Optimized transection: a prelude to oriented sectioning

A technique is described which permits blocks of tissue to be flat-embedded in euhedral plastic castings and then to be transected along a plane so that sections may be cut which are optimally oriented to the internal ultrastructure of the block. In the transection procedure a hollow plastic cylinder is placed on the specimen trimming block. The cylinder's top prescribes a plane to which the tissue block is accurately oriented and clamped at a predetermined level. Two hand files and a burnisher are worked across the cylinder's top to 1) remove extraneous material above the plane of transection, 2) expose the tissue for sectioning and 3) smooth the block face. The clear plastic at the periphery of the exposed tissue is then easily trimmed away with a razor blade. The result is a block face with a flat, reflective surface which may be quickly aligned to the knife on the ultramicrotome. The effort needed to transect, align and face the block is minimal and 1-micron or semithin sections produced will be precisely parallel to, and at, the plane of transection. Dust produced by the transection procedure is easily eliminated from the work area by use of a small disposable vacuum cleaner. The technique of producing optimally oriented light microscope sections, using the transector, is enhanced by application of solvents to the block face which cause it to develop a temporary low relief, exactly matching the structural detail of sections cut from the block face.(ABSTRACT TRUNCATED AT 250 WORDS)     

Holding Plastic Embedded Specimens During Dissection: Two Improvements

Sectioning of tissue specimens of aligned cells (e.g., muscle, cochlea, retina) for micrographs, often requires the capsule containing the tissue to be positioned at a precise angle during sectioning. The correct angle can be set by trial and error, but the process can be shortened if the gross anatomy of the cell system is used as a guide to orient the embedded sections as closely as possible in the optimal plane. Thick sections are then cut in this plane with a razor blade, and these sections are re-embedded in preparation for thin sectioning. This technique eliminates the large angles of the capsule in the microtome which occur when the gross tissue is poorly aligned in the first embedding.     

Plastic Embedding, Orientation in the Mold and Tape-Supported Sectioning of Sclerotized Insects and Other Hard Objects

Sections of large specimens such as whole honeybees or beetle adults embedded in plastic usually are difficult to cut with a constant thickness. The sections also compress and roll. Sections of even thickness have been obtained by using a mixture of methacrylates (ethyl, 1:butyl, 3) and by firmly supporting the block in the microtome with a special holder. Scotch tape #810 applied to the block before each section is cut eliminates section compression and rolling. The sections are attached to slides with 2% celloidin in an absolute alcohol-methyl benzoate mixture (5:5-7:3); and the tape is removed with heptane. Large sections can also be cut from blocks of styrene mixed with butyl methacrylate. The specimens are oriented in the monomer in gelatin capsules by directing them into the desired plane among the fibers of a wad of absorbent cotton previously placed in the bottom of the capsule. The cotton is sectioned with the specimen but its fibers do not interfere, and remain outside the tissue.     

Electron Microscopy of Mummified Material

It has proved possible to cut ultrathin sections of mummified material obtained from an American Indian burial (approximate age unknown). Small pieces of tissue were placed for 48 hr in a softening fluid consisting of 96% ethyl alcohol, 30 vol.; 1% aqueous formalin, 50 vol.; 5% aqueous Na₂CO₃, 20 vol. During this period the fluid was changed twice. The tissue was then cut with a razor blade into cubes of 1 mm per side or less, dehydrated in graded ethanols, infiltrated and embedded in methacrylate and the plastic polymerised by placing in the oven at 58°C overnight. The blocks were trimmed to a truncated cone leaving a surface area of 0.5 mm² or less, and cut on a Porter Blum ultramicrotome using a glass or a diamond knife.     

Measuring the resolution of uncompressed plastic sections cut using an oscillating knife ultramicrotome

Thin sections of biological tissue embedded in plastic and cut with an ultramicrotome do not generally display useful details smaller than approximately 50 A in the electron microscope. However, there is evidence that before sectioning the embedded tissue can be substantially better preserved, which suggests that cutting is when major damage and loss of resolution occurs. We show here a striking example of such damage in embedded insect flight muscle fibres. X-ray diffraction of the embedded muscle gave patterns extending to 13A, whereas sections cut from the same block showed only approximately 50 A resolution. A possible source of this damage is the substantial compression that was imposed on sections during cutting. An oscillating knife ultramicrotome eliminates the compression and it seemed possible that sections cut with such a knife would show substantially improved preservation. We used the oscillating knife to cut sections from the embedded muscle and from embedded catalase crystals. Preservation with and without oscillation was assessed in Fourier transforms of micrographs. Sections cut with the knife oscillating did not show improved preservation over those cut without. Thus compression during cutting does not appear to be the major source of damage in plastic sections, and leaves unexplained the 50 A versus 13A discrepancy between block and section preservation. The results nevertheless suggest that improvements in ultramicrotomy will be important for bringing thin-sectioning and tomography of plastic-embedded cells and tissues to the point where macromolecule shapes can be resolved.     

