6.10.
"SANDWICHED" SECTIONS
211
(1957) to propose that both surfaces of methacrylate sections be protected
by supporting films.
Watson originally used two carbon films for support. However, for convenience, and for maximum transparency, one of the films can be of Parlodion or Formvar. (Do not, though, evaporate carbon through the grid
mesh to the back side of a section. No carbon reaches the parts of the section protected by grid wires, and this permits different tensions to be set
upon the two sides of the section when it is subsequently examined. The
usefulness of the preparation is apt to be destroyed by numerous ripples
which then may develop.)
Sandwiched sections preserve at least surface detail elegantly (Figs. 24
and 38), and thus somewhat resemble the preservation achieved with
Araldite and Vestopal W embedding. Naturally the second supporting
film reduces contrast significantly, and herein lies the principal difficulty
with the method. Under almost all circumstances it is necessary to use
"stained" material. But this need not present any problems with modern
alkaline lead hydroxide stains [Chapter 7.3, Fig. 28(d)]. It is also essential
that the supporting films be as truly thin as possible. It may prove desirable to use a high accelerating voltage (100 kv) with relatively thick sections to achieve a good contrast differential.
Roth (1959) has suggested recently that a thin methacrylate film be
used as the top layer of the sandwich, for this has the advantage of decomposing somewhat in the electron beam, and thus becomes quite transparent. Yet, apparently, it is adequate to preserve intact the top surface of
the section. He indicates that suitably thin methacrylate films can be
made if 1-2% prepolymerized methacrylate (Chapter 4.8) is dissolved in
methylmethacrylate monomer. A drop of this solution is allowed to
spread in an unimpeded fashion on a clean water surface. Grids prepared
FIG. 24. Methacrylate embedded kidney, mounted sandwiched between two layers of
evaporated carbon to avoid the collapse of structures at exposed surfaces which otherwise occurs as methacrylate decomposes and sublimes in the electron beam, (a) A
transverse section of a glomerular capillary showing the urinary space (ur.), part of an
epithelial cell and its feet (epi.), the basement membrane and fenestrated endothelium
(end.), (b) The apical portion of a cell of a proximal tubule showing the basal portion
of the brush border. This should be compared with Fig. 7 which had a supporting
film on only one side of the section. Although there is a better preservation of detail
in the sandwiched section, much contrast is lost, and electron "stains" are apt to be
necessary. These sections were floated upon a 0.5% solution of phosphotungstic acid
in 50% alcohol before mounting.
"SANDWICHED" SECTIONS
211
(1957) to propose that both surfaces of methacrylate sections be protected
by supporting films.
Watson originally used two carbon films for support. However, for convenience, and for maximum transparency, one of the films can be of Parlodion or Formvar. (Do not, though, evaporate carbon through the grid
mesh to the back side of a section. No carbon reaches the parts of the section protected by grid wires, and this permits different tensions to be set
upon the two sides of the section when it is subsequently examined. The
usefulness of the preparation is apt to be destroyed by numerous ripples
which then may develop.)
Sandwiched sections preserve at least surface detail elegantly (Figs. 24
and 38), and thus somewhat resemble the preservation achieved with
Araldite and Vestopal W embedding. Naturally the second supporting
film reduces contrast significantly, and herein lies the principal difficulty
with the method. Under almost all circumstances it is necessary to use
"stained" material. But this need not present any problems with modern
alkaline lead hydroxide stains [Chapter 7.3, Fig. 28(d)]. It is also essential
that the supporting films be as truly thin as possible. It may prove desirable to use a high accelerating voltage (100 kv) with relatively thick sections to achieve a good contrast differential.
Roth (1959) has suggested recently that a thin methacrylate film be
used as the top layer of the sandwich, for this has the advantage of decomposing somewhat in the electron beam, and thus becomes quite transparent. Yet, apparently, it is adequate to preserve intact the top surface of
the section. He indicates that suitably thin methacrylate films can be
made if 1-2% prepolymerized methacrylate (Chapter 4.8) is dissolved in
methylmethacrylate monomer. A drop of this solution is allowed to
spread in an unimpeded fashion on a clean water surface. Grids prepared
FIG. 24. Methacrylate embedded kidney, mounted sandwiched between two layers of
evaporated carbon to avoid the collapse of structures at exposed surfaces which otherwise occurs as methacrylate decomposes and sublimes in the electron beam, (a) A
transverse section of a glomerular capillary showing the urinary space (ur.), part of an
epithelial cell and its feet (epi.), the basement membrane and fenestrated endothelium
(end.), (b) The apical portion of a cell of a proximal tubule showing the basal portion
of the brush border. This should be compared with Fig. 7 which had a supporting
film on only one side of the section. Although there is a better preservation of detail
in the sandwiched section, much contrast is lost, and electron "stains" are apt to be
necessary. These sections were floated upon a 0.5% solution of phosphotungstic acid
in 50% alcohol before mounting.