Search

A number of techniques are presented which permit precise selection and efficient preparation of individual cultured cells for electron microscopy. Techniques described include marking of the living and embedded cells, drilling and mounting cores of embedded material, and improved technique for marking of the selected area on the block face before trimming.

Light from the fluorescent lamp on an ultramicrotome can be reflected from a mirror located beneath the knife face so that the knife and edge can be 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, so 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-2 ultramicrotomes are also described.

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.

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)

The organization of the fibers in tissues of laminated fibrous structures constitutes an obstacle to obtaining ultrathin sections for transmission electron microscopic observations routinely. This paper deals with the orientation of the specimen block face in a way that greatly improves sectioning of these tissues.

Sucrose in the tissues of the sugarbeet (Beta vulgaris L.) can be shown as follows. Fresh or stored roots are cut into pieces having a block face of about 1 × 2 cm, and sections of about 150 μ thickness prepared from these. The sections are rinsed 15–30 sec in iced distilled water, placed in Ba(OH)2-saturated methanol for 3 min and then rinsed twice in methanol for 1 and 5 min respectively. They are then transferred to ethanol through a graded series consisting of 80, 60, 40, and 20% methanol in ethanol, 5 min in each, with evacuation as necessary to remove bubbles. Temporary mounting is in ethanol and examination made by incident light or darkfield illumination. Gradual replacement of ethanol with xylene permits mounting in a resinous medium. An opaque granular precipitate of barium saccharate shows the location of the sucrose within the cells.

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.