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Preparation of biological tissues for electron microscopy by freeze-drying.

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Preparation of Biological Tissues for Electron
Microscopy by Freeze-dryi ng
5
H. DAVID COULTER AND LOUIS TERRACI03
Department of A n a t o m y , Lrnruerstty of blrnnesotcz.
Mznneapolzs, Minnesota 55455
ABSTRACT
A dependable method for freeze-drying tissues for electron microscopy has been developed. Thin slices of fresh tissue were frozen by bringing
them into direct contact with a polished copper bar at liquid nitrogen temperature. The tissue was transferred to a copper specimen block equipped with a
thermocouple and heating circuit for accurate control of the environmental
temperature of the tissue, and evacuated i n a glass freeze-drier using clean
high vacuum techniques for keeping the system free of hydrocarbons. The tissue
was dried by increasing the temperature of the specimen block 1 0 ° C each hour
while monitoring the rate of water removal from the tissue with a partial pressure
analyzer. The dry tissue was fixed with Os04 vapor, vacuum embedded i n a low
viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Tissue processed i n this manner exhibits excellent morphological preservation at both cellular and organellar levels without prefixation or the use of
cryoprotective agents. The results of the experiments using the partial pressure
analyzer indicate that small blocks of tissue can be dried i n a short time at low
temperature.
Intermittent but serious attempts to
freeze-dry biological tissue for electron
microscopy have been made since the
earliest years of electron microscopy (Richards et al., '42, '43; Sjostrand, '43). At
that time most electron microscopists were
concerned primarily with obtaining presentable micrographs, and freeze-drying was
one of many possible methods worthy of exploration. Although the micrographs obtained were as good as many others that
appeared in the literature at that time,
it gradually became apparent that fixation in aqueous solutions was superior
for general application, and interest in
freeze-drying faded as electron microscopic
cytology developed in the 1950s.
More recent efforts to freeze-dry tissue for
electron microscopy have utilized the technique for specialized applications, such as
radioautography of soluble tracers (Stirling
and Kinter, '67), electron microscopic cytochemistry (Gersh, '73), and maintenance
of cellular relationships thought to be distorted by aqueous fixation (Van Harreveld
and Malhotra, '66).The quality of preservation exhibited has always been poor, however, when compared with standard methods, and methodological difficulties have
been serious and often intractable. For
ANAT. REC., 187: 477-494
these reasons, the technique has never come
into routine use despite its theoretical appeal.
Satisfactory freeze-drying of tissues for
electron microscopy requires first, that the
tissue be frozen in such a way that ice
crystal formation is minimized, and second,
that the tissue be dried at low temperature. This paper presents in detail a technique for obtaining well frozen material
and for drying it in a controlled environment. The superiority and efficacy of the
method is proven by the most important
morphologic standard, the excellent preservation of fine structure. Additionally,
data on drying rates and theoretical considerations of the drying process make it
clear that drying can be accomplished in
a short period of time, and that a very
simple and inexpensive drier (Terracio
and Coulter, '75) is all that is needed for
good results.
Received June 15, ' 7 6 . Accepted Oct. 14, '76.
'Work o n this technique was initiated at t h e University of Tennessee Medical Units, where i t was supported by USPHS Training Grant GM 00202.
At the University of Minnesota, this work was supported by the Minnesota Medical Foundation. the Graduate School, and USPHS Training Grant GM114.
Dr. Terracio's present address is the Department
of Anatomy, Hahnemann Medical College, Philadelphia.
Pennsylvania 19102.
477
4 78
H. DAVID COULTER AND LOUIS TERRACIO
MATERIALS AND METHODS
Freezing method
This freezing technique was developed
from a method described first by Van
Harreveld and Crowell ('64). A copper
bar 8 inches high by 3 inches in diameter
with a mirror finish on one end was
polished with jeweler's rouge, rinsed with
xylene, and dried gently with a flannel
cloth, The bar was placed in a 10-liter
Styrofoam ice chest fitted with a lid containing a centrally placed 3-inch diameter
hole. The mirror surface of the bar was
about 2 inches below the lid of the ice
chest (fig. 1). The ice chest was filled with
liquid nitrogen and kept full for two and
one-half minutes. Approximately 11 more
minutes were required for the bar to reach
liquid nitrogen temperature, at which time
the level of the liquid nitrogen was 2 inches
below the freezing surface. Adding nitrogen
to the container after that time was avoided
because it remains on the upper surface of
the bar if it is added after the bar is chilled
and possibly retards freezing by limiting
direct contact of the tissue with the copper
surface.
Fresh tissue was cut by hand with a
razor blade into slices about 1/4 mm in
thickness and were placed on a No. 5 cork
stopper covered on its small end with thin
gauge (0.0007 in) aluminum foil. The
stopper was grasped with a pair of Russian
tissue forceps so that the surface of the
tissue remained perpendicular to the long
axis of the forceps. The tissue was blotted
gently to remove excess blood and promptly frozen by bringing it into direct contact with the surface of the bar and holdi n g it firmly for 20 seconds. If the tissue did
not meet the bar squarely, or if it slipped
or rotated on the bar after the initial contact, the tissue was discarded. After 20
seconds the aluminum foil with its adhering tissue was slipped from the stopper and
dropped into the surrounding liquid nitrogen. It was possible to freeze 20 or more
slices of tissue in this manner on the 3inch diameter surface before having to
warm and repolish the bar. The area where
each piece of tissue had been frozen was
clearly visible, arid care was always taken
to freeze only on a clean area of the surface. When a sufficient amount of tissue
had been frozen, it was removed from the
aluminum foil and transferred to a 10 ml
Fig. 1
Styrofoam ice chest (about 7 X 9 X 10
inj c u t away to show copper bar.
beaker immersed in liquid nitrogen. The
tissue was then dissected into pieces about
1 mm square by chipping i t with dissecting
needles chilled with liquid notrogen.
Drying method
The freeze-drying apparatus (fig. 2 ) was
constructed and designed to minimize the
accumulation of hydrocarbons and to
achieve a vacuum in the
torr range.
