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.     

An Illuminator for Ultramicrotome Knife Orientation and Block Approach

The construction and operation of a simple, inexpensive illuminator that produces high quality illumination of the ultramicrotome knife edge and the edge to block face gap resembling dark field is described. Use of the illuminator greatly speeds knife adjustment and reduces the likelihood of specimen or knife edge damage. The illuminator uses a grain-of-wheat light bulb and an adjustable bulb holder fashioned from bent paper clips. The holder permits both lateral and axial adjustment of the bulb position, which is necessary to achieve satisfactory illumination with different specimens and knives. The illuminator, with slight modification, can be adapted for use on any ultramicrotome.     

Simple and Rapid Block Face Alignment Methods for the Ultramicrotome

Light from the fluorescent lamp on an ultramicrotome can be reflected from a mirror located beneath the knife face 50 that the knife and edge can he imaged on the block face. It is well known that this image can be used to accurately align the block face to the knife edge and cutting direction. A method is described of pre-aligning the lamp, stereomicroscope, knife, and the mirror, which is fixed with respect to the knife face, 80 that a bright reflection of the knife face on the block face is obtained only when the block face is brought close to alignment. This initial alignment is an extremely rapid procedure, and is followed by slower, more accurate manipulation of the block and knife for precise alignment.

The mirror, easily mounted to a Porter-Blum MT-2 ultramicrotome knife holder, is very simple in design and readily adaptable to any ultramicrotome. Methods to permit small movements of the block for the MT-1 and MT-P ultramicrotomes are also descrihed.     

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.     

A Ralph Knife Holder Assembly for Use with the Sorvall “Porter-Blum” MT-1 Ultramicrotome

The superiority of plastic embedding for the production of high quality sections for light microscopy is well known, but the use of conventional glass knives with a cutting edge of approximately 4 mm has severely restricted the size of specimens in the past. Ralph knives provide a much longer cutting edge and adapters are available for certain models of microtomes and ultramicrotomes. A modified knife holder for use with the Sorvall “Porter Blum” MT-2 microtome was described by Gorycki and Sohm (1979); however, this is not suitable for the MT-1 model. We have therefore designed and made an adapter which enables Ralph knives to be used with this instrument. The design allows approximately 18 mm of cutting edge to be used on each knife, allowing larger specimens to be sectioned than with a conventional glass knife and reducing the frequency with which the knife needs to be changed when working with smaller blocks.     

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)     

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. Areas of interest can be accurately located and isolated on the block face, using only a hand-held razor blade, so that oriented ultrathin sections of important regions can be routinely cut and examined in the electron microscope.     

An Inexpensive Trimmer for Paraffin Blocks

One of the minor difficulties in cutting serial sections with the rotary microtome is the accurate trimming of the block of paraffin so that the upper and lower edges facing the knife are parallel to each other and to the knife edge; this is necessary to ensure a straight ribbon. Several block trimmers have been described,¹ but they are all rather complicated and expensive to make. The device described below can be made in a short time at little or no expense in any laboratory.     

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.     

Cover Picture

Imaging biological tissue in 3D with a serial block‐face electron microscope. In an automated acquisition cycle, a thin layer of sample surface is removed with a diamond knife and imaged at high resolution with the microscope's electron beam.     

Trimming Selected Areas of Embedments for Electron Microscopy—Dead Simple

Precision trimming of specific areas of plastic embedments can be accomplished without having to find the specimen detail in the block face itself. The desired area is located in a thick section of the pretrimmed block. The position of the area in the section is evaluated using an ocular micrometer. The block is then mechanically trimmed in the ultramicrotome in such a way that the amount of plastic removed from each of its sides is determined from the magnitude of the knife advance. The procedure can be used in connection with inclined blocks if the microtome head can be rotated behind the specimen orientation arc.     

Some observations on glass-knife making

The yield of usable knife edge per knife (for thin sectioning) was markedly increased when glass knives were made at an included angle of 55 degrees rather than the customary 45 degrees. A large number of measurements of edge check marks made with a routine light scattering method as well as observations made on a smaller number of test sections with the electron microscope indicated the superiority of 55 degrees knives. Knives were made with both taped pliers and an LKB Knifemaker. Knives were graded by methods easily applied in any biological electron microscope laboratory. Depending on the mode of fracture, the yield of knives having more than 33% of their edges free of check marks was 30 to 100 times greater at 55 degrees than 45 degrees.     

Improved Methods for Molding, Facing and Sectioning Glycol Methacrylate Embedded Tissue Blocks

Improvements in glycol methacrylate embedding, block facing, trimming, and sectioning are described. The improvements are derived from a novel molding system, a multipurpose instrument for rapid block facing, trimming and examination, and a device for removing unwanted sections from the microtome knife while sectioning is in progress. Together, these methods facilitate specimen preparation and result in a significant reduction of the time required to prepare high resolution, very thin sections for light microscopy.     

Polyester Resin Embedding for Sectioning of Hard and Soft Tissues

Soft and calcareous tissues embedded in polyester resin may be cut on a sledge microtome to produce thin sections of 3-4 β thickness. Fixed tissues, dehydrated in ethyl alcohol, cleared in methyl benzoate and chloroform, are taken into a wide-necked bottle containing equal parts of polyester resin and chloroform with 0.75% catalyst. The bottle kept in water bath at 37°C is connected to a vacuum pump. With the evaporation of the chloroform under reduced pressure (approximately 10 mm Hg) infiltration is complete. Tissues transferred into a blocking form containing pure polyester resin with 1.5% catalyst are polymerized at 37° C until blocks are firm (48 hr or more). Blocks are prepared with at least 5 mm margin of plastic surrounding the tissue. The edge of the block adjacent to the knife is then filed at an angle of 45° to the cutting movement. Sections are cut with a wide-backed biplanar knife having a cutting edge of 40-44° positioned at an angle of 30° to the plastic block. As the resin is permeable to most stains, staining is carried out through the plastic Sections carried through staining procedures in wire baskets are floated onto slides and mounted in polystyrene; the cover-glass is compressed with a spring-clamp. Microscopic examination shows no staining of plastic, minimal shrinkage and good cellular detail.     

