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.     

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.     

Direct sectioning of unembedded cartilage: a simple method for microscopical and histochemical studies on chondrocytes and extracellular matrix

On account of the rigidity and compact structure of the hyaline cartilage, unfixed or formaldehyde fixed samples of this tissue can be directly sectioned by using a conventional ultramicrotome and a glass knife. This simple method allows to obtain microscopical sections from unembedded cartilage blocks, which show a well preserved histological structure and are very suitable to carry out morphological and histochemical studies on chondrocytes and cartilaginous matrix.     

Monitoring of Epoxy Block Trimming by the Use of a Laterally Mounted Mirror

Mounting a small mirror on the side of the knife holder of an ultramicrotome permits the monitoring of selected areas during epoxy block trimming without removing the block from the chuck. This technique is particularly useful for trimming toward preselected areas of in situ embedded cell cultures for subsequent sectioning. When the visibility of the embedded cells is improved by staining with Paragon-1301 or toluidine blue before embedment, such mirror monitoring can be carried out at magnifications up to 40 times during the trimming procedure.     

The ultramicrotome superstage: a versatile aid for section manipulation

The design and properties of a rigid, box-like device to be placed on the knife stage of a Porter-Blum MT-2 ultramicrotome are described. This "superstage" acts as a hand and tool rest, and a wind screen for the knife boat. The superstage easily permits tight rafts of ultrathin sections to be centrally located on grids. Additionally, it aids in placing 1 micron thick sections at the same relative location on glass slides to facilitate their examination in the light microscope.     

The Ultramicrotome Superstage: A Versatile Aid for Section Manipulation

The design and properties of a rigid, box-like device to be placed on the knife stage of a Porter-Blum MT-2 ultramicrotome are described. This “superstage” acts as a hand and tool rest, and a wind screen for the knife boat. The superstage easily permits tight rafts of ultrathin sections to be centrally located on grids. Additionally, it aids in placing 1 μm thick sections at the same relative location on glass slides to facilitate their examination in the light microscope.     

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.     

A simple technique for supporting single cells in electron microscopic immunocytochemistry and embedding.

Cell suspensions of rat anterior pituitaries were filtered with a polycarbonate filter (pore size 3 micron) and fixed on the filter. After fixation the cells were adherent to the filter and immunocytochemical staining could be accomplished by simply dipping the filter into the different incubation media. The cells could be dehydrated and embedded in Epon 812 on the filter. After polymerization the embedded filter was sawn into small blocks and the cell layer was sectioned tangentially on an ultramicrotome. This method also seems to be applicable to other histochemical studies on single cells.     

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.     

辣根过氧化物酶标记与样品后染色联用在微观脑联结组学中的应用

三维电子显微成像(volume electron microscopy)是微观尺度脑联结组学(micro-brain connectomics)研究的主要研究手段.自动卷带式连续超薄切片-收片系统(automated tape-collecting ultramicrotome,ATUM)及扫描电镜序列成像系统的联合应用,是三维电镜成像中一种重要的技术手段.该方法利用卷带式收集超薄切片,形成完整的样品切片库,进而在后续成像中形成样品三维电镜成像数据库.根据ATUM系统镜外切片-收片、保留样本切片库等特点,对相应的生物样品制备方法进行改进,包括树脂配方及树脂渗透方式改良,神经元细胞膜辣根过氧化物酶(horseradish peroxidase,HRP)稀疏化标记与样本切片库后染色联用——从而很大程度上减少样品切片的褶皱率,并使得在整体高衬度的样品图像库中高效地识别神经元身份成为可能,最终获得符合脑联结组学所需的高质量成像数据.     

A simple technique for supporting single cells in electron microscopic immunocytochemistry and embedding

Summary Cell suspensions of rat anterior pituitaries were filtered with a polycarbonate filter (pore size 3 m) and fixed on the filter. After fixation the cells were adherent to the filter and immunocytochemical staining could be accomplished by simply dipping the filter into the different incubation media. The cells could be dehydrated and embedded in Epon 812 on the filter. After polymerization the embedded filter was sawn into small blocks and the cell layer was sectioned tangentially on an ultramicrotome. This method also seems to be applicable to other histochemical studies on single cells.Supported by Deutsche Forschungsgemeinschaft, SFB 87/B2     

