Ultrathin sectioning

Ultramicrotomy is an obvious example of a preparation method developed by biologists and now in general use by polymer microscopists. In the early 1950s Porter and Blum [48], Sjostrand [49] and Haanstra [43] were constructing microtomes to provide sections, about 0.1 jum thick (or less), for TEM study. Today, ultramicrotomes are available from companies such as LKB and Reichert that permit ultrathin sections of polymers to be obtained on a routine basis. 

Sectioning is one of the most widely used methods in the preparation of polymers for electron microscopy. Microtomy permits the observation of the actual structure in a bulk material which is not possible by methods such as thin film casting or surface replication. Polymers (in common with biological tissues) require care in handling, embedding in resins for support and addition of stains to enhance contrast in the TEM. 

A general method will be described for the preparation and sectioning of polymers, pointing out those areas of difficulty and citing some of the excellent references available. The aim of the preparation is sections that truly represent the original bulk structure and which are thin, flat, not deformed, have contrast and can be used to form images that can be interpreted. Experience is required to obtain such sections of polymer specimens consistently. Fortunately, the tedious fixation and dehydration steps required for bio- 

Staining is often required in order to increase the electron scattering of polymers selectively and, thus, aid contrast and resolution of details. Staining is performed either on small pieces of the material before embedding or by post-staining the sections themselves, or both. This topic is so vast and important to the polymer microscopist that it will be treated separately (Section 4.4). 

There are many embedding media available, almost too many for the novice to choose one over another for a given specimen. Glauert [51] is an excellent reference that includes a description of the various media and the reasons for their use. There are three media in general use epoxy 

Recently, biologists have suggested the use of microwave techniques for more rapid embedding [66-70], and recipes have been developed for a reduction in curing from 48 h to 15 min or less at a power of 700 W in a 2540 MHz microwave oven [67]. 

Glass knives are used to face the block up to the specimen and for final trimming either in the 

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Fig. 6.8 Electron photomicrograph of mouse kidney mitochondria. The structure of both the cytoplasmatic membrane (centre) and the mitochondrial membranes is visible on the ultrathin section. Magnification 70,000x. (By courtesy of J. Ludvik)... 

Goode NP, Shires M, Crellin DM, et al. Post-embedding double-labeling of antigen-retrieved ultrathin sections using a silver enhancement-controlled sequential immunogold (SECSI) technique. J. Histochem. Cytochem. 2004 52 141-144. 

IV. Cutting Sections Ultramicrotomes are designed to cut ultrathin sections, semithin sections and ultrathin frozen sections if suitably... 

Fig. 1. An ultrathin section of an untreated fresh protoplast with 4% uranyl acetate and 0.4% lead citrate. A line profile of gray level (below) was taken along the double-headed arrow through the Golgi cistemae (G) and the endoplasmic reticulum (ER). (From ref. 40.)...

Roth J. The preparation of 3 nm and 15 nm gold particles and their use in labeling multiple antigens on ultrathin sections. Histochem J 1982 14 791-801. 

Apply epoxy ultrathin sections on nickel or gold grids coated with Coat-Quick G Pen (Structure Probe, Inc.) and correspondingly air dry. 

Preparation of biological material for electron microscopy still required fixation, dehydration, and ultrathin sections. Araldite and other resins were used in place of paraffin wax for blocking. At first, specially sharpened steel knives were employed to cut the sections, but from 1950 glass or diamond knives were used which could cut slices 100-200 nm thick. By 1952, Palade and others were obtaining sections... 

Takahashi, H., M. Nagayama, H. Akahori and A. Kitahara. 1973. Electron-microscopy of porous anodic films on aluminum by ultrathin section technique. Part 1. The structural change of the film during the current recovery. J. Electron Microscopy 22(2) 149-57. 

Lesion in bovine dentin with tubules protruding from degraded intertubular matrix (left degraded matrix right intact matrix). Demineralization in 0.1 M acetic acid pH 4.0, with subsequent exposure to bacterial collagenase. Fixed and demineralized with glutar-dialdehyde-acetic acid, post-fixed with osmium tetroxide ultrathin sections stained with uranyl acetate - lead citrate. 

We shall first examine the microscopic techniques which allowed us to study these transformations and to show the striking analogy between the images obtained by optical microscopy in polarized light and by electron microscopy with ultrathin sections, despite the difference of the absorption mechanisms of light and electrons. Once this analogy was established, we sought to use electron microscopy and electron microdiffraction to learn more about the texture and structure of the anisotropic areas. 

Figure 1. Electron micrograph of an ultrathin section of a dull attrital layer (durain) in a high volatile A bituminous (hvab) coal. V—vitrinitey E—exinite, M—granular micrinite. X 10,500...

Since the work reported by McCartney et al. (9), ultrathin sections of other, more heterogeneous components and mixtures of components of coals of different rank have been prepared and observed. Procedures for minimizing artifacts have been learned and followed, and experience in observation has led to avoiding obvious faults. These sections were often not as large and continuous as those of homogeneous vitrinites, but adequate areas were available for electron microscopy. Observations of these various components revealed ultrafine structures of different size and form. Some of the structures can be correlated with those deduced from other direct or indirect study techniques others are unfamiliar and novel, and suggested interpretations are tentative. 

The general appearance in the electron microscope of an ultrathin section of a heterogeneous area of dull coal (high volatile A bituminous) is illustrated in Figure 1. Components readily identifiable as vitrinite, exinite, and granular micrinite are indicated. Distinguishing the other variously shaded areas is not so positive, but identification can usually be established from shape, density, and association. 

An ultrathin section of a resin inclusion in vitrain from a subbituminous coal is shown in Figure 4. This variety of resin appeared to have a very fine granular structure in the light microscope. The micrograph shows that this consists of nearly spherical particles ranging from 300 to 700 A. in diameter. 

Figure 10. Electron micrograph of an ultrathin section of exinite in hvab coal. X 42,000...

The micrographs presented here illustrate the potentialities of electron microscopy of ultrathin sections in revealing extremely fine details of crystallite,... 

In general, details of cell structure in vitrinite are distinguished only with great difficulty in ultrathin sections in the electron microscope, probably because of insufficient contrast between cell parts. If cell spaces are filled with another component the cellular structure becomes evident. 

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