Experiments on fixation for electron microscopy 2. Alkalized

16 Pages·2006·4.32 MB·English

28?
Experiments on fixation for electron microscopy
2. Alkalized osmium tetroxide
By S. K. MALHOTRA
(From the Cytological Laboratory, Department of Zoology, University
Museum, Oxford)
With 6 plates (figs. I to 6)
Summary
The effect of fixation with osmium tetroxide solution, made alkaline by the addition
of potassium acetate, was studied by electron microscopy. Exocrine cells of the pan-
creas and the cells of the first ('proximal') convoluted tubules of the kidney of the
mouse were used as test-objects. Partially prepolymerized methacrylate was used
for embedding. The preservation of the various cell inclusions was similar to what is
generally produced after fixation with Palade's buffered osmium tetroxide.
Introduction
OSMIUM tetroxide has come to take a predominant place as a fixative for
studies of biological materials by electron microscopy. It is considered as the
most suitable substance for preservation of the whole of the cellular consti-
tuents. Potassium permanganate, which is nowadays rather widely used for
studies of membranous structures (Robertson, 1959, i960; Mollenhauer,
1959; Zebrun and Mollenhauer, i960), removes ribonucleic acid and histones
from the tissue (Luft, 1956; Bradbury and Meek, i960). Osmium tetroxide is
mostly used in buffered solutions, because it has been suggested that the
quality of fixation by osmium tetroxide is largely dependent on the concen-
tration of hydrogen ions. It is believed that when tissues are fixed in an un-
buffered solution of osmium tetroxide, a wave of acidity precedes the osmium
into the tissue, and that the structure of cells is best stabilized if the fixing
solution is buffered slightly on the alkaline side of neutrality (Palade, 1952a).
Palade recommended the use of Michaelis's (1931) sodium veronal/ sodium
acetate buffer (without sodium chloride) for this purpose, probably because it
is not harmful to most cells. Palade experimented with phosphate buffer at
the same pH as the veronal/acetate buffer, but found it less satisfactory.
Dalton (1955) used a chromate buffer (K Cr 0 /K0H) in osmium tetroxide
2 2 7
solution for fixation at pH 7-2. He considered that his fixing solution had
some advantages over Palade's fluid.
However, there is some evidence that the quality of preservation is not
necessarily determined by the concentration of the hydrogen ions in fixing
solutions (Malhotra, 1962). Sjostrand and his associates (see Sjostrand, 1956)
have obtained comparable preservation of a wide variety of animal cells by
[Quart. J. micr. Sci., Vol. 103, pt. 3, pp. 287-96, 1962.]
2421.3 X
288 Malhotra—Experiments on fixation for electron microscopy
using unbuffered osmium tetroxide made isotonic by the addition of in-
different salts. They regard the tonicity of the fixing fluids as of vital impor-
tance for ideal preservation of cell structures. It may be remarked that
Palade's fluid is greatly hypotonic to the body-fluids of mammals.
I have elsewhere stated (1962) that I am studying the effect on fixation of
the osmotic pressure due to those non-fixative ions that are commonly added
to fixing solutions for electron microscopy; and it is intended to complete
this work in a series of papers. In the first paper of this series I have described
the results of fixation with a simple solution of osmium tetroxide in distilled
water. In the present paper, the effect on fixation by an alkalized solution of
osmium tetroxide is described.
Material and method
The exocrine cell of the pancreas and the cells of the first ('proximal')
convoluted tubule of the kidney of the mouse have been used as test-objects
for this series of experiments on fixation. The reasons for the selection of these
cells have been given in the first paper of the series.
A 1% solution of osmium tetroxide in 'deionized' water (see Malhotra,
1962), made alkaline by addition of potassium acetate (pH 7-3 to 7*5), was used
as fixative. Potassium acetate is hygroscopic, and therefore a small amount
from the stock supplied by the manufacturer was dried for several days over
phosphorus pentoxide in a vacuum desiccator before use. The pH of a 0-22%
solution of the dried salt varies between 7-3 and 7-5, probably because of
impurities in the salt; 'Analar' grade salt is not available. A stock solution of
the salt was prepared at 0-44% and mixed with an equal volume of a 2%
solution of osmium tetroxide. The mixture was used for fixation. The de-
pression of the freezing-point of this fixing solution, as determined by the use
of a Beckman thermometer, varies between 0-143° and 0-148° C. The depres-
sion of the freezing-point due to osmium tetroxide alone in this fixing solu-
tion varies between 0-055° an(i 0-06° C.
