Glass Knives verses Diamond Knives

In regards to the purchase of a histology diamond knife for preparing light microscopy sections, I was one of those folks, in possession of a nice glass knife maker, who was very comfortable with breaking, making and using glass knives. So the question was—did I really need a costly diamond knife for making light microscopy sections?Once I finally tried a diamond histo knife, I never went back to glass. There are some very good incentives for this: long lasting cutting surface, much time saved in sample preparation, larger block faces can be prepared, and more consistent cutting results obtained. A histo diamond knife is also a big aid in preparing specimens for diagnostic or biological microscopy. These very important bonuses make a good list to have in hand when justifying the purchase to your organization. However, are they worth the price? Yes, I think so—even so far as providing each tech with their own diamond histo knife.

Practical methods for TEM

Multiple sectioning: Two or more core specimens from a kidney needle biopsy can be embedded in single block using an 8-facet silicone rubber embedding mold. Tissue blocks are softened by placing them face down on an 85°C hot plate for one minute and then trimming with a Teflon coated razor blade. This prevents the block face from being deeply fractured which can affect section quality. If glomeruli are found within different cores in a single block, the block face can be retrimmed into multiple mesas and simultaneously thin-sectioned. This method greatly increases the efficiency of both thick and thin sectioning which is extremely helpful when handling more than 1600 cases of renal biopsies in our EM laboratory each year.Knife Spacer: Due to the difference in thickness between a glass and diamond knife, and also depending on which portion of the diamond knife is being used, the knife stage has to be moved laterally over a long distance when changing from one knife type to the other. Placing a L-shaped spacer, 3 to 5 mm in thickness, on the left side of the stage when sectioning a glass knife will greatly reduce this lateral adjustment.

A new method for characterization of domain morphology of polymer blends using RuO4 staining and LVSEM

Ruthenium tetroxide (RuO4) staining for TEM is a well proven technique for the characterization of crystalline polyolefins. Blend morphology has also been studied using RuO4 staining and SEM and low voltage SEM. A new method has been developed which uses RuO4 staining and LVSEM in the characterization of polyolefin blend morphology, specifically blends of polypropylene modified by the addition of elastomers or plastics. This method is often preferred over TEM for the characterization of blends and is applicable to many problems encountered in commercial and industrial laboratories including the analysis of domain morphology in molded parts, extruded films and fibers, failure analysis, and the analysis of layer morphology in certain coextruded films.Three sample preparation steps are required prior to the LVSEM analysis. (1) The sample face is cryogenically sectioned on a glass knife in the cryomicrotome. (2) This face is stained in RuO4 vapors for 2.5 hours. The staining solution is prepared in situ by a modification of the method of Montezinos.

Preparation of successive thin sections of fine oxide particles by an ultramicrotome

A large number of monodispersed fine oxide particles, which are uniform in shape, size and composition, have been developed as ideal advanced materials. Recently, by the newly developed gel-sol method, various shapes of micron-sized monodispersed hematite particles (α-Fe2O3) were produced, such as peanut-type, ellipsoidal and pseudocubic shapes. To understand the growth mechanism of these particles, it is necessary to investigate their internal structures. In this study, in order to analyze three dimensional internal structure of these particles, we have prepared their successive thin sections with an ultramicrotome and carried out HREM study on these successive thin sections.Monodispersed peanut-type and pseudocubic particles were prepared by the standard procedures of the “gel-sol method. For carrying out ultramicrotomy, these particles were embedded with acrylic resin (methyl methacrylate: n-butyl methacrylate=3:7 or 4:6, benzoyl peroxide 1.5-1.7%) in a gelatin capsule. After trimming with a glass knife, successive thin sections of the particles were obtained by a diamond knife with the optimum slicing condition. The slicing speed and thickness were set to be 0.6 mm/sec and 70 nm, respectively.

The discovery of the glass knife

We celebrate 500 years of development of the new world following its discovery by Columbus. We also celebrate 50 years of the Electron Microscope Society of America and the exploration of the new world of electron microscopy. A major part of this exploration was the development of techniques for the preparation and sectioning or biologic materials for electron microscopy, which depended on improvements in fixation, staining, embedding, microtomes, and microtome knives.The story of the glass knife involves serendipity and a Japanese sword. It begins in 1949 when a group of research fellows at the Massachusetts Institute of Technology (MIT) were trying to section tissue for electron microscopy with conventional microtome knives sharpened by hand. One day I had to park my car at the far end of the MIT lot and walk back through several buildings. On my way I passed a metallurgy exhibit which displayed a Samurai sword.

LM and SEM of Epoxy Embedded Lung Sections Containing Hard Particles

Presence of hard particles in tissues destined for morphological analysis precludes TEM because of sectioning difficulties. Frequent occurrence of focal lesions requires observation of tissue areas larger than those imposed by TEM limitations. SEM has no specimen size limitation. Secondary electron (SE) and backscatter electron (BSE) imaging modes allow observation of specimens with thicknesses comparable to those used in LM. Following Humphrey's observations (1979) on SE/SEM imaging of RF/O2-plasma etched epoxy embedded sections, we have developed protocols that allow (1) imaging of 1 cm2 sections containing glass, coal, or other particles; (2) alternate LM and SEM of the same section; (3) cutting thin sections of the same embedment for TEM; (4) LM and SEM imaging and X-ray analysis in any desired sequence on the same section cut with a 2.5 cm wide glass knife on an LKB Ultrotome IV; and (5) an AC oxygen plasma etching mode.