A Remodeled Tissue Flattener for Use with the International Microtome Cryostat

The antiroll plate is cut from a standard microscope slide, a 2 cm length, to give a 2 × 2.5 cm piece. This is fitted into inside grooves of a movable metal frame which is held by a hinge joint parallel to the back of the microtome knife. A stationary frame, which supports the hinged member, has spring clips welded to its sides for attachment to the knife. Clearance between the antiroll plate and knife is obtained by applying Scotch tape to the edge of the plate that is adjacent to the knife edge. The hinge permits the plate to be swung back and thus clear the knife surface.     

Electron tomographic analysis of frozen-hydrated tissue sections

Electron tomography of frozen-hydrated tissue sections enables analysis of the 3-D structure of cell organelles in situ and in a near-native state. In this study, 160-200-nm-thick sections were cut from high-pressure frozen rat liver, and improved methods were used for handling and mounting the sections. Automated data collection facilitated tilt-series recording at low electron dose (approximately 4000 e(-)/nm(2) at 400 keV). Higher doses (up to 10,000 e(-)/nm(2)) were found to increase contrast and smooth out surface defects, but caused section distortion and movement, with likely loss of high-resolution information. Tomographic reconstruction showed that knife marks were 10-40 nm deep and located on the "knife face" of the section, while crevices were 20-50 nm deep and found on the "block face." The interior of the section was normally free of defects, except for compression, and contained useful structural information. For example, the topology of mitochondrial membranes in tissue was found to be very similar to that in frozen-hydrated whole mounts of isolated mitochondria. In rare cases, a 15-nm banding pattern perpendicular to the cutting direction was observed in the interior of the section, most evident in the uniformly dense, protein-rich material of the mitochondrial matrix.     

Herstellung und Verwendungsmöglichkeiten semidünner Kryostatschnitte unfixierten Gewebes

Zusammenfassung Es wird ein einfacher Glasmesserhalter beschrieben, der anstelle des üblichen Mikrotommessers in wohl jedes Kryostatmikrotom eingebaut werden kann. Zum Schneiden semidünner Schnitte wird die Glasmesser-Schneide nach unten gerichtet und der Gewebsblock gegen die Messerschneide angehoben. Dadurch wird verhindert, daß sich die semidünnen Schnitte aufrollen und am Glasmesser kleben bleiben; denn durch das Eigengewicht der Schnitte hängen sie von der Messerschneide und strecken sich. Bänder von 6–8 Schnitten können ohne Schwierigkeiten hergestellt werden. Das Zurechtschneiden der Gewebsblöcke auf Messerbreite ist überflüssig.Die Kryostattemperatur während des Schneidens sollte um –30°C liegen. Diese Temperatur reicht für die Erhaltung der Gewebsstruktur sowie die Lokalisation von Enzymen und wasserlöslichen Verbindungen völlig aus. Der relative Verlust von Enzym aus semidünnen Schnitten an das Inkubationsmedium scheint nicht größer zu sein als beim Kryostatschnitt normaler Dicke. Auch das Anschneiden von Zellorganellen, z.B. Lysosomen, scheint nicht not-wendigerweise zur Enzym Verlagerung aus diesen Organellen zu führen. So ist die Verwendung semidünner Kryostatschnitte nichtfixierten Gewebes in der Histochemie uneingeschränkt möglich.

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Semithin cryostat sections of nonfixed tissue

Summary A knife-holder for glass knifes is described that instead of the ordinary knife can be fastened on the microtomes of all cryostat models. Sectioning happens with the glass knife edge showing down and the tissue block moving upwards against it. By this semithin sections are prevented from curling and adhering to the knife because due to their weight they tend to hang down from the knife edge and need not be flattened. Ribbons of 6–8 sections may be cut without any difficulty; trimming of the tissue block to knife thickness is not necessary.During cutting the cryostat temperature should be about –30°C. This temperature is fully sufficient for the preservation of tissue structure and the localization of enzymes as well as of water soluble compounds. The relative loss of enzyme into the incubation medium seems not to exceed that from cryostat sections of normal thickness. The sectioning of cell organelles, e. g. lysosomes, too, seems not necessarily lead to enzyme diffusion from these organelles. So there are no restrictions for the use of semithin sections of nonfixed tissue in histochemistry.

     

A Method for Making Ribbons of Semithin Plastic Sections

Epoxy resin sections form strong, heat resistant ribbons if, prior to sectioning, contact cement has been painted onto the leading and trailing faces of the block. The forming ribbon floats onto a drop of water held in place by a wax line drawn across the back of the glass knife parallel to the cutting edge. A long trough made from stainless steel tubing is inserted horizontally into the drop, and as the ribbon lengthens it is directed into the trough. The ribbon can be carried in the trough to a hot plate for expansion and then poured onto a slide for mounting. The serial ribbons obtainable by this simple procedure greatly facilitate three dimensional reconstruction of fine tissue structures.'     

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.