  316 . This surface next comes in contact with two adhesive strip applicator heads  318  that, with the help of a smoothing and drying roller mechanism  320 , lay down two strips of adhesive on the block face  322 . This section of the block&#39;s surface with TEM support film and adhesive strips applied is now ready to accept the hole-cut tape  309  for blockface taping via the pressure roller  330 . 
         [0153]      FIG. 6A  shows the composition of the tissue-tape  338 .  FIG. 6B  shows the backside of the tissue-tape. In these figures, each layer in the composite sandwich has been peeled away and labeled. The tissue tape  338  consists of a composite tape-sandwich where the microtomed ribbon cut off the tissue block  402  is secured and protected between top  408  and bottom  410  base tapes. A multitude of 1 mm 2  microtomed tissue slices  400  (each 100 nm to 1 μm thick) are seen to be embedded in the ribbon  402 . Further, this ribbon  402  is covered by a TEM support film coating  404  providing support for each tissue slice  400  across the viewing slots (holes) in tapes  408  and  410  (these holes are labeled  409  in the top tape  FIG. 6A, and 411  in the bottom tape  FIG. 6B ). The adhesive strips  406  laid down by the applicator heads  318  just before blockface taping by pressure roller  330  are seen clearly in  FIG. 6A . Notice how these strips avoid obstructing the view of the tissue slices  400  but still provide adherence between the tissue ribbon  402  and the top tape  408 . 
         [0154]    Seen in the close up view offered by  FIG. 6A , one can appreciate the tissue-tape  338 &#39;s similarity to the film in a movie projector. Each tissue slice  400  resides in its own frame, acting as a TEM slot grid. This analogy to the film in a movie projector can be taken further. In this form, the tissue-tape  338  can be reeled up without damage to the delicate tissue slices  400  since the slices are protected on both sides by the base tapes  408  and  410 . These reels of tissue-tape can be handled and stored efficiently, and can be fed into an electron tomography tape cassette  500  (shown in  FIG. 7A ) for fast random access ultrastructure imaging in a standard commercial TEM. 
       Electron Tomography Tape Cassette Mechanical Design and Operation 
       [0155]      FIG. 7A  shows the electron tomography tape cassette  500 . Side panels have been removed to reveal two tissue-tape reels  506 . The electron tomography tape cassette  500  is designed to act like a standard TEM specimen stage, and thus can slide into the specimen port of a standard TEM  530  (see  FIG. 8B ). The main difference between the electron tomography tape cassette and a traditional TEM specimen stage is the addition of a set of tape reels and motors  506  for mounting the tissue tape  338  on, and the addition of internal mechanisms that allow the tissue tape  338  to be fed all the way out to the specimen stage&#39;s tip  508  and thus into the TEM&#39;s electron beam for ultrastructure imaging of the tissue slice  510  clamped at the stage&#39;s tip  508 . There is a TEM mounting flange  502  which secures the body of the electron tomography tape cassette  500  to the side of a TEM  530 . There is also a cylindrical specimen stage body  504  which slips into the vacuum port on the TEM  530  and forms a tight vacuum seal with it, yet simultaneously allows rotation around the long axis of the cylindrical specimen stage body  504 . This rotation allows the incidence angle at which the electron beam impinges upon the tissue slice  510  to be varied by rotating the entire assembly of the cylindrical specimen stage body  504  and the cassette reels and motors  506  relative to the mounting flange  502  (see  FIGS. 8C, 8D, and 8E ). This rotation of the cylindrical specimen stage body  504  relative to the flange  502  is driven by a drive motor  512 . Changing this angle of incidence allows for 3D reconstruction of the tissue slice having better resolution in depth than the slice thickness would allow if only 2D (non-tilt series) imaging were performed, and is a standard technique in electron microscopy today. 
         [0156]      FIG. 7B  shows a close-up view of the specimen stage tip  508 .  FIG. 7C  is a close-up view of the specimen stage tip  508  where the sides of the tip have been removed to reveal the tape path and clamping mechanism within. The tissue-tape  338  wraps around a pulley  524  at the very front of the tip  508 . During operation, the tape drive motors  506  reel the tissue tape  338  such that the tissue slice to be imaged  510  is centered between two top clamps  520  and is thus inline with the TEM&#39;s electron beam. These two top clamps  520  then engage, securing that section of tissue-tape containing the slice to be imaged  510  stably in position. The pulley  524 &#39;s position is then adjusted electronically to lengthen or shorten the section of tape  338  between the top clamps  520  and a pair of bottom clamps  521  in order to bring a blowout hole  522  into position between the two bottom clamps  521 . These bottom clamps  521  are then engaged to secure the entire tape  338  for imaging. 