The material was also fixed in a 1 % solution of osmium tetroxide that had
been made alkaline by the addition of potassium acetate at pH 7-2 to 7-3, but
at lower concentration than 0-22%. This was done by considerably diluting
the stock solution of potassium acetate and noting its pH. The diluted salt
solution was mixed with an equal volume of 2% osmium tetroxide. The
depression of the freezing-point of this solution was 0-08° C.
The tissue was fixed, dehydrated, and embedded according to the pro-
cedure standardized for fixation with the unbuffered solution of osmium
tetroxide, so that the results could be closely compared. The tissue was
FIG. 1 (plate). Micrograph showing the general appearance of the exocrine cells of the
pancreas fixed by osmium tetroxide (alkalized with higher concentration of potassium acetate
at pH 73 to 7"S)- Parts of three cells appear in the micrograph ,cm, limiting cell membrane;
er, endoplasmic reticulum; v, vacuoles found in association with the non-granular membranes
(y-cytomembranes); m, mitochondria; n, nucleus; z, zymogen granules.
FIG. I
S. K. MALHOTRA
FlC. 2
S. K. MAI,HOTRA
Malhotra—Experiments on fixation for electron microscopy 289
fixed for a short time (less than an hour) in a solution previously cooled to just
above freezing-point. It was then dehydrated through a graded series of
ethanols and embedded in partially prepolymerized w-butyl methacrylate.
A reference may be made to the first paper of this series (Malhotra, 1962) for
details of the preparative technique.
Sections were cut on the Huxley ultramicrotome manufactured by the
Cambridge Instrument Co. Ultrathin sections were mounted on grids coated
with formvar, and examined in the Akashi 'tronscope' (model TRS 50 Ei)
operated at 50 kV, with anode aperture of 100 fx and objective aperture of
50 or 100 fx.
Results
It is the purpose of this paper simply to record the general appearance of
the various cell structures seen after fixation with alkalized osmium tetroxide,
according to the experimental technique, and to compare this appearance with
the results produced by the unbuffered and unbalanced solution of osmium
tetroxide (Malhotra, 1962). It is also intended to compare these results with
what is already known about the exocrine cell of the pancreas and the cells of
the first convoluted tubules of the kidney.
The appearance of the various cell inclusions is essentially similar with
lower or higher concentrations of potassium acetate used to alkalize the
fixative, and it will therefore not be necessary to give separate accounts of the
results.
Exocrine cell of the pancreas
A general survey of the various inclusions of this cell fixed with alkalized
osmium tetroxide solution shows that the addition of potassium acetate has
not produced any gross alterations suggestive of poor preservation. Fig. 1
shows the general quality of fixation. It would appear that the cell inclusions
are satisfactorily preserved, and there is no difficulty in recognizing their
identity. The nuclear contents are more or less evenly dispersed and appear
finely granular. The nuclear membrane does not seem to be disrupted and is
sharply delimited. The two nuclear membranes are closely apposed to each
other (fig. 2, A). The maximum thickness of the nuclear membrane is about
FIG. 2 (plate). A, from exocrine cell of the pancreas fixed by osmium tetroxide (alkalized
with higher concentration of potassium acetate at pH 7-3 to 7-5); showing the endoplasmic
reticulum and ribonucleoprotein granules in the basal region. The arrow indicates a pore in
the nuclear membrane (nm). A mitochondrion is also seen in the middle of the field of view.
B, from exocrine cell of the pancreas fixed by osmium tetroxide (alkalized with lower
concentration of potassium acetate at pH 7-2 to 7P3); showing a part of the apical region.
er, endoplasmic reticulum with its associated ribonucleoprotein particles; g, non-granular
paired membranes (y-cytomembranes) in association with large vacuoles and small vesicles;
2X and^2 are probably different stages of condensation of protein in the vacuoles leading to the
formation of zymogen granules (z).
290 Malhotra—Experiments on fixation for electron microscopy
the same as after fixation with unbuffered osmium tetroxide, about 60 m/z.
The small dense particles, of the size of the ribonucleoprotein granules, often
seen on the outer nuclear membrane, are easily seen in the micrographs (fig. 2,
A). The nuclear pores (fig. 2, A) are quite evident and similar to those seen
after fixation with buffered (Watson, 1955, 1959) or unbuffered (Malhotra,
1962) osmium tetroxide.
The characteristic ultrastructure of the mitochondria seems to be re-
markably well preserved, and there is no evidence of any marked swelling
•due to the hypotonic nature of the fixing solution used. The limiting double
membrane and the cristae are clearly seen. The mitochondrial matrix appears
almost structureless in the micrographs (figs. 2, A; 3).