Both of these features are necessary for
analysis of drying rates. Figure 2 shows
the freeze-drier in the prepurnping stage
of operation. It was supported at such a
height that 7-inch O.D. glass Dewars could
conveniently be lifted into place under
the specimen tube (E) and pumping chamber (K). Supporting hardware, clamps for
the O-ring joints, electrical connections,
mechanical pump, and large glass Dewars
for the pumping tube and specimen tube
are not shown.
Starting with the system at atmospheric
and room temperature, the pumping
chamber (K) was preheated at least 12
hours at 150" C. With N r blowing through
the system, the specimen tube (E) was
lowered allowing the specimen block (G)
to hang below the rest of the system supported only by the heater wires. The specimen block was brought to liquid nitrogen
temperature by surrounding it with a
small, stable container of liquid nitrogen.
FREEZE-DRYING OF TISSUES FOR ELECTRON MICROSCOPY
The level of liquid nitrogen around the
specimen block was maintained carefully
after equilibration so that it just covered
the surface, and the small pieces of frozen,
chipped tissue were transferred to it with
chilled fine forceps. The actual transfer of
tissue from the beaker to the circular depressions in the specimen block was effected over as short a distance and as
quickly as possible. Nitrogen in liquid
form was visible on the blocks during
transfer. The tissue was always kept immersed in liquid nitrogen at other times to
obviate concern about heat transfer directly from atmosphere to the specimens.
The bottom one-fourth of the specimen
tube (E) was precooled for about 30 seconds
with liquid nitrogen, the container of liquid
nitrogen for the specimen block was lowered, and the specimen tube was promptly
attached with an O-ring clamp to the freezedrier, A small Dewar (H) with liquid
nitrogen was then placed around the bottom of the specimen tube to maintain the
copper block temperature for the next one
to two hours. Next, in a sequence of three
steps, the mechanical pump (not shown
in the diagram) was connected to the system by way of the U-tube (B), the pumping
chamber (K) was removed from the oven
and connected to the system with an O-ring
and clamp, and the bottom 3 4 inches
of the U-tube was immersed in liquid nitrogen (Dewar C ) for a few seconds. Then the
mechanical pump was started. As the
system pumped down over the next 30
minutes, liquid nitrogen was gradually
added to the Dewar (C) until it was nearly full. A considerable volume of water
came off the hot molecular sieve (K), and
if the Dewar trap (C) was filled immediately, water sometimes blocked the U-tube
completely on the upper right hand side.
If the Dewar was filled gradually, water
was trapped over the entire extent of the
right side of the tube, and it remained patent. Maintaining the system in this way,
with nitrogen in both Dewars (C and H),
the vacuum dropped to about 5 X lo-'
torr in an hour, the temperature of the
block rose to about - 160"C, and the molecular sieve pump gradually cooled off.
Valve A was then closed and the system
appeared as shown in figure 2.
The mechanical pump and the U-tube
trap were now removed from the system
and a large glass Dewar (7-inch O.D.) was
479
placed around the pumping chamber (K)
and filled with liquid nitrogen. A vacuum
of about 1 X
torr was achieved in
about two hours. The small Dewar (H)
around the specimen tube was then replaced with a large Dewar, the partial pressure analyzer was turned on, and the entire system was allowed to equilibrate
overnight .
The copper block achieved a steady state
temperature of about - 130"C, the total
pressure dropped to approximately 1 X 10 torr, and the partial pressure of water
torr. The temperareached about 6 X
ture of the copper block was then raised
10°C per hour until it reached room temperature, and the concomitant changes in1
the partial pressure of water were recorded
on a strip chart. For achievement of reproducible results on drying rates it was
necessary to maintain the levels of liquid
nitrogen in both Dewars for the duration
of the experiment. This precaution was
not necessary for routine use of the system for freeze-drying without the use of
the partial pressure analyzer.
Fixation a n d embedding
After the copper block reached 2 5 ° C the
partial pressure analyzer was turned off
and the pressure in the system was brought
to atmosphere through the isolation valve
(fig. 2 : A) with dry nitrogen. The specimen
tube was lowered and the dry tissue was
immediately transferred to a small glass
fixing chamber (fig. 3). This chamber had
been prepared earlier. With valves B and
D closed, a molecular sieve pump had
been attached to the evacuation valve (D)
and chilled for two hours with liquid nitrogen. This pumping chamber was used only
for this purpose and was somewhat smaller
than the one for the freeze-drier. After
transferring the tissue to the fixing chamber the lid was securely clamped into
place and valve D was opened cautiously.
To evacuate the atmospheric gases from
the tube below valve B and yet retain the
fixative, tube (A) was first chilled with liquid
nitrogen for a few minutes. Then valve
B was opened, the evacuation valve (D) was
closed, and tube (A) was allowed to come
to room temperature. The tissue was fixed
4 to 12 hours with OsOl vapor. Following
fixation valve B was closed, and the pressure in the chamber was brought to atmo-
480
H. DAVID COULTER A N D LOUIS TERRACIO
itJ
Fig. 2 Freeze-drying apparatus i n prepumping stage, one-fifth actual size. A high vacuum
bakeable glass valve (Kontes, Vineland, New Jersey 80360) with 17-mm O.D. side arms, teflon
plunger, a n d Viton O-rings (A) isolates the freeze-drier from a liquid nitrogen trapped mechanical pump, a n d also serves a s a n up-to-atmosphere valve. A 17-mm O.D. U-tube (B)
with a n O-ring joint for coupling to the valve serves a s a trap for the mechanical pump. A
Dewar flask (C) chills the U-tube with liquid nitrogen. Heater wires a n d thermocouple wires
from a temperature controller that can regulate temperatures to - 196OC (Leeds and Northrup,
North Wales, Pennsylvania 19454) enter the system at D. Short segments of ceramic thermocouple tubes (Omega Engineering, Stamford, Connecticut 06907) were glued into holes
bored in a Kovar closed-end cap (Latronics Carp., Latrobe. Pennsylvania 15650) with epoxy
glue. The heater a n d thermocouple wires were then threaded through the ceramic tubes a n d
adjusted to a height that would barely allow the specimen block (G) to rest on the bottom
of the specimen tube ( E j . They were then glued into place at that height. The specimen tube
(E) is made from 4 5 m m O.D. Pyrex tubing, closed off and flattened at t h e bottom a n d fused
to a n O-ring joint at the top. The thermocouple wires and heater wires a r e shown at F. The
thermocouple wires are 0.010-inch bare copper and constantan wires (Omega Engineering)
.joined with a bead that is snugly fitted into the specimen block. (If it is not tightly fitted,
the temperature regulation will be erratic). The thermocouple bead a n d the hole for accommodating it are disproportionately large in the drawing. The heater wires that pass through
the ceramic tubes are 0.018-inch copper. They are attached to fiber glass insulated wires t h a t
connect to the cartridge heater i n the specimen block. The copper specimen block (GI is 1 by
2 314 inches with a central li4-inch hole. sized precisely for accommodating a tightly fitted
100-watt cartridge heater 1/4 inch from the bottom of the hole. One-fourth inch holes are
bored all around the periphery of the specimen block for accommodating the specimens. The
pumping chamber (K) is made from 100 m m Pyrex tubing a n d contains 5 A molecular sieve.