Sandwich Embedding of Monolayers of Cells in Epoxy Resin for Ultrathin Sectioning

In this procedure for embedding monolayers of cells, the usual glass slides are replaced by plates of resin 1-1.5 mm thick. Unlike the open-face embedding technique, the present procedure uses only a few drops of unpolymerized resin, which are applied to the fixed and dehydrated cells. During polymerization this small amount of liquid resin spreads across a relative large area, leaves the cells covered by a very thin layer, and permits phase contrast observations through it. Ultrathin sections of a particular cell encircled by a rotary scriber can be obtained by sectioning the resin slide, which has been trimmed and mounted directly in the specimen holder of the ultramicrotome.     

Development and new design of a semiautomatic cryo-ultramicrotome for histopathological transmission electron microscopy on large ultrathin cryosections: Description of a prototype

In a previous publication, we described new devices and results for diagnostic histopathological transmission electron microscopy on large, ultrathin cryosections from several human tumours. As reported, cryo-ultramicrotomy is gaining in significance and use as technical improvements are made. In comparison with epoxy resin procedures, cryo-preparations are more rapid, there is negligible loss of material and thus antigenicity is well preserved. This work contains some newly developed details for a new type of cryo-ultramicrotome, especially adapted for histopathological investigations on large ultrathin cryosections. It is a software-controlled ultramicrotome advance drive system with feedback regulation and various advancement sensors. Some of these developments could also be applied to existing microtome systems.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.     

KINETICS OF NUCLEOSIDE INCORPORATION INTO NUCLEAR AND CYTOPLASMIC RNA

The difficulties in sectioning frozen biological objects for electron microscopic investigations are overcome by Steere's freezing-etching method. In order to test this method and to open up a wide field of application, the new freezing-ultramicrotome has been designed. The apparatus consists of the combination of an ultramicrotome with freezing-drying and shadow-casting installations in the same vacuum container. The preliminary results show, on the one hand, the practicability of all preparational steps and, on the other, that it is possible to resolve internal structures of cell organelles and even macromolecular patterns.     

Electron Microscope Observations on the Behavior of the Bacterial Cytoplasmic Membrane During Cellular Division

Bacterial cells were fixed in OsO₄, washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on a Porter-Blum ultramicrotome and were examined in an RCA electron microscope, type EMU-2D. The sections revealed that the cytoplasmic membrane undergoes a centripetal growth to form a membrane septum. This septum is formed as a double structure. Constriction of the daughter cells and deposition of cell wall material lead to the separation of the daughter cells. The bacterial cytoplasm appears to consist largely of 200 A granules and occasionally reveals arrays of parallel dense lines.     

Plastic sectioning for light microscopy of Pyrenomycetes.

In preparation for light microscopy, ascocarps of Sordaria fimicola Ces. & DeNot. were embedded in Spurr's medium and sectioned at 1-1.5 mum on an ultramicrotome. Sections were floated on Giemsa staining solution at 60 C for 10-30 min, washed in distilled water, affixed to slides by drying, and mounted in immersion oil. Best preservation of the delicate sterile tissues of the centrum was obtained by fixation in 1% KMnO4 for 2.5-3 hr, followed by the Giemsa stain. This method is suggested for future studies on the morphology of perithecial ascomycetes.     

Inner-ear structure in Morganucodon, an early Jurassic mammal

The shapes and dimensions of the cochlear cavities from four petrosals of the genus Morganucoden obtained through sectioning and reconstruction. Morganucodon dates from the early Jurassic and represents many of the earliest known mammal specimens. Each Morganucodon petrosal fossil was embedded in Araldite and shaved with an ultramicrotome to expose the internal structure. Line drawings of the exposed cross-sections were digitized and used to produce three-dimensional reconstructions. The reconstructed Monganucodon cochlear cavities differ from extant mammalian cochleas in several respects: they are uncoiled, shorter in length, and lack the bony lamina which supports the basilar membrane. These three features ar characteristic of extant Aves and Reptilia.     

On some improvements in the preparation of high-resolution radioautographs by the flat substrate method

Summary The flat substrate method of preparing high-resolution radioautographs is generally preferred for quantitative studies. Some experience with this method is reported. A glass slide holding device for the ultramicrotome is described. With it the transfer of sections to the easily damaged collodion-membrane is possible under microscopical control.     

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.