2. Tissue Exposure
2.1. Introductory Remarks
Every piece of tissue that is finally examined in the electron microscope
represents a substantial investment of time and nervous energy. One
cannot afford to waste this investment on haphazardly preserved material.
In the long run any amount of effort that may be expected to improve
preservation must be regarded as an economy.
There are two aspects to quality tissue preservation. First, and obviously,
is the fixative itself. Second, and often unappreciated, there is the problem of getting it to the tissue.
It can be said at the present time that we have two excellent general
purpose fixatives for electron microscopy. One of these is suitably buffered
osmium tetroxide which has been in common use since the earliest days
of electron microscopy. However, it requires considerable technical
knowledge and skill on the part of the investigator to use this most
effectively as a primary fixative in a particular situation. Even the most
experienced investigators expect difficulties as they approach some new
tissue or organ. Within the past few years, it has slowly come to be
realized that certain aldehydes also are excellent fixatives if the tissue
subsequently is dehydrated and embedded properly. Additionally, they
can be particularly useful if they are employed as primary fixatives before
secondary fixation with osmium tetroxide (Chapter 3.9). As primary
fixatives,
aldehydes have the virtue of rapidly and easily penetrating
tissue.
The problem of obtaining optimal preservation was a difficult one so
long as we were virtually dependent upon osmium tetroxide as a fixative
of choice. For many years it has been realized by conventional microscopists that this fixative penetrates poorly. If one expects to bring substantial concentrations of it in contact with living cells, the latter must
be very near an exposed surface, in most instances within a half a millimeter of a surface. It might be thought that perfusion would solve the
problem by bringing the fixative to the tissue. However, it remains a
very difficult problem to achieve adequate perfusion with osmium tetroxide although Palay and his co-workers (1962) finally have shown that it is
14
2.1. Introductory Remarks
Every piece of tissue that is finally examined in the electron microscope
represents a substantial investment of time and nervous energy. One
cannot afford to waste this investment on haphazardly preserved material.
In the long run any amount of effort that may be expected to improve
preservation must be regarded as an economy.
There are two aspects to quality tissue preservation. First, and obviously,
is the fixative itself. Second, and often unappreciated, there is the problem of getting it to the tissue.
It can be said at the present time that we have two excellent general
purpose fixatives for electron microscopy. One of these is suitably buffered
osmium tetroxide which has been in common use since the earliest days
of electron microscopy. However, it requires considerable technical
knowledge and skill on the part of the investigator to use this most
effectively as a primary fixative in a particular situation. Even the most
experienced investigators expect difficulties as they approach some new
tissue or organ. Within the past few years, it has slowly come to be
realized that certain aldehydes also are excellent fixatives if the tissue
subsequently is dehydrated and embedded properly. Additionally, they
can be particularly useful if they are employed as primary fixatives before
secondary fixation with osmium tetroxide (Chapter 3.9). As primary
fixatives,
aldehydes have the virtue of rapidly and easily penetrating
tissue.
The problem of obtaining optimal preservation was a difficult one so
long as we were virtually dependent upon osmium tetroxide as a fixative
of choice. For many years it has been realized by conventional microscopists that this fixative penetrates poorly. If one expects to bring substantial concentrations of it in contact with living cells, the latter must
be very near an exposed surface, in most instances within a half a millimeter of a surface. It might be thought that perfusion would solve the
problem by bringing the fixative to the tissue. However, it remains a
very difficult problem to achieve adequate perfusion with osmium tetroxide although Palay and his co-workers (1962) finally have shown that it is
14