The thermocouple gauge (I) a n d partial pressure analyzer (J) (Ultek Division, Perkin-Elmer,
Palo Alto, California 94303) are mounted with standard ultra-high vacuum flanges to the
rest of the system by means of a glass to Kovar seal.
FREEZE-DRYING OF TISSUES FOR ELECTRON MICROSCOPY
n
48 1
infiltration floated back to the surface,
and the others were properly infiltrated at
the end of six hours. They were usually
oriented at a n angle in the bottom of the
capsules, and the surface which had come
into direct contact with the bar during
freezing was easy to locate and preserve
during sectioning.
RESULTS
b'reezing
The gross appearance of the tissue after
freezing depended upon the force with
which the tissue struck the bar, the type
of tissue, and the manner in which it was
sliced. A 114 mm slice of highly parenchymatous tissue such as kidney or liver
brought down firmly against the copper
bar had about the same size and shape as
before freezing. Tissues which were difficult to slice evenly and which did not
retain their shape after slicing such as
brain or intestine were flattened. Also, a
Fig. 3 Fixing chamber. Os04 is shown at A
small cube of tissue was more likely to
i n the bottom of a 7-mm tube fused to a n O-ring
joint. A small, high vacuum glass valve (Kontes) become pressed into a sheet. A n impression
isolates the Os04 from the fixing chamber (C) of the tissue was left on the bar in the
except during fixation. The fixing chamber w a s
form of a clear area surrounded by a
made from a 45-mm O-ring joint, flattened and
small area of frost condensation, providclosed on the bottom, and is prepumped through
anotherhigh vacuum glassvalve (D) by a molecular
ing an immediate record of whether it
sieve pump which is used only for this purpose.
has been properly frozen.
sphere. OsO, was left in the tube at A and
used repeatedly.
After vapor fixation the tissue was
vacuum embedded in a low viscosity epoxy
resin (Spurr, '69). The resin was mixed
carefully to avoid excess bubbles, placed
in polyethylene capsules, and degassed
in a glass desiccator with a water aspirator or a positive displacement pump.
For infiltrating the tissue, a single block
of tissue was placed on the surface of the
degassed resin in each capsule, and the
desiccator was evacuated cautiously to
avoid bubbling. As soon as the pump
reached equilibrium, it was removed and
the tissue was allowed to infiltrate under
vacuum for two to six hours. Completion
of infiltration was indicated by the sinking
of the blocks in the resin. If some of the
blocks did not sink in two hours, the system was brought to atmosphere, the blocks
were mechanically pushed into the resin,
and the desiccator was re-evacuated.
Blocks which were still too large for good
Drying
Drying characteristics of a group of
about 100, 1/4 X 1 X 1 mm blocks of
kidney are shown in figure 4. For the first
four hours, each time the copper block
temperature was raised by 10°C, the partial
pressure of water in the system rose sharply and then declined gradually. Also, the
maximum peak heights declined each
hour. These sharp peaks and characteristic declines ended abruptly at - 8 0 ° C .
From - 70°C to room temperature, the
partial pressure of water remained near
the base level, indicating that no further
water was being sublimed from the tissue
after the first five hours of drying.
The blocks of tissue retained their
shape during the drying process. They had
a vaguely fluffy appearance when fully
dry at room temperature but were not
visibly distorted or shrunken. Much larger
blocks became misshapen and distorted,
even when long drying times were employed.
482
11. DAVID COULTER
AND LOUIS TERRACIO
I
lo
9
8
7
I-
LL
3
3
2
I
TIME (HRS.)
I
0
DEGREES(C1 -120
I
-110
2
3
-100 -90
4
5
6
7
8
9
10
-80 -70 -60 -50 -40 -30 -20
II
12
-10
O
Fig. 4 Partial pressure analysis of the drying time for 114 mni blocks of kidney. At time
0. after pumping the system overnight with the molecular sieve pump. water w a s equilibrated
at about G X 1 0 - 9 torr. a n d the copper block temperature was about -130OC. Each time the
temperature of the copper block was raised by lO"C, the partial pressure of water i n the system
rose sharply, well into the 10-8 torr range, a n d then declined to about 1 X l o - * torr. A n
imaginary line drawn from point to point at the top of each peak would also show a decline,
with a rather sudden termination of the s h a r p peaks at -80°C. The tissue was considered essentially dry after about five hours. when the copper block temperature w a s raised to - 70°C .
Fixation arid e?nbcdding
During vapor fixation with OsO, the
tissue developed a slightly greyish cast.