The endoplasmic reticulum is also very well shown. Its form and distri-
bution throughout the greater part of the cytoplasm do not suggest any
noticeable disorganization (figs. 1; 2, A). The membranes bounding the ele-
ments of the endoplasmic reticulum are well defined. The ribonucleoprotein
particles are well preserved (figs. 2; 3). Their distribution on the surfaces of
the cisternae of the endoplasmic reticulum at the base of the cell is clearly
depicted in the micrographs (fig. 2, A).
The highly organized membranous complex, consisting of y-cytomembranes
(paired non-granular membranes), vacuoles, and small vesicles (or granules),
generally present in cells that actively secrete protein, is clearly seen (figs. 2,
B; 3). This complex is commonly referred to as the Golgi apparatus, after its
first description by Dal ton and Felix (1954) in the epithelial cells of the
mammalian epididymis (see Dalton, 1961, for details). The structure and
appearance of this complex, and of the zymogen granules, is essentially similar
to that described after fixation with unbuffered osmium tetroxide (Malhotra,
1962), and no additional remarks seem to be necessary. Some of the vacuoles
associated with the non-granular membranous complex appear more or less
empty, while others have contents of higher electron density (fig. 2, B). This
appearance has been interpreted as suggestive of a gradual condensation
of protein in these vacuoles, leading to the formation of zymogen granules
(see Palay, 1958; Hirsch, 1961; Palade, 1961; Malhotra, 1962). The vacuoles
associated with the non-granular membranous complex have sometimes been
seen in close proximity to the elements of the endoplasmic reticulum (fig. 3).
This appearance has been interpreted as suggestive of an organic association
of the endoplasmic reticulum with the non-granular membranous complex
(Palade and Siekevitz, 1956).
The boundaries between each cell and the next are easily recognized. They
are represented by thin, sharply demarcated, dense lines in the micrographs
(fig- 1).
FIG. 3 (plate). Apical region of exocrine ceil of the pancreas fixed by osmium tetroxide
(^aallkkalized with higher concentration of potassium acetate at pH 7-3 to 7-5). Endoplasmic
reticulum (er) and ribonucleoprotein particles are seen throughout the cytoplasm. The arrow
indicates a close contact between the endoplasmic reticulum (er) and a vacuole belonging to
the non-granular membranous complex (g), m, mitochondria; z, zymogen granules.
FIG. 3
S. K. MALHOTRA
Fie. 4
S. K. MALHOTRA
Malhotra—Experiments on fixation for electron microscopy 291
Cells of the convoluted tubules of the kidney
The general preservation of the various structures of these cells is essentially
similar to what is seen after fixation by unbuffered (Malhotra, 1962) or buf-
fered (Sjostrand and Rhodin, 1953; Rhodin, 1954; Pease, 1955 a, b) osmium
tetroxide. The micrographs clearly show this resemblance. The nuclear
contents are fairly and uniformly granular (figs. 4; 6, B). The nuclear mem-
brane is sharply delimited and seems to be well preserved. Its double nature
is clearly retained, and the two membranes are not pulled apart. The inner
nuclear membrane often appears thicker than the outer (fig. 4), as described
by Porter (1961).
The characteristic infoldings of the cell membrane into the cytoplasm at
the basal end of the cell are very well preserved (figs. 4; 5, A). The membranes
of the infoldings are clearly demarcated and appear as thin, dense lines. The
two membranes of an infolding (j3-cytomembranes of Sjostrand, 1956, 1959)
are regularly disposed, parallel to each other, and more or less parallel to the
major axis of the cell. They are also closely apposed to each other, about
15 m/x to 20 m/u. apart (see Rhodin, 1954; Porter, 1959; Forster, 1961). In
micrographs at high resolution, each of the thin, dense lines is seen to be
composed of two very thin, dark lines, separated by a thin, pale line (fig. 5, B).
Rough estimates of the total thickness of this tripartite membranous complex
give a value between 7 mju. and somewhat less than 9 m/x; that is to say, about
the thickness of the 'unit membrane' of Robertson (1959, i960). Porter (1959)
has given 8 ntyi as the thickness of this tripartite complex of the infoldings of
the tubules of the kidney, as compared to 6 m/x given by Rhodin (1954). The
tripartite complex is not so well seen after fixation in osmium tetroxide as
after potassium permanganate (Robertson, 1959, i960). The double mem-
branes of the infoldings are formed by invaginations of the cell membrane
(Rhodin, 1954; Pease, 19556; see Porter, 1959; Sjostrand, 1959). The cell
membrane at the base of the cell is seen as a single dark line in fig. 6, A.