         [0157]    This blowout hole  522  is one of a multitude of blowout holes spaced periodically throughout the tape  338 . These holes are made within the automatic taping lathe microtome&#39;s bottom tape applicator and blowout hole mechanism  336  by simply directing a puff of air at the fragile section of sliced ribbon  402  in periodically spaced frames of the tissue tape  338 . Recall that a few tooth-indentation cavities  132  are specifically left empty of tissue cubes  140  during the embedding process for this reason. Thus, the final axle-mounted tissue block  160  had three tissue-free regions  162  around its periphery (see  FIG. 3C ). These holes  522  are purposely blown out to allow the wrapped around section of the tissue tape  338  which resides between the bottom clamps  521  to not obstruct the imaging of the tissue slice  510  directly above it. The cutaway view of the specimen stage tip  508  in  FIG. 7C  shows both sets of clamps  520  and  521  engaged securely holding a single slice of tissue  510  in position inline with the TEM&#39;s electron beam. Directly below this tissue slice  510  is a blowout hole  522  in the tissue tape  338  and thus only the particular slice to be imaged  510  will be seen by the TEM&#39;s electron beam. 
         [0158]      FIG. 8A  shows the electron tomography tape cassette  500  with arrows drawn to display the main degrees of freedom of movement allowed by the mechanism. The reels of tissue tape  506  can rotate in synchrony to bring any desired slice of tissue in the tape out to the specimen tip and thus into the electron beam for imaging. Exact positioning of the field of view is set by driving the whole tip mechanism along the two degrees of freedom perpendicular to the electron beam&#39;s cavity (depicted by arrows shown near tip). Also, the entire cassette and stage body  504  can rotate relative to the TEM mounting flange  502  as described below. 
         [0159]      FIG. 8B  depicts a stylized transmission electron microscope (TEM) with the electron tomography tape cassette inserted into its specimen port. The tape cassette (with cassette covers, which were removed in previous view, installed) is hermetically sealed and can thus share the TEM&#39;s vacuum via its seal along the stage&#39;s body  504 . The tissue tape  338  within the tape cassette  500  is electronically advanced using reel motors  506  to bring a particular tissue slice  510  to be imaged inline with the TEM&#39;s electron beam. Clamps ( 520  and  521 ) engage to allow stable unobstructed viewing of the slice  510 . Any X-Y motions of the stage are now performed to address a small section within the slice (using standard X-Y specimen stage motors present in the electron tomography tape cassette  500  but, for clarity, not depicted here). A tomographic tilt-series (a set of 121 2D electron micrograph images of the tissue slice  510 ) can be taken by stepping the incidence angle in 1° increments from −60° to +60°. 
         [0160]    In  FIGS. 8C, 8D, and 8E  the manner in which the body of the electron tomography tape cassette rotates relative to the TEM mounting flange  502  is depicted. Those three figures show the tape cassette mechanism at three different incidence angles (−60°, 0°, and +60° respectively). 
         [0161]    At each angle, a 2D electron micrograph is produced and all 121 of these images are fed into a standard electron tomographic volume reconstruction algorithm in order to compute a 3D voxel volume digital image of the particular piece of tissue  510  under examination. The system is designed such that any of the multitude of tissue slices in the tissue-tape  338  loaded into the electron tomography tape cassette  500  can be randomly and automatically accessed for 2D or 3D tomographic imaging (at ultrastructure resolution) without ever cracking the vacuum of the TEM. Thus, this avoids any time-consuming manual intervention in the imaging process. 
       Summary of Preferred Embodiment 
       [0162]    This concludes the description of the preferred embodiment of the automatic taping lathe-microtome and associated methods and apparatuses for the automated production, collection, handling, and imaging of large numbers of serial tissue sections. Hopefully it can now be appreciated that this disclosed set of methods and apparatuses addresses many (if not all) aspects pertaining to the true automation of the process of 3D serial reconstruction of biological (especially neural) tissue. The methods and apparatuses disclosed cover changes to the standard practices ranging from the initial tissue embedding process, through the microtomy process, and all the way to the TEM imaging process. They form an integrated set of process and mechanical design changes meant to allow 3D ultrastructure reconstructions of large volumes of neural tissue to be reliably and efficiently produced. The ability to automate the production of such large-scale ultrastructure reconstructions of neural tissue would prove incredibly important to the neuroscience research community just as the ability to automate the sequencing of large stretches of DNA has already proved incredibly important to the genetics research community over the past decade. 