This greyish appearance was slightly more
pronounced on the side of the tissue that
came into initial contact with the copper
bar. A smoothness, almost a glossiness,
was apparent on this surface which was
in clear contrast to the rougher appearance on the side of the tissue which had
been in contact with the aluminum foil
during the freezing procedure. Large,
shrunken blocks turned black during
vapor fixation rather than grey.
Upon infiltration, the grey tissue turned
completely black. The tiniest slivers of tissue became infiltrated and turned black
the moment they came into contact with
the resin. The other blocks turned black as
they sank into the resin during vacuum
infiltration. Some osmium-containing materials leached from the blocks during infiltration, especially when the longer
periods of vapor fixation had been employed.
Light microscopy
Inspection of the frozen-dried tissue with
the light microscope revealed whether or
not the material would be useful for electron microscopy. If ice crystals were visible
in 1/2 Fm sections under oil immersion
near the surface of the block that first
contacted the bar, the material was invariably unsatisfactory for electron microscopy. In blocks of tissue that had been
FREEZE-DRYING OF TISSUES FOR ELECTRON MICROSCOPY
483
block, and no drying or compression artifacts of the type discussed above are present,
Electron microscopy
Fig. 5 One-fourth micron section of fiozendried mouse kidney. No ice crystal formation.
compression artifact. or gross artifacts of drying a r e
present. X 1,000.
well frozen, that contacted the bar squarely and remained in position without subsequent movement, ice crystals were found
on the far surface of the tissue and disappeared as one scanned toward the side
that had been frozen initially. If the block
of tissue had slipped or rotated on the bar
during freezing, the cellular integrity
was disrupted, and ice and compression
artifacts were evident throughout the
block.
Attempting to dry blocks which were too
thick yielded characteristic artifacts. The
blocks were not completely infiltrated if
they were thicker than 2 mm, and blocks
in the 1-2 m m range often contained a
core of densely stained, macerated and unrecognizable material which was surrounded on all sides by intact tissue which had
been satisfactorily dried.
Figure 5 illustrates a 1/4 pm section of
frozen-dried mouse kidney. A small arteriole is shown in the center of the field,
surrounded by kidney tubules. Nuclei,
mitochondria, and cytoplasmic vacuoles
are all prominent, Occasional breaks in the
tissue between tubules probably occurred
during drying or subsequent handling. No
ice crystal formation is evident, even at
the bottom of the print, 180 p m into the
Tissue processed by this method yields
excellent morphological preservation (figs.
6-9). Except for obvious shrinkage spaces,
morphological relationships are intact at
all levels of organization, tissue, cellular,
and subcellular. Cells retain their normal
shapes, and plasma membranes and intracellular membranes are always intact.
The inherent contrast of the material is
remarkably high. Membranes have nearly
as much contrast in unstained frozen-dried
sections that have been vapor-fixed with
OsO4 as they do in double-stained aqueousfixed specimens.
Two types of characteristic artifacts were
always present, ice crystal damage and
shrinkage spaces. The ice crystal reticulations were very predictable, increasing in
size with increasing distance from the surface which was initially frozen. Easily
recognizable shrinkage spaces occurred
most commonly around mitochondria.
Figure 6 is a micrograph of renal proximal tubule exhibiting excellent morphological preservation. No discontinuities are
seen in plasma membranes or intracellular
membranes. The basal infoldings, microvilli, and Golgi apparatus all appear
normal. The mitochondria are of special
interest. Two clearly defined populations
are found. One group of very dark mitochondria has extremely electron dense
matrices in which the cristae are barely
discernible, and always seem to be surrounded by prominent shrinkage spaces.
A smaller group of mitochondria has light
matrices with concomitantly prominent
cristae, and are seldom if ever surrounded
by shrinkage spaces. At this magnification,
cytoplasmic reticulations due to ice crystal
growth become evident in the upper portion of the cell, which is several microns
from the surface initially frozen by the
bar. Inexplicably, they are also found in
the basement membrane, which is only
about 112 pm from the surface of the
block.
Figure 7 is a micrograph of surface epithelial cells of rabbit gallbladder, frozen
by bringing the luminal surface into contact with the copper bar. A characteristic
lateral intercellular space is seen between
484
H. DAVID COULTER AND LOUIS TERRACIO
Fig. 6 Renal proximal tubule cells. frozen-dried, fixed i n O s 0 4 vapor, stained bv uranvl acetate arid
lead citrate. Basal infoldings are seen below and microvilli at the top of the print. Most of the mitochondria have a dense matrix (DM), and are surrounded by a shrinkage space. A few others have a
light matrix (LM), and lack a shrinkage space. Many of them are connected with one another by small
buds (light arrow). An array of 30-50 nni electron dense reticulations due to ice crystal growth are
seen in the cytoplasm at the top of the print a n d in the basement membrane below (dark arrows).
x 22.000.
FREEZE-DRYING OF TISSUES FOR ELECTRON MICROSCOPY
485
Fig. 7 Rabbit gallbladder, luminal epithelial c emils, frozen-dried, fixed by OsO, vapor, stained by
uranyl acetate and lead citrate. X 27,000.
adjoining cells, The mitochondria shown in
this particular micrograph are almost all
of the dark matrix variety.
Figure 8 illustrates the circumstance
that commonly occurs at the edge of the
block. The nucleus of an unidentified kidney cell is at the interface that was initially frozen. Nuclei seem to be especially
susceptible to ice crystal formation. No de-
tectable ice crystal growth occurs at the
initially frozen surface, down to the limit
of resolution in this micrograph of about
2 nm. Extremely small ice crystal reticulations occur on the other side of the nucleus.
The section was unstained, illustrating
the high inherent contrast of vapor fixed
blocks.
Figure 9 shows a high resolution stereo-
486
€1. DAVID COULTER AND LOUIS TERRACIO
Fig. 8 Frozen-dried kidney. fixed by Os04 vapor, n o additional staining. The cytoplasm of the
cell in the lower part of the print is disrupted because the razor blade passed directly through the cell.