Oriented Embedding of Biological Materials and Accurate Localization for Ultrathin Sectioning

Gelatin capsules with rounded ends clipped off and open ends moistened, affixed to a glass slide and sealed with a 15% gelatin solution are used to embed blocks of tissue in plastic. The surface of the slide serves as an orientation plane for structures of the tissue. The plane end of capsules of polymerized plastic containing no tissue is used in embedding frozen tissue sections. The plastic-infiltrated section is flattened against the capsule end under the weight of a 3/4 inch square of plate glass so that larger sections may be cut and surveyed. Embedding cultured cell monolayers grown on coverslips is accomplished in a comparable manner, but the square of plate glass is not needed as a weight. Block-face localization methods depend on the type of material embedded. With blocks of tissue it is achieved by moistening the face with xylene to develop relief. Thin tissue sections are examined by transmitted light, while cell monolayers are stained on the capsule end with methylene blue.     

Orienting Minute Objects for Sectioning in a Definite Plane

Minute objects can be prepared for sectioning in a definite plane by a method which reembeds them directly on the cutting block under a dissecting microscope. By melting the paraffin immediately around the specimen, the latter can be oriented with reference to the planes of the block. After trimming, the block can be oriented squarely with reference to the microtome knife. Objects as small as 0.2 mm. have been cut successfully. The material sectioned included carpel primordia of Lathyrus, and young embryos, shoot apices and young axillary buds of Pinus. The technic is simpler than most methods previously suggested and it permits good control over the plane of sectioning.     

Aligning Block Faces by Reflected Light for Precise Sectioning

A microscope substage mirror is mounted by means of a channeled lucite block on the base of a Porter-Blum knife holder. The plane face of the mirror, centered on, and about 2 inches below the edge of the knife, reflects light to the front edge of the knife so that the formation of an image of the knife edge on the face of the tissue block results. Alignment of the edge of the knife with the edge of the block to parallelism is accomplished by rotating the block. Alignment of the face of the block to the cutting plane is done by tilting the block until the image of the knife neither approaches the knife nor recedes from it with up and down movement of the block. Ultrathin sections of an area within a 1-2 μ section examined by light microscopy can thus be obtained from a previously established cutting face without loss of material.     

Sections from Double Mesas Obtained at the Same Tissue Plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3° to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6° in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

Sections from double mesas obtained at the same tissue plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3 degrees to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6 degrees in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

Light microscopic identification and photography of Golgi impregnated central nervous system neurons during sectioning for electron microscopy.

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin sections of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which part of the neuron and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Photographic Mapping of a Tissue Surface to Locate Fields for Electron Microscopy; Mouse Lung

Precise sampling from whole lobes of mouse lungs fixed in the inflated state and embedded in epoxy resin can be not only feasible but also efficient. A 1 μm section is cut from an embedded lobe with a rotary microtome and a steel knife. This section is stained and photographed, and from it a 35 × enlarged print is prepared. A grid of transparent plastic scored with 35 mm squares, lettered vertically and numbered horizontally, is superimposed over the photograph. The area chosen for electron microscopy thus becomes identifiable by a letter-number designation obtained from the grid. This area is then located by light microscopy on a 2 mm slice taken from the block from which the 1 μm section was cut, by use of oblique illumination and the calibrated mechanical stage of the light microscope. A block of 1.3 mm diameter is removed for electron microscopy from the tissue by a rotatable circular spring-loaded punch screwed into the objective turret of the microscope. The removed cylinder is mounted on a metal stub and ultrathin sections cut from the faced tissue. The method is as equally suitable for the examination of other tissues, particularly when large areas and multiple sampling may be required.     

An Inexpensive Vibrating Microtome for Sectioning Fixed Tissue

A tissue sectioner which uses a vibrating razor blade and a simple mechanism for the elevation of the tissue can be constructed for less than fifty dollars. The razor blade is fixed to the vibrator of a hair clipper and a nut and bolt serve as the tissue advance mechanism. A metal disc attached to the nut is used for the stage. The tissue advance mechanism is placed inside a machined teflon cylinder which provides a smooth surface upon which the razor assembly is moved. Fixed tissue may be sectioned uniformly at a thickness of 50 μm or more. This device has the advantages of portability, rapidity of sectioning, and inexpensive construction.     

Light Microscopic Identification and Photography of Golgi Impregnated Central Nervous System Neurons During Sectioning for Electron Microscopy

A method is described for a rapid and systematic light microscopic documentation of Golgi impregnated neurons while they are being sectioned for electron microscopy. A drawing under the light microscope of a Golgi impregnated neuron is made first; subsequently thin door of the tissue containing this neuron are cut in the same plane as for light microscopy. During thin sectioning the chuck containing the block is taken out of the ultramicrotome at regular intervals and placed in a special device under a light microscope. The neuron is photographed to record the stage of sectioning. Comparison of the micrographs indicates which put of the and its dendritic tree are contained in the thin sections. No semithin sections are used and therefore no material is lost for reconstruction.     