       Alternative Embodiments of the Automatic Taping Lathe-Microtome 
       [0163]    The following describes some alternative embodiments for the automatic taping lathe-microtome. The following descriptions of alternative embodiments of the invention are presented for the purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed. 
         [0164]    These alternative designs involve variations on the blockface taping and tissue collection processes. These designs are depicted in a series of schematic side views in  FIGS. 9A  through  9 D. The preferred embodiment is re-represented in  FIG. 9E  in this same schematic form to further promote ease of comparison. 
         [0165]      FIG. 9A  shows a minimalist core design (alternative design #1) of the lathe-microtome. The axle-mounted cylindrical tissue block  160  is rotated against the knife  214  in order to liberate a thin ribbon of tissue slices in embedding medium  402 . There is no blockface taping in this design (just thin-film support film deposition by  312  and  314 ) and so a boat  600  filled with water  602  must be attached to the knife  214  in order to collect the fragile un-taped tissue ribbon  402  as it comes off the knife. In this design, once the tissue ribbon becomes longer than the water boat, manual collection of the tissue ribbon is required, thus this design cannot be considered truly automated. 
         [0166]      FIG. 9B  show alternative design #2. This is a modification to the minimalist core design in which a submerged conveyor belt  610  is made up of the bottom base tape  410  looped around a pulley  612  firmly attached to the knife&#39;s water boat and submerged in its water  602 . This arrangement allows the fragile floating tissue ribbon  402  to be gently and continuously lifted out of the water by the conveyor belt  610  as shown in the figure. A bottom base tape hole punching mechanism  614  punches viewing holes in the bottom base tape, and its punches are synchronized with the angle of the tissue block  160  such that each tissue slice  400  in the tissue ribbon  402  resides over a viewing hole. The top tape  408  after having similar viewing holes punched in it by hole puncher  306  is aligned and pressed onto the top of the conveyor belt by pressure roller  616 . This produces a tape-sandwich which can be sealed by a sealing mechanism (e.g. a heated pressure roller)  618  before finally being reeled up as a finished TEM-ready tissue tape. This design is fully automated and produces a tissue tape-sandwich capable of automated imaging using the electron tomography tape cassette  500 . This design ( FIG. 9B ) does not employ blockface taping meaning that there is a place in the mechanism where a fragile, freely-floating ribbon of tissue  402  is unsecured by any base tape. This reduces the reliability of the design, but it also reduces its complexity by eliminating blockface taping. 
         [0167]      FIG. 9C  shows alternative design #3 which simply adds blockface taping to the submerged conveyor-belt design. This design is similar to the preferred embodiment&#39;s in its use of a blockface taping pressure roller  330  adhering the top base tape  408  directly to the blockface before cutting. In this way there is never a fragile, unsupported tissue ribbon. This design has both the advantage of blockface taping tissue support at the knife and the advantage of a knife water boat to prevent friction-induced damage with the knife. It, however, suffers from the complexity of both a water boat and blockface taping mechanism. 
         [0168]      FIG. 9D  shows alternative design #4 in which the water boat has been removed and the conveyor-belt formed by the bottom base tape is no longer submerged. The blockface taping has provided enough support for the tissue ribbon  402  coming off the knife  214  such that the water boat support can be eliminated. 
         [0169]    Finally,  FIG. 9E  shows the preferred embodiment in the same schematic manner as the just described alternative designs. In it, the conveyor-belt made up of the bottom tape is replaced by a printing head  336  that manufactures the bottom base tape  410  in situ. This simplifying change can be tolerated if the final tissue tape-sandwich still has sufficient strength provided now only by the top tape. The in situ manufactured bottom tape is then only acting as a relief to protect the tissue from friction damage during reel-up operations. 
         [0170]    Another alternative embodiment, which is not depicted in the figures, is to forgo cutting viewing holes in the top and/or bottom base tapes within the microtome, and instead, as a later step, etch these holes using an acid to reveal the tissue slices within. If the top and bottom base tapes are made of a solid material (preferably a metal such as copper) and no holes are cut in the microtome in these tapes, then the composite tape sandwich taken-up on the final take-up reel  302  will not be ready for imaging since the tissue slices between the top and bottom tapes will be hidden by the overlying tapes. This tape-sandwich can then be put through an etching machine where a mask is placed around each section of tape covering up all areas of tape except those having tissue directly beneath. Then the tape is exposed to an etchant (acid in the case of metal tapes) that will dissolve the parts of the top and bottom tape directly above and below each tissue slice. The etchant is chosen so as not to damage the delicate tissue slice which is revealed via the etching process. The advantage of this viewing hole etching method is that it allows the blockface taping step to proceed with a solid tape instead of one with viewing holes. This implies that the tissue slice being cut can be supported across its entire width during the cutting procedure. 
         [0171]    Once again, these descriptions of alternative embodiments of the invention were presented for the purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible. It is intended that the scope of the invention be limited not by this detailed description of the preferred embodiment or the alternative embodiments, but rather by the claims section to follow. 
       ADVANTAGES AND CONCLUSIONS 
       [0172]    We will now summarize the key advantages of the present invention over the current state-of-the-art in serial section production, collection, handling, and imaging. 
         [0173]    Advantage #1: The present invention allows for the processing of multiple tissue cubes simultaneously by embedding a multitude of tissue cubes in a single axle-mounted tissue block. 
         [0174]    Advantage #2: The novel design of the automatic taping lathe-microtome departs most radically from current sliding, rotary, and disc microtome designs in that it makes possible a smooth, continuous slicing motion where the knife never disengages from the tissue block during operation. This offers a distinct improvement in the fundamental reliability of the slicing operation in that a continuous, non-disengaging, lathing action can be made much more stable than current microtomes which use a discontinuous ratcheting motion. This is a crucial improvement if thousands of thin tissue slices are to be produced without error in a fully automatic manner. 
         [0175]    Advantage #3: Because the slicing action of the automatic taping lathe-microtome proceeds in a smooth, continuous manner (where the knife never disengages from the tissue block during operation) it is now possible to include in the design continuous blockface taping where the tape never disengages from the block. This represents a dramatic simplification in the process of blockface taping over any proposed automation of blockface taping in discontinuous motion microtomes (which by their nature necessitate the tape to engage and disengage during any blockface taping operation as well). 
         [0176]    This blockface taping technique, along with the novel technique of blockface deposition of the TEM support film, strengthens the tissue sections before cutting by making the thin slice required for TEM work appear thicker and stronger before the time of slicing. As importantly, continuous blockface taping also allows the freshly liberated tissue slice (ribbon) to be collected automatically as part of the tissue tape-sandwich. 
         [0177]    Advantage #4: All aspects of the current invention are centered on producing and using the TEM-ready tissue tape-sandwich  338 . This novel collection, storage, and imaging medium has no direct analogy in current electron microscopy practice. It has the potential to replace the water boat as a slice-collection device and the TEM slot grid as a sample holding device thus eliminating the arduous manual steps of water boat slice retrieval and grid mounting. Unlike the storage tape proposed by Bolles which only allows light-microscopic imaging, the tissue tape-sandwich in this invention allows for TEM viewing by integrating a series of viewing holes into both top and bottom base tapes and by using the novel technique of blockface deposition of the TEM support film. 
         [0178]    Advantage #5, Finally, this TEM-ready tissue tape-sandwich containing many thousands of individual tissue slices (representing perhaps many cubic millimeters of neural tissue volume) can be loaded, all at once, into a single TEM using the novel electron tomography tape cassette. That invention allows the tissue tape to be threaded through a standard TEM&#39;s electron beam in a fashion similar to film in a movie projector. This allows any tissue section on the entire tape (i.e. any point within the sliced volume) to be randomly accessed for ultrastructure imaging without forfeiting the time needed to crack the TEM&#39;s vacuum and repumping. 
         [0179]    The truly automated production, handling, storage, and random-access imaging of TEM-ready serial tissue sections made possible by the disclosed innovations could even make possible new ways of thinking about how TEM imaging is applied to neuroscience research. For instance, it might make possible the establishment of tissue-tape libraries storing the volume of entire mammalian brains and containing vastly more tissue slices than could ever be methodically imaged in total. Such tape libraries would instead be made available online to the entire research community for random-access imaging on request. One could as well imagine automated image-sequence direction algorithms putting the random-access imaging capability to use for following and imaging multi-scale structures of interest such as the torturously long, thin, and complexly intertwined axonal and dendritic processes of the brain. These could be traced and imaged across multiple tissue slices contained on the same tape all without user intervention. The benefits to the neuroscience research community of such efficient multi-scale (ultrastructure to whole brain) imaging could be enormous.