.just missing the niicleus and allowing the contents of the cell to spill away before it was brought into
contact with the bar. X 30.500
scopic view of renal proximal tubule cells
contacting basement membrane. The inner lamina of the trilaminar membrane is
especially prominent, The basement membrane shown on the left is only about 200
nm from the surface that was initially
frozen. The section was about 30 n m in
thickness, and the field that is shown
here in stereo is about 300 nm in width,
The 10:1 ratio of width to depth accounts
for the limited appreciation of depth perception viewing the micrographs with a
stereo viewer. No evidence for ice crystal
reticulation is evident, even in the basement membrane, where ice crystal formation often seems especially prominent,
DISCUSSION
Successful execution of this technique
requires meticulous attention to detail;
even slight modification of the technique
may affect the results adversely.
Frwzing technique
The freezing operation is the step that
always limits quality if the tissue is dried,
fixed, and embedded according to the
protocols outlined in this paper. The crucial
moment is the initial contact of the tissue with the bar and the maintenance
of that contact for 20 seconds without
movement. The glass transition temperature of water is about - 137°C (Rasmussen and MacKenzie, '71), and if the tissue
is not brought to that temperature quickly, ice crystals will form. Experience will
soon direct the operator to a reasonable
balance of force and velocity in making
contact with the bar, not squashing the
tissue, yet not being too delicate with it.
The use of a cork stopper provides a slight
cushioning effect that is very helpful, absorbing minor unsteadiness and keeping
the tissue placed where it initially made
contact with the bar. The use of a more
rigid supporting material such as a wooden
dowel makes the operation much more
critical. The slightest movement of the
hand is transmitted to the contact area
between the metal and the tissue, re-
FREEZE-DRYING OF TISSUES FOR ELECTRON MICROSCOPY
487
Fig. 9 Frozeri-dried kidney. fixed by OsO, vapor, double stained by a n aqueous solution of
uranyl magnesium acetate at 5OoC a n d lead citrate. The micrographs were taken at plus a n d
minus 10" of tilt on a n AEI 801 at 60 kV, using a n initial magnification of X 160,000. Final
magnification is X 220,000.
sulting in slipping or rotation of the sample
on the bar and invariably spoiling the tissue for electron microscopy. At the moment
of freezing, heat is being rapidly conducted from the sample to the bar. If the
process is interrupted or delayed before
both the tissue arid the aluminum foil
reach a low temperature, the tissue will
warm up due to conduction of heat from the
aluminum foil, resulting in devitrification of
the water and extensive ice crystal growth.
Drying technique
Theoretical consideration of the variables
that are important in freeze-drying of tissue blocks is a n invaluable aid to the design of a simple and inexpensive drying
apparatus, and to understanding why
tissue can be dried in a relatively short
time. There are five major variables that
must be considered, (1) the thickness and
(2j the temperature of the dry shell of
tissue that develops on the outer surface
of the block during the drying process,
( 3 ) the initial surface area of the sample
per unit of volume, (4) the temperature
of water at the drying boundary between
the frozen core of tissue and the surrounding shell of dry tissue, and (5) the efficiency of the drier in condensing water
that escapes from the sample. Stephenson
('53) first considered in detail the occurrence and growth of a shell of dry tissue on
the outer surface of frozen blocks. This
shell contains all cytoplasmic materials
with the exception of water and dissolved
gases, and is of great importance in impeding the flux of water molecules from
the interior core of frozen tissue to the
surface of the block. Each molecule will
make a vast number of collisions with
organelles and other structures in the
cytoplasm, and will be adsorbed and released at a temperature-dependent rate at
488
H . DAVID COULTER AND LOUIS TERRACIO
each site, so the number of molecules that
finally escape from a unit area of dry
surface per second will depend on both the
thickness of the shell and its temperature
gradient.
The most easily manipulated variable
is surface area. A sheet of tissue, having a
larger initial surface area than a cube
of equal volume, will obviously be more
easily dried, Not only will the availability
of a larger initial surface area yield an
increased initial drying rate, but the block
will dry to completion before the shell becomes as thick as it will be for the same
volume of tissue in a cube. Next, the rate
of drying will increase as the temperature
and vapor pressure of water in the frozen
core increases, provided the increased water
that is sublimed can move through the
shell. As Meryman ('60) has pointed out,
the equilibria that occur at the drying
boundary are crucially affected by the resistance of the dry shell to vapor flow, Last,
it is important that the water that does
finally escape from the outer surface of
the dry shell be trapped efficiently by the
freeze-drier.
The partial pressure analysis of drying
rates has aided considerably in removing
some of the unknowns from the technology
of freeze-drying of tissue samples for electron microscopy, and provides a clear rationale both for tissue drying time and for
temperature regulation of the copper specimen block. The base level of 6 X 10-9
torr for the partial pressure of water (fig.
4) represents a steady state value, Water
is being continually desorbed from all
inside surfaces of the vacuum system,
and adsorbed onto them. When water is
adsorbed onto a surface at liquid nitrogen
temperature, such as the inside surfaces
of the pumping tube and the specimen
tube, or the molecular sieve, it is trapped
there, or pumped, for as long as the temperature is maintained, Each time the temperature of the copper block is raised
10°C, a small fraction of the escaping water
leaves the samples at such an angle that
permits access to the orifice of the specimen
tube, where it escapes into the rest of the
system and upsets the equilibrium, The
upward limb of each peak in figure 4 occurs when an increase in conductive and
radiative heat from the specimen holder
causes increased vaporization of water
from the drying boundary in the samples
and an increased flux of water to their
surfaces. It is important to understand
that the temperature of the water in the
core of the sample may not have an obvious
relationship to the temperature of the
specimen holder, Increased heat is no
doubt radiated from the copper block
each time its temperature is raised by a
1 0 ° C increment, but the increase in temperature of water inside the specimen will
depend on many factors, the most important of which is the degree to which the
dry shell insulates the core. This insulating effect may be considerable, and presumably becomes more important as the
thickness of the shell increases. Indeed,
the argument will be developed later that
the primary effect of the increasing copper
block temperature is on the shell rather
than on the frozen core, The descending
limb of each peak (fig, 4) occurs for two
reasons: first, the sublimation of water
produces evaporative cooling, causing the
temperature of the core of the sample to
drop and thereby checking the rate of
sublimation; and second, the dried shell
of the sample becomes slightly thicker
and more effectively insulates the frozen
core from its heat source, decreasing the
amount of heat available for driving sublimation at the drying boundary.