Film tomography as a tool for three-dimensional image construction and gene expression studies

In order to observe three-dimensional (3D) expression patterns of genes in whole animals, whole organs, or whole tissues, in situ hybridization (ISH) of many sections must be carried out and then used to construct a 3D image. For this purpose, we have developed an automatic microtome to prepare tissue sections with an adhesive film. We used commercially available film suitable for sectioning and ISH. We constructed a microtome and, after adherence of the film to a paraffin-embedded tissue block, cut the block with a blade to prepare sections on film. Then, the sections-on-film were automatically set in a plastic frame that was the same size as a conventional glass slide. With this automatic microtome, tissue sections can be made for ISH or immunohistochemistry in addition to conventional hematoxylin and eosin staining without specific training. We demonstrate that we can construct 3D images of gene expression patterns obtained by ISH on sections prepared with this automatic microtome. We have designated this method as 'Film Tomography (FITO)'.     

A Simple Technique for Osmicating and Flat Embedding Large Tissue Sections for Light and Electron Microscopy

Many investigators now use thin hand-sliced, tissue chopper, or Vibratome sections of fresh tissue in various procedures. In our experience brain and nerve sections varying in thickness from less than 40 to more than 300 μm, with or without prior embedding in agar, have a tendency to roll up or curl during aldehyde fixation and buffer washes. Once osmicated, such curled sections cannot be flattened. When the entire cut face of such thin slices is to be studied, sufficiently flat embedding so that some regions are not completely sectioned before others are even sampled is critical. This report describes fixation and flat embedding procedures, developed for light and electron microscopic autoradiographic studies of plastic embedded brain slices about 200 μm thick (Schwartz 1981), which can be applied to any comparable thin slice of nervous tissue (or potentially of many other tissues) to achieve maximally flat tissue faces. Since osmicated tissue slices are usually too thick to be transilluminated for direct examination with the light microscope, the methods described simplify preparation of the semithin sections required for this purpose.     

A SIMPLE FREEZE-FRACTURE REPLICATION METHOD FOR ELECTRON MICROSCOPY

A simple method to achieve results similar to the freeze-etching technique of Moor et al. (1961) is described. The frozen tissue is cut under liquid nitrogen with a razor blade outside the evaporator rather than inside with a cooled microtome. The conditions of the experiment do not favor sublimation, and it is proposed that the structure of the replica be explained by local faults in the cleavage plane which leaves structures, such as membranes, standing above the ice. Micrographs of replicas of glycerol-protected frozen small intestine of mouse prepared by the method are presented and the structural details they show are discussed. The problem of vapor-deposited contamination is discussed. It is concluded that this is a practical method for obtaining electron micrographs that are relatively free of artifact, and that further improvements may be expected from the use of rapidly frozen fresh tissue and a clean vacuum system, possibly of the ion-pumped type.     

A Rapid Method for Resectioning 0.5-4 μ Epoxy Sections for Electron Microscopy

A simple and rapid method is described for resectioning semithin Epon sections which have been stained for light microscopy, mounted on slides, and examined under immersion oil. The immersion oil is removed with xylene and the section is air dried. A drop of distilled water is applied to the slide and a razor blade is slid under the section. Freed from the slide, the section floats on the surface of the water and is transferred to another drop of water on the surface of a smooth, newly prepared Epon block face. The water under the section is withdrawn with bibulous paper. The section is thoroughly dried and bonded to the block surface by briefly heating in a 60 C oven. The tissue may then be re-sectioned and stained for electron microscopy in the conventional manner. This method has been used by several different technicians to produce ultrathin sections equal in quality to those produced by conventional methods and it greatly facilitates the selection of critical areas for examination by electron microscopy.     

Methods for Precisely Trimming Block Faces for Ultramicrotomy

Two devices are described to aid in trimming block faces of embedded tissue for ultramicrotomy. The first, a reticle to fit the ocular of a stereomicroscope, can be manufactured by the ultramicrotomist and is designed to outline the edges of the block face so that it can be trimmed to a standard size and shape with the area of interest centered in it. The second, a rectangular “trim-align” block mounted in the knife holder of the uitramicrotome, is, with the block face, aligned to the plane of sectioning, and aids in retrimming the top and bottom edges of the block face. This is the simplest trimming device yet described and the first which will, from any sort of embedded material, produce a block face with parallel top and bottom edges even if the block face is not perpendicular to the axis of the specimen holder. If the edge of the diamond knife used for sectioning is parallel to the axis of rotation of the knife holder, the block face has also been automatically aligned to the knife as a consequence of this aligning and trimming procedure. As a result, sectioning can begin immediately without further adjustments.