The mitochondria do not show any obvious defects of preservation (figs. 4;
5, A; 6, c): their characteristic cristae and outer double membrane are clearly
shown, and the cristae are densely packed and arranged in a regular fashion,
especially in the mitochondria towards the base of the cell. In this situation
the mitochondria are known to be disposed more or less perpendicularly
to the base of the cell (Sjostrand and Rhodin, 1953; Rhodin, 1954). This
arrangement is quite evident in the micrographs (fig. 4). In the micrographs
at high resolution, each of the outer double membranes and also the mem-
branes of the cristae have been seen to be tripartite, but the images are not
quite clear enough for reproduction in print. These observations, and the
FIG. 4 (plate). Micrograph showing basal region of a cell from the first convoluted tubule
of the kidney fixed by osmium tetroxide (alkalized with higher concentration of potassium
acetate at pH 7-3 to 7-5). cm, infoldings of the cell membrane; er, endoplasmic reticulum;
I, dense bodies, which may be lipid globules; m, mitochondria; n, nucleus. Small particles
of the size of ribonucleoprotein granules are scattered about in the cytoplasm.
292 Malhotra—Experiments on fixation for electron microscopy
demonstration of the tripartite nature of the membranes of the infoldings,
suggest that alkalized osmium tetroxide reveals essentially the same structure
as is discernible after fixation with buffered osmium tetroxide.
The cylindrical processes of the cytoplasm, forming the brush border at the
apical end of the cell (Rhodin, 1954; Pease, 1955 a, b), are of fairly uniform
diameter. Rhodin gives 50 mju, to 70 m/x for the diameter of these processes.
In my micrographs they are mostly closely packed (fig. 6, c, D). It has been
pointed out that this close packing is quite characteristic of the material that
has been removed from the animal in small pieces and subsequently immersed
in the fixing solution. In tissue fixed in situ the cytoplasmic processes are
wide apart (Pease, 19556). Pease thinks that the apical region of this cell is
particularly sensitive to osmotic changes and becomes swollen during fixation.
At the base of the brush border, vacuoles of various sizes appear scattered
about in the cytoplasm. They are delimited by a membrane, which is seen as
a thin, dense line in the micrographs (fig. 6, c). Pease (1955 a, b) has demon-
strated that these vacuoles are connected with small ducts (fig. 6, c), situated
between the cytoplasmic processes of the brush border. Novikoff (1961) has
also described similar ducts as canalicular structures.
Small, dense granules of the size of ribonucleoprotein granules (Pease,
19556) are scattered throughout the cytoplasm. Some of these granules are
associated with elements of the endoplasmic reticulum, as in figs. 4 and 5.
Fig. 6, B shows what appears to be a continuation of the endoplasmic reti-
culum into the outer nuclear membrane (see Porter, 1959,1961 for references).
y-cytomembranes (Golgi apparatus of Rhodin, 1954; Forster, 1961) have
very seldom been seen in my micrographs of the cells of the first convoluted
tubules. They appear to be confined to the cytoplasm in the vicinity of the
nucleus (fig. 6, c). It would appear that their preservation is satisfactory.
The large vacuoles and granules (or small vesicles) that are usually associated
with y-cytomembranes in a wide variety of cells have not been encountered
in my micrographs of these cells of the kidney.
In addition to the inclusions described above, there are nearly spherical
bodies, about 0-14 /x to 0-2 p in diameter, dispersed in the cytoplasm at the
basal end of the cell. In the micrographs they appear as homogeneously
dense structures (fig. 4). They may perhaps be minute lipid globules.
The basement membrane that surrounds the tubules appears to be rather
uniformly dense in the micrographs (fig. 6, A). The appearance is the same as
after fixation by unbuffered osmium tetroxide.
FIG. 5 (plate). From a cell of the first convoluted tubule of the kidney fixed by osmium
tetroxide (alkalized with higher concentration of potassium acetate at pH 7'3 to 7"s).
A, showing infoldings [cm) of the cell membrane into the cytoplasm disposed more or less
parallel to the mitochondria (m). er, endoplasmic reticulum in association with ribonucleo-
protein particles.
B, magnified view of a part of fig. 5, A, showing tripartite nature of the double membranes
of the infoldings. Arrows indicate where the tripartite appearance is easily seen, m, mito-
chondrion.