To consider the drying process more
rigorously, it is helpful to model the ideal
drying characteristics of a single block
of pure ice, 1 X 1 X 114 mm, a special
case of 1 X 1 X h, finding the decrease
in volume with respect to time, -dV/dt,
keeping all other variables constant. No
shell is present, the initial surface area (S)
of 3 mm2 (212
4hl) and volume (V) of
114 mm:1 (h12) are given, the temperature
of the block is kept constant with whatever
heat input is necessary to counteract evaporative cooling, and the condensing process
is 1 0 0 8 efficient. Water sublimes from
all surfaces equally so that all dimensions
of the block decrease by equal amounts as
drying occurs.
To find the necessary expressions, consider first that the new volume of ice
after a short time of sublimation or
V(t
A t), will be equal to the old volume
minus the water that comes off the surface area during that period of time, or
V(t) - k A t S(t). As A t approaches 0, it
+
+
389
FREEZE-DRYING OF TISSUES FOR ELECTRON MICROSCOPY
can be shown that dVldt = -kS. This
simply states that volume is decreasing at
a rate proportional to surface area, a relationship that is readily appreciated intuitively.
At t = 0, V = hl? and S = 212 + 4hl.
Since all dimensions decrease equally, h
can be defined in terms of 1 throughout
the course of drying as 1 -314. For example, initially 1 - 3/4 = 1/4, and at a
later time when the block has lost water
to a depth of 1/16 mm from all surfaces h
is still 1 - 314, or in that case 718 - 3/4 =
118. Now V and S can be defined in terms
of 1, not only at t = 0 but for the entire
course of drying, as V = 13 - 31'/4, and
S = 612 - 31.
Now, stating dVldt = - kS in terms of 1,
it can be shown that dl/dt = -2k, which
is simply a statement that the decrease
in any given dimension with respect to
time is a constant, Again, this can be appreciated intuitively by understanding that
the molecules of water would come off a
unit of surface area at a constant rate
dependent only on vapor pressure, and
that the constant depletion of water from
any two opposite surfaces dictates a constant decrease in the distance remaining
between them.
Integrating the expression dl = - 2k dt
yields 1 = - 2kt
C, and since at time 0,
1 = 1, it follows that C = 1, and 1 = 1 2kt. Finally, since V = 1s - 312/4, the
equation showing the relationship V =
f(t) is found to be a cubic with the form
V = (1 - 2kt)3 - 3(1 - 2kt)'/4. The
meaningful part of the curve between V =
0.25 and V = 0 is shown in figure 10.
The height of the curve at any time t
represents the volume of ice present at
that time, and the slight change in the
slope of the curve as the volume approaches
0 represents a slightly decreased rate of
drying as surface area decreases. This
is the pertinent function. A little introspection will make it clear that if a n ideal
partial pressure analyzer were being employed to study the model, it would be measuring a function of the rate of drying,
not the volume of ice remaining in the
block. It actually would be measuring
small decreases in volume (the molecules
that are sublimed away) over small periods
of time at any given moment, or the negative of AVlAt versus time. So the equation
Fig. 10
V = f(t).
I .5
--dvd t
+
t
Fig. 11
0.125
-dV/dt = X t ) .
needed to compare the model with the
data is not V = f(t), but the negative of
its first derivative, -dV/dt = f(t), or
-dV/dt = 24k3t2 - 18k2t
3 k (fig. 11).
Again, the curve has no physical meaning after the block is dried at t = 0.125,
and the drying rate becomes zero at that
time.
The analysis given holds for t between
to and t I where t I is the time at which the
volume becomes 0 . At t , , 1 = 314, 1 2kt1 = 3/4, and k = 1/8t,. For figures 10
and 11, units of time are chosen so that
t , = 1/8,making k = 1 .
Examining the model further, it is
+
490
H. DAVID COULTER AND LOUIS TERRACIO
clear that the reason the drying rate is
still quite substantial just before t = 0.125
is that there is still considerable area on the
top and bottom surfaces of the block as
the thickness approaches 0 . The behavior
of a model of a cube of ice is quite different, with the drying rate (and surface area)
decreasing gradually as all dimensions decrease identically toward 0. In that case
there is no sharp cutoff of - dV/dt as drying is completed.
The plot of - dVldt = f(t) shows a definite qualitative resemblance to the data
shown in figure 4. Since the upward limbs
for the individual peaks in figure 4 represent a n approach to steady state for each
new temperature, and since the downward limbs result from evaporative cooling,
a tentative comparison can be made by
drawing a curve through the tops of the
peaks in figure 4 and superimposing that
over the curve of the model. If that is done
the shapes of the curves coincide sufficiently to require explanation. Only the
first two peaks in figure 4 are slightly higher than predicted from the model.
The resemblance of - dV/dt = f(t) for a
block of ice to the drying characteristics
of the tissue blocks suggests that the five
variables discussed earlier either behave
as constants using the drying technique described in this paper or that they cancel
each other, The third and fifth variables,
in fact, are certainly constant. The efficiency of the drier in condensing water
that escapes from the sample is purely a
function of freeze-drier design, and the
initial surface area of the blocks per unit
of volume is set as soon as the blocks are
prepared. Both of these variables are important, but they cannot change after the
drier is built and the tissue is prepared.
The first and second variables tend to
cancel each other. The drying rate is decreased with increasing thickness of the
shell and increased by the warming of
the shell by the copper block. A temperature gradient is established in the shell,
as cold as the ice at the drying boundary
and as warm as the copper block on the
outer surface. If, as will be discussed next,
the temperature of water remains relatively constant in the core, the temperature gradient in the shell must become
very steep as drying proceeds. Each water
molecule is condensed on innumerable
surfaces in its course across the shell, and
its release in each case is dependent upon
vapor pressure. The occurrence and steepness of the temperature gradient in the
shell therefore becomes a n extremely important factor in facilitating vapor flow.
