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The inadequacy of microwave radiation as a means of fixation for electron microscopy

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Quiimipiac
College
THE INADEQUACY OP MICROWAVE RADIATION
AS A MEANS OF FIXATION FOR
ELECTRON MICROSCOPY
By
STEVEN ?. SCHMIDT
B.S. Wheaton College,
1983
A THESIS
Presented to the School of Allied Health and Natural Sciences
and Quinnipiac College
in partial fulfillment of the requirements
for the degree of
Master of Health Science
June 1985
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ABSTRACT
THE INADEQUACY OF MICROWAVE RADIATION
AS A MEANS OF FIXATION FOR
ELECTRON MICROSCOPY
Steven P. Schmidt
Master of Health Science
Quinnipiac College
June 1935
The aim of this project was to evaluate the use of
microwave radiation as a means of fixation for electron
microscopy.
Routine methods,
though adequate, have failed
to provide an all inclusive method of fixation without ser­
iously damaging the fragile cellular ultrastructure.
Two variables
(temperature and fixative mediums) were
manipulated to determine the optimal temperature-medium
combination that gave the best results.
Temperatures ranged
o
o
o
from 50 - 80 C in 5 C increments and glutaraldehyde,
formalin and saline were the mediums used.
From this study it can be stated that microwave radia­
tion cannot be used as a fixative for electron microscopy.
The effects of the high, rapid, random heating characteris­
tic of microwave radiation, destroyed the delicate ultra-
QU1NNIPIAC COLLEGE
U A M P kC M
f* T
LIBRARY
n e rto
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16167
structure leaving the tissue inadequate for diagnostic
interpretation.
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THE
INADEQUACY OF MICROWAVE RADIATION
AS A MEANS OF FIXATION FOR
ELECTRON MICROSCOPY
This thesis
is approved as a creditable and independent
investiga­
tion by a canidate for the degree of Master of Health Sciences,
acceptable as meeting the thesis
out
implying that the conclusions
requirements
and is
for this degree, but with
reached by the canidate are necessar­
ily the conclusions o f the major department.
r
Quinnipiac College Thesis A dvisor
2al
'<
U
Mary Jane Clarke, M.S., R.T.
Assistant Professor, Director of Radiography
Quinnipiac College
Clinical
Thesis Advisor
David S. Papermaster, M.D.
Director, EM Facility
West Haven Veterans Administration Medical Center
Associate Professor o f Pathology, Yale University
Medical Director
P athologists1 Assistant Training Prog
Rosa E. Enriquez, M.D.
Director of Pathologists' Assistant Training Program,
West Haven Veterans Administration Medical Center
^tholoftyy, yQ\e Univei^ity
/?
Assistant Professor,
Director, Graduate Studies_
Quinnipiac College
Kent S. Marshall,
Associate Professor ofyChemistry
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Ac knowledgement s
I would like to thank the following people for their
support in this endeavor:
Lillemor Wallmark whose technical expertise, assist­
ance and patience were integral to the completion of this
proj e c t .
Dr. Papermaster and Mary Jane Clark who were of im­
measurable assistance in the preparation of this work.
My mother and father who not only made this education
economically feasible, but who have also taught me the
value of finishing each project that is begun.
To my patient wife, Tamara, whose friendship, motivation
and affection are an inspiration daily.
iii
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TABLE OP CONTENTS
PAC-E
INTRODUCTION
Statement of the P r o j e c t ............................
1
Literature Review ....................................
2
Microwave R a d i a t i o n ................ ...... .
5
Chemical Fixation Proc es s.......................
~
Physical F i xa tion ...............................
Microwave F i x at io n.............................
Ra tio nale ...............................................22
MATERIALS AMD MET HODS ....................................... 23
R E S U L T S ...................................................... 25
D I S C U S S I O N ................................................... 46
Data Analysis.......................................... 4&
Overall Conclusions.................................. 51
RE FE R E N C E S ..................................................
52
A P P E N D I X E S ..................................................
55
iv
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LIST OP TABLES
Page
Table 1.
Energies of Electromagnetic Radiati ons ........ 6
v
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LIST OF PLATES
Page
Plate 1.
Rat kidney controls fixed in two stan­
dard fixatives and examined by light microscopy,
a) formalin,
b) glutaraldehyde
Plate 2.
Rat kidney fixed in saline with m i c r o ­
wave radiation at various temperatures examined
by
light microscopy, a) 50 C. 1800x, b) 55 C,
1800x, c)60 C, 1800x, d) 65°C, 1800x.
Plate 5.
Rat kidney fixed in formalin with micro­
wave radiation at various temperaturesand examined
bv
light m i c r o s c o p y , a) 50 C, 1800x, b) 55 C, 1800x,
c) 60 C , 1800x, d) 65 C , 1800x.
Plate 4.
Rat kidney fixed in glutaraldehyde with
microwave radiation at various temperatures and
examined bv light microscopy, a) 50 C, 1800x, b)
55 C, 1800x, c) 60 C , 1800x, d) 65°C, 1800x.
26
28
50
52
Plate 5. Rat kidney fixed in saline at two temperatures
with microwave radiation and examined by light
55
microscopy, a) 70 C, 1800x, b)80 C, 1800x.
Plate 6.
Rat kidney fixed in formalin with m ic ro ­
wave radiation at two temperatures and examined
by
light microscopy, a) 70 C, 1800x, b) 80 C, 1800x.
Plate 7.
Rat kidney fixed in glutaraldehