Adaptation of a sliding microtome for sectioning of material embedded in epoxy resins]

The employment of a sliding microtome of sectioning plastic embedded material with glass knives is described. Using a new knife holder and a modificated device for fixing plastic blocks succeeded in cutting sections 1--10 micron thick of relatively large pieces of tissue.     

Improved Methods for Making and Using Glass Knives

We have observed over time that the right side of a glass knife is the optimal cutting edge for microtomy if the counterpiece (heel opposite the edge) is controlled within 1 mm. The right cutting edge has been considered the “saw toothed” side and has not been used for ultrathin sectioning. We have observed that the right cutting edge is sharper and more durable than the left. Light and scanning electron microscopy were used to observe the cutting edge, and transmission electron microscopy was used to examine semithin and ultrathin sections of animal and plant tissues cut by the right and left sides of the cutting edge. The results indicate that the cutting edge becomes sharper and more durable from left to right. Both the quality and efficiency of ultrathin sectioning is improved by using the right cutting edge.     

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.     

Vitreous Carbon: A New Material for Making Microtome Knives

The quality of sections obtained by microtomy depends to a large extent on the quality and characteristics of the microtome knife itself. Despite the need for improved microtomy techniques, there have been few significant developments since the introduction of glass and diamond knives in the 1950's. The manufacture of microtome knives from vitreous carbon provides new possibilities for developing both improved methods and improved equipment for specimen sectioning. Vitreous carbon has unique physical properties that lend themselves to the generation of precision cutting edges. Such an edge can be obtained either by breaking a piece of vitreous carbon or by using lapidary techniques. The resultant edge seems well adapted to both thick and thin sectioning. The introduction of vitreous carbon as a sectioning tool offers a significant alternative to metal, glass and diamond knives.     

New perspectives for wood anatomical analysis in dendrosciences: The GSL1-microtome

The variability of wood anatomical features in the rings of trees and shrubs is known to be dependent on multifaceted environmental parameters. The ability to determine anatomical variations over longer time periods as decades or centuries is a step forward in dendroecological and dendroclimatological research. In this regard micro sectioning is still one of the basic requirements but sectioning devices (microtomes) designed for wood anatomical purposes are rarely available. We present an affordable heavy duty, but light-weight microtome operated with removable blades. This portable device enables the production of large sections for dendroclimatological reconstructions as well as various types of common sections for wood anatomical studies.     

Improved Paraffin Sectioning with Etched Microtome Knife Edges

Carbon steel microtome knives etched with 0.1 N HNO₃ for 2 min display a very sharp cutting edge. Over a period of 3 yr no damage to the steel has been detected. The effect on paraffin sectioning was observed by comparing acid-treated knives with nonetched but well-sharpened ones. Sections of whole eyes cut with an etched blade showed approximately 15% less compression of the parffin matrix than those sectioned with an untreated knife. Tissues selected from routine autopsy material presented approximately 9% reduction in compression. As a result, excellent ribbons of sections could be cut from 5-7 μ and floated on water at 42—46 C with a minimum of folds or distortion. Etching improved sectioning when knife edges having bevel angles in a range of 31-39° were used, and also when the bevel was decreased to 20°, but the 20° edge gave impractically short service.     

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.     

A Thermoelectrically Cooled Microtome Table and Knife

Thermoelectric cooling units (Frigistor thermoelements) have been used to replace CO₂ gas and solid CO₂ for microtome stage and knife cooling. These units consist of assemblies of series-connected thermoelements, functioning by the Peltier effect. Cooling is controlled by the direct current supply to the units. Current supply is from a double power pack giving 15 amp at 4.8 v for the knife cooling units, and 15 amp at 1 v for the stage. By varying the current flow, the optimum cutting temperature can be obtained and held indefinitely. An 8-couple Frigistor unit replaces the CO₂ stage of a Lipshaw freezing microtome. The stage temperature may be lowered to -36 C in 40-60 sec and at the optimum cutting temperature, 5 μ serial sections of fixed frozen tissue are obtainable. Four 12-couple units are used to cool a 160 mm Jung plane wedge microtome knife fitted to a Reichert sledge microtome, with the stage cooled by one 8-couple unit. The knife temperature can be lowered to -20 C in 5 min; the stage in 1 min. The apparatus has been used to cut a variety of unfixed rat and mouse tissues. The optimum sectioning temperature for such unfixed liver, kidney, spleen, lymph glands, heart, testes, small intestine, pancreas, skin and lung was -20 to -22 C.     