For example, if the temperature of the ice
were - 100°C and the surface temperature were - 74"C, the vapor pressure at
the dry surface would be approximately
100 times that at the drying boundary, 1 X
10 3 torr compared to 1 X
torr.
The temperature of water at the drying
boundary, the fourth variable, is difficult
to evaluate. It may be relatively constant,
shifting only slightly higher each time the
radiative heat from the copper block is increased and dropping back to its base
value over each ensuing hour until the
tissue is dry. The comparison of the drying
data obtained with the partial pressure
analyzer with the model developed for pure
ice at constant temperature suggests that
this is indeed the case, except during the
first hour or two of drying, when the shell
is so thin that it is not yet a very efficient
insulator, Actually measuring the temperature of the core of frozen tissue is difficult.
It is a simple matter to embed a fine
thermocouple in a block of tissue and
then freeze it, but an insulating shell
quickly forms around the thermocouple
as well as on the outer edge of the block,
and the temperature of the block appears
to rise even when the surrounding environmental temperature is kept constant.
Stumpf and Roth ('67) measured drying rates with a n electrobalance, obtaining decreasing weight versus time, a
measure equivalent to V = f(t) rather
than dV1dt = f(t). The above hypothesis
lends itself to a n explanation for why they
found a n exponential decline in loss of
water rather than a decline based on a
cubic equation. Without a controllable
source of external heat in their drier, the
growing shell insulated the core of ice more
and more effectively during the course of
drying, and the temperature of the remaining frozen tissue dropped as a function of evaporative cooling. The vapor
pressure of water at the dry surface remained the same, and that at the drying
boundary dropped concomitantly with the
drop in temperature. An exponential decline in the loss of water from the blocks
would be expected under those circumstances.
FREEZE-DRYING OF TISSUES FOR ELECTRON MICROSCOPY
Apart from theoretical considerations,
our technique is eminently practical, The
unit shown in figure 2 can be constructed
in any high quality glass shop and used
according to the time schedule indicated,
A partial pressure analyzer is unnecessary,
A n even simpler drier can be constructed
using Erlenmeyer vacuum filtering flasks
for accommodating the specimen holder
and molecular sieve, and a low temperature thermometer for monitoring the temperature of the copper block (Terracio and
Coulter, '75). Tissue can be dried routinely in one day. Correctly executed, the technique is entirely reliable.
Fixation and embedding
Investigators have occasionally prepared
frozen-dried tissue and examined it without fixation (Sjostrand and Kretzer, '75)
hoping to get some image of the material
in as natural a state as possible. The rationale for fixing the tissue in this work
with Os04 vapor was to facilitate comparisons with ordinary methods of fixation
in aqueous media, so that a critical evaluation of morphological preservation could
be obtained. As laudable as the efforts
have been to study unfixed tissue, it seemed
a more pressing priority to evaluate the
technique first with fixed tissue.
There are two phases to the process of
fixation. First, Os04vapor diffuses throughout the dry block, accumulating at all exposed interfaces and presumably dissolving in the lipid components and reacting
with double bonds. The greyish cast of
the tissue at this stage may occur as a
result of those reactions. There is probably little chemical interaction with
proteins, carbohydrates, and the polar ends
of lipid molecules during this period.
The second phase of fixation occurs during infiltration with resin, during which
time the tissue turns completely black,
taking on the typical appearance of 0smicated tissue, The presence of the liquid
environment seems to be the key factor in
permitting the reduction of the OsO,, because any epoxy resin, alcohol, or even
paraffin causes the tissue to turn black.
This is not due merely to a change in refractive index. If the resin is removed from
the infiltrated blocks and they are redried,
they remain black, although they have a
porous appearance.
The fact that extremely large blocks
49 1
turned black rather than grey during exposure to vapor is probably due to the
fact that water remains in the cores of
those blocks and thaws when the freezedrier is brought to atmosphere. Then with
the liquid phase present, the OsO, goes
into solution and the tissue is fixed in an
aqueous medium, reducing the OsO, in a
conventional manner. Then, as the block
remains in the fixing chamber, it gradually becomes desiccated.
Light microscopy
Light microscopic examination of blocks
is very helpful in finding tissue that is
satisfactory for electron microscopy. The
best blocks can be located, and the surface
of the tissue that was initially frozen can
be found. Then, the block can be oriented
longitudinally and trimmed so that each
thin section will be 1 mm or more in length
and include 100 pm of good tissue plus
several micrometers of excess plastic. The
full length of the block can then be evaluated in the electron microscope. Often one
segment has better tissue in it than the
rest, and that part can be isolated for
critical work.
The densely stained and compressed tissue found in the central parts of the 1-2
mm blocks probably results from the same
process that occurs when extremely large
blocks turn black during vapor fixation.
The central core of ice does not dry completely during initial freeze-drying, and
violent surface tension effects occur subsequently during thawing and vapor fixation,
Electron microscopy
The most meaningful standard for evaluation of a technique of tissue preparation for
electron microscopy is the reproducible
preservation of fine structure. The reason
that freeze-drying for electron microscopy
has been so difficult is that useful tissue
and reproducible results are quite difficult
to obtain. Considered with the fact that
a week or more of drying time has frequently been spent for a single experiment,
it is not surprising that the electron microscopic literature of fkozen-dried tissue is
very limited.
