3. Fixation
3.1. Introductory Remarks
Biological electron microscopy has depended much upon osmium
tetroxide fixatives. It is historically interesting that these were used from
the very first by electron microscopists attempting to work with tissues.
There was, of course, some reason for this since discerning cytologists
long had recognized osmium tetroxide as a superior preservative of
cytological detail, and it was common practice to use its vapors to fix
delicately protozoa and tissue cultures. As electron microscopes became
generally available, after the Second World War, several laboratories
made efforts to grow tissue culture cells in such a flattened manner that
they could be used as whole mounts in an electron microscope [cf. Porter
(1953) for early references]. Osmium tetroxide was quite logically their
fixative of choice.
During this period a few ultrathin sections of tissue were obtained
by somewhat heroic means. Claude and Fullam (1946) published the most
revealing micrographs of sectioned material to that date, and these had
been preserved with osmium tetroxide. Pease and Baker were able to cut
substantial numbers of moderately thin sections in 1948, and followed the
lead of Claude and Fullam in using osmium tetroxide. Before two more
years passed, three different laboratories had completed comparative
studies of osmium tetroxide, and a variety of other more or less common
fixatives. All three laboratories were in common agreement that osmium
tetroxide was clearly the fixative of choice (Dalton et al., 1950; Porter,
1950; Pease and Baker, 1950). Subsequent work has amply confirmed this
decision.
In those early days, of course, it was not clear just what quality might
be obtained in thin sections of preserved material; and, in fact, osmium
tetroxide often seemed to behave quite erratically. It remained for
Palade in 1952 to demonstrate what might be done. He developed a
slightly alkaline, buffered osmium tetroxide solution that gave reasonably consistent results, and which was clearly superior to acidic fixatives
or unbuffered osmium tetroxide (which produces an acidic wave of injury as it penetrates). This is a solution which has since come to be known
34
3.1. Introductory Remarks
Biological electron microscopy has depended much upon osmium
tetroxide fixatives. It is historically interesting that these were used from
the very first by electron microscopists attempting to work with tissues.
There was, of course, some reason for this since discerning cytologists
long had recognized osmium tetroxide as a superior preservative of
cytological detail, and it was common practice to use its vapors to fix
delicately protozoa and tissue cultures. As electron microscopes became
generally available, after the Second World War, several laboratories
made efforts to grow tissue culture cells in such a flattened manner that
they could be used as whole mounts in an electron microscope [cf. Porter
(1953) for early references]. Osmium tetroxide was quite logically their
fixative of choice.
During this period a few ultrathin sections of tissue were obtained
by somewhat heroic means. Claude and Fullam (1946) published the most
revealing micrographs of sectioned material to that date, and these had
been preserved with osmium tetroxide. Pease and Baker were able to cut
substantial numbers of moderately thin sections in 1948, and followed the
lead of Claude and Fullam in using osmium tetroxide. Before two more
years passed, three different laboratories had completed comparative
studies of osmium tetroxide, and a variety of other more or less common
fixatives. All three laboratories were in common agreement that osmium
tetroxide was clearly the fixative of choice (Dalton et al., 1950; Porter,
1950; Pease and Baker, 1950). Subsequent work has amply confirmed this
decision.
In those early days, of course, it was not clear just what quality might
be obtained in thin sections of preserved material; and, in fact, osmium
tetroxide often seemed to behave quite erratically. It remained for
Palade in 1952 to demonstrate what might be done. He developed a
slightly alkaline, buffered osmium tetroxide solution that gave reasonably consistent results, and which was clearly superior to acidic fixatives
or unbuffered osmium tetroxide (which produces an acidic wave of injury as it penetrates). This is a solution which has since come to be known
34