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.'     

Preparing Single or Serial Sections of Calcified Bones and Teeth

An apparatus for cutting single or serial sections of calcified bone and teeth consists of a motor-driven shaft on which is mounted one saw (for single section cutting) or a gang (for serial sectioning at one cutting operation). The plastic-embedded specimen is attached to a cylindrical plastic holder which is in turn mounted on the machine and fed into the saw. Prior to cutting the specimen may be oriented in two planes, as well as rotated, with respect to the cutting edge. Single or serial sections made by means of repeated cuts with a single saw, may be 0.3 mm or more thick as determined by the setting of a micrometer screw. For serially sectioning a tooth or bone specimen at one cutting operation, the thickness of the separators between adjacent saws (0.5 mm or more) determines the section thickness. After sectioning, specimens may be ground and polished, with or without reimbedding in fresh plastic.     

Paraffin Section Thickness—A Direct Method of Measurement

Paraffin section thickness may be directly measured by re-embedding the sections wider consideration, cutting them again at right angles to the original plane of sectioning, and taking direct measurements with a filar micrometer after staining and mounting. Conditions and materials with which relatively un-distorted 3 and 5 μ sections were secured include (a) a hand-honed knife with a 23° facet bevel, set at a clearance angle of 7°, and (b) a hard paraffin (56-58°) embedding medium, preferably with 5% beeswax and 5% bayberry wax added. By taking direct measurements, it was found that bull testis tissue cut at a microtome setting of 10μ averaged 10.82 μ in thickness. Settings of 5 μ and 3 m resulted in sections averaging 5.25 and 3.31 μ in thickness respectively. Stages in sporogenesis of Onoclea sensibilis, Lewitsky fixed, were examined after sectioning at settings of 10, 5, and 3 μ to determine necessity for thin sections. For this material, it was indicated that mitochondrial preparations as thick as 10 μ were worthless, regardless of good fixation and proper staining. Three-micron sections give the best results.     

A New Method for Reducing Static Electricity During Paraffin Sectioning

Static electricity interferes with the production of good ribbons of thin paraffin sections. Sections tend to stick to the knife leading to compression, shredding and paraffin sections. Sections ribbon disintegration. the static electricity that builds up is caused by friction between the knife and the tissue block and by the rubbing together of the operator's clothing and sectioning table (Mattheij and Dignum 1975, Bryan and Hughes 1976).     

A New Method for Reducing Static Electricity During Paraffin Sectioning

Static electricity interferes with the production of good ribbons of thin paraffin sections. Sections tend to stick to the knife leading to compression, shredding and paraffin sections. Sections ribbon disintegration. the static electricity that builds up is caused by friction between the knife and the tissue block and by the rubbing together of the operator's clothing and sectioning table (Mattheij and Dignum 1975, Bryan and Hughes 1976).     

A method of mesa trimming with glass knives for obtaining large series of ultrathin sections

Ultrastructural analysis of tissue based on 3D reconstruction from serial ultrathin sections is one of the most adequate methods in studies of spatial organization of bio-objects. The sample preparation technique for 3D reconstruction includes the two most technically difficult procedures: an obtaining of stable ribbon of serial sections and mounting of this ribbon onto a slot grid coated with a support film. To mount the ribbon, special approaches and technical tools have been proposed and well evaluated. Much attention has also been paid to obtaining a large and stable ribbon, but this attention deals mainly with the choice of epoxy embedding media. The critical condition of obtaining the straight and stable ribbon is the precise parallelism of trailing and leading edges of mesa falling onto the knife cutting edge. The mesa trimming with dry diamond knife for cryoultratomy allows this condition to be maintained. In the present communication, the way of obtaining parallel sides of the mesa has been proposed with the aid of two forms of glass knives.     

Ultra-Thin Sectioning for Electron Microscopy

A technic is described for obtaining thin sections of animal tissue suitable for electron microscopy. Fixation is accomplished by perfusion of the whole animal with neutral formalin or alcohol formalin followed by immersion of pieces to be examined in neutralized osmium tetroxide. The embedding medium is a mixture of equal parts of n-butyl and ethyl methacrylate polymerized by ultra-violet light. Sectioning is done by means of a glass knife on an International ultra-thin sectioning microtome set at 0.1 μ. The sections are floated on warm water to spread, then placed on Formvar-coated grids, dried, and put into toluene to dissolve the plastic. The technic produces routinely usable, thin sections that show a minimum of damage owing to fixation, embedding, and sectioning.     

The diamond knife "semi": a substitute for glass or conventional diamond knives in the ultramicrotomy of thin and semi-thin sections

The diamond knife "semi" for ultramicrotomes was originally designed by its manufacturer (DIATOME S.A.), for cutting semi-thin sections from 0.2 micron to 2.0 micron. Cutting tests of Epon-embedded material (nervous system, myelin sheat) with this knife have shown that the quality of semi-thin sections is equivalent or better than that obtained with a glass knife, and much time could be saved during the microtomy of serial sections. The quality of thin sections (0.07 micron to 0.12 micron) is excellent and comparable to that obtained with a conventional diamond knife. Furthermore, when adjacent sections are cut thin and semi-thin (for immunochemistry or high voltage microscopy), both are excellent in that they are of uniform thickness. In conclusion, this tool has advantages for both light microscopy and transmission electron microscopy.     

Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining

Background

For decades, the Vibratome served as a standard laboratory resource for sectioning fresh and fixed tissues. In skilled hands, high quality and consistent fresh unfixed tissue sections can be produced using a Vibratome but the sectioning procedure is extremely time consuming. In this study, we conducted a systematic comparison between the Vibratome and a new approach to section fresh unfixed tissues using a Compresstome. We used a Vibratome and a Compresstome to cut fresh unfixed lymphoid and genital non-human primate tissues then used in situ tetramer staining to label virus-specific CD8 T cells and immunofluorescent counter-staining to label B and T cells. We compared the Vibratome and Compresstome in five different sectioning parameters: speed of cutting, chilling capability, specimen stabilization, size of section, and section/staining quality.

Results

Overall, the Compresstome and Vibratome both produced high quality sections from unfixed spleen, lymph node, vagina, cervix, and uterus, and subsequent immunofluorescent staining was equivalent. The Compresstome however, offered distinct advantages; producing sections approximately 5 times faster than the Vibratome, cutting tissue sections more easily, and allowing production of larger sections.

Conclusions

A Compresstome can be used to generate fresh unfixed primate lymph node, spleen, vagina, cervix and uterus sections, and is superior to a Vibratome in cutting these fresh tissues.     

Paraffin Compression Due to the Rotary Microtome

The extent of compression of microtome sections has been studied for blocks with tissue and also blocks of clear paraffin. Thick sections are commonly compressed 15% or more, while in sections below 5 or 10 μ, compression may exceed 50%. Compensatory thickening of sections occurs. The degree of compression for various paraffin samples and for various conditions of knife edge, temperature, etc., is compared. Microscopical work, particularly where quantitative data or reconstructions are involved, is often seriously unpaired by unrecognized artifacts of sectioning. The present work indicates the magnitude of such artifacts. Compensation for distortions of sections is not easy because tissues, particularly dense tissues, may compress less than the paraffin matrix. Section corrugation is due to this inequality in compression. Absorption of water in section flattening causes some tissue readjustment, but this varies with different tissues and different fixations.