By far the most troublesome problem
with any freeze-drying technique is the
formation of cytoplasmic reticulations
caused by the growth of ice crystals. The
492
H . DAVID COULTER AND LOUIS TERRACIO
physical aspects of ice crystal formation
have been discussed in detail by Luyet
(‘60) and will not be reviewed here. The
tissue damage occurs when solutes and
insoluble components of the cytoplasm
are pushed to the periphery of the growing
crystal. During lyophilization these components do not move back to their original
sites in the cell but remain as a shell
surrounding the area where the ice crystal
grew. These shells of protoplasm are then
fixed with OsO4 vapor with uncompromising clarity, and appear as reticulations of
electron dense material scattered throughout the micrograph. They are absent only
when the tissue was brought to a low temperature so quickly that water passes from
a liquid to a frozen vitreous state. Such a
field of tissue was shown at high resolution in figure 9. For intermediate magnifications and resolution, 1-10 nm reticulations are of no practical significance, and
at even lower magnifications, 10-100 nm
reticulations can be tolerated.
The mitochondrial shrinkage spaces observed in frozen-dried tissue are puzzling.
Their exclusive correspondence with the
dense mitochondria may simply be an
accident of freeze-drying, resulting from
some minor biological variation, or they
may be reflective of significant biological
differences in the two mitochondrial populations. The dense mitochondria may have
a higher content of electrolytes, or their
membranes may have low permeabilities.
These conditions could occur intermittently in relation to normal variations in functional state or as a result of hypoxia due
to handling prior to freezing. In either
case, an increased level of electrolytes
would be expected to reduce the vapor pressure of the associated water, thereby slowing its removal. A reasonable hypothesis is
that the dense mitochondria contain water
whose outward diffusion is markedly hindered as the other water is removed. This
remaining water becomes liquid when the
drier is brought to atmosphere at room
temperature, and the shrinkage results
when the tissue is subsequently evacuated,
shrinkage resembling in microcosm the
distortions that occur when one tries to
dry blocks that are too large.
Frozen-dried Os04-fixed tissue has very
high inherent contrast. This is not surprising, when the nature of the fixation
process is considered. With no solvent interference, OsOI diffuses easily through
the tissue and accumulates at every interface in the cell, dissolving in the lipids to
the point of saturation, and coating the
surface of every exposed protein and carbohydrate molecule. Probably not all of the
G O 4 is reduced when the tissue is infiltrated, but i t is physically bound at every
potentially reactive site, and is reduced
immediately when the solvent becomes
available. During aqueous fixation, on
the other hand, the penetration of a01
is significantly limited, its accumulation
on various structures requires chemical
interactions, and many structures or molecules which might have reduced i t are
lost by solvent extraction before the reactions can occur.
The inherent contrast of membranes
in this tissue is especially noteworthy.
The O s 0 4 is taken up quite selectively by
the inner lamina of the trilaminar membrane. The outer lamina is not usually
visible unless the sections are stained with
uranium and lead salts. Even then it is
much thinner and less electron dense
than the inner lamina (fig. 9).
Successful application of the technique
of freeze-drying for electron microscopy
is of considerable importance to many
problems in cell biology. Enzyme cytochemistry at the electron microscopic level,
radioautography of water soluble tracers,
and delineation of labile organellar and
supramolecular relationships are but a
few of the important potential applications.
ACKNOWLEDGMENTS
The authors thank Doctor William Sudderth for assisting with the mathematical
derivations, and Doctors Morris Smithberg and Stanley Erlandsen for critically
reviewing the manuscript.
LITERATURE CITED
Gersh, I. 1973 Submicroscopic Cytochemistry.
Chap. 1. I. Gersh, ed. Academic Press, New
York and London, pp. 1-15.
Luyet, B. 1960 On various phase transitions
occurring in aqueous solutions at low temperatures. Ann. N . Y. Acad. Sci., 85: 5 4 9 4 6 9 .
Meryman, H . T. 1960 Principles of freeze-drying. Ann. N. Y. h a d . Sci., 85: 630-640.
Rasmussen, D. H.,and A. P. MacKenzie 1971
The glass transition in amorphous water. A p
FREEZE-DRYING OF TISSUES FOR ELECTRON MICROSCOPY
plication of the measurements to problems arising in cryobiology. J. Phys. Chem., 75: 967973.
Richards, A. G., Jr., T. F. Anderson and R. T.
Hance 1942 A microtome sectioning technique for electron microscopy illustrated with
sections of striated muscle. Proc. Soc. Exp.
Biol. Med., 51 : 148-152.
Richards, A. G., Jr., H. B. Steinbach and T. F.
Anderson 1943 Electron microscopic studies
of squid giant nerve axoplasm. J. Cell. Comp.
Physiol., 21: 129-143.
Sjostrand, F. S. 1943 Fixering och preparering
for elektronmikroskopisk undersokning av vavnad. Nord. Med., 19: 1207-1212.
Sjostrand, F. S., and F. Kretzer 1975 A new
freeze-drying technique applied to the analysis
of the molecular structure of mitochondria1
and chloroplast membranes. J. Ultrastruct. Re%,
5 3 : 1-28.
Spurr, A. R. 1969 A low-viscosity epoxy resin
embedding medium for electron microscopy.
J. Ultrastruct. Res., 26: 31-43.
Stephenson, J. L. 1953 Theory of the vacuum
493
drying of frozen tissues. Bull. Math. Biophys.,
1 5 : 411429.
Stirling, C. E., and W. B. Kinter 1967 Highresolution radioautography of galactose-aH accumulation in rings of hamster intestine. J.
Cell Biol., 3 5 : 585-604.
Stumpf, W. E., and L. J . Roth 1967 Freezedrying of small tissue samples and thin frozen
sections below -6OOC. A simple method of
cryosorption pumping. J. Histochem. Cytochem., 1 5 : 243-251.
Terracio, L.,and H. D. Chulter 1975 Preparation of frozen-dried tissue for transmission electron microscopy: A simple and dependable
method. 33rd Ann. Proc. Elect. Microscopy
Soc. Amer., pp. 614415.
Van Harreveld, A,, and J. Crowell 1964 Electron microscopy after rapid freezing on a metal
surface and substitution fixation. Anat. Rec.,
149: 3 a 1 3 a 6 .
Van Harreveld, A., and S. K. Malhotra 1966
Demonstration of extracellular space by freezedrying i n the cerebellar molecular layer. J.
Cell. Sci., 1 : 223-228.
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