4. Embedding
4.1. Introductory Remarks
The earliest attempts at ultrathin sectioning failed principally because
of an inadequate embedding medium. Pease and Baker (1948) achieved
a measure of success because they combined the tough properties of the
plastic "Parlodion" with the hardness that could be obtained with a
carnauba-paraffin wax mixture. Material "double-embedded" in this
way sections well. It seemed necessary at the time to extract the wax
component with an organic solvent after sectioning in order to gain the
contrast between specimen and background necessary for satisfactory
micrographs. Unfortunately this reduced the support of the specimen to
such an extent that surface tension forces came into play and destroyed
much of the fine structure present. It has since come to be recognized
that one must never chemically extract the embedding medium.
Newman et al. (1949) introduced butylmethacrylate embedding which
quickly became the standard method. From the point of view of sectionting, this was a fortunate choice for even today we know of nothing that
cuts better. It also impregnates tissue easily and reliably.
One of the chief characteristics of methacrylate is that it partially
volatilizes in the electron beam, which thus removes "background"
material, and greatly enhances specimen contrast (Fig. 9). Therefore,
ordinarily there is no need for any electron "stain" when tissue fixed in
osmium tetroxide is examined (Fig. 7), whereas stains become essential
when non-volatilizing plastics are employed. Much highly important
electron microscopy was completed between 1949 and 1958, before Watson developed the first really effective general purpose stain—lead
hydroxide (Chapter 7.3). This would have been impossible without taking
advantage of the properties of methacrylate as an embedment. Yet it is
now realized that one must pay for this gain in contrast by accepting an
artifact that occurs at macromolecular levels. Apparently once again
surface tension forces are the villains. As methacrylate is heated by the
electron beam and decomposes, liquid interfaces are formed transiently,
and fine order detail in the specimen may collapse.
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4.1. Introductory Remarks
The earliest attempts at ultrathin sectioning failed principally because
of an inadequate embedding medium. Pease and Baker (1948) achieved
a measure of success because they combined the tough properties of the
plastic "Parlodion" with the hardness that could be obtained with a
carnauba-paraffin wax mixture. Material "double-embedded" in this
way sections well. It seemed necessary at the time to extract the wax
component with an organic solvent after sectioning in order to gain the
contrast between specimen and background necessary for satisfactory
micrographs. Unfortunately this reduced the support of the specimen to
such an extent that surface tension forces came into play and destroyed
much of the fine structure present. It has since come to be recognized
that one must never chemically extract the embedding medium.
Newman et al. (1949) introduced butylmethacrylate embedding which
quickly became the standard method. From the point of view of sectionting, this was a fortunate choice for even today we know of nothing that
cuts better. It also impregnates tissue easily and reliably.
One of the chief characteristics of methacrylate is that it partially
volatilizes in the electron beam, which thus removes "background"
material, and greatly enhances specimen contrast (Fig. 9). Therefore,
ordinarily there is no need for any electron "stain" when tissue fixed in
osmium tetroxide is examined (Fig. 7), whereas stains become essential
when non-volatilizing plastics are employed. Much highly important
electron microscopy was completed between 1949 and 1958, before Watson developed the first really effective general purpose stain—lead
hydroxide (Chapter 7.3). This would have been impossible without taking
advantage of the properties of methacrylate as an embedment. Yet it is
now realized that one must pay for this gain in contrast by accepting an
artifact that occurs at macromolecular levels. Apparently once again
surface tension forces are the villains. As methacrylate is heated by the
electron beam and decomposes, liquid interfaces are formed transiently,
and fine order detail in the specimen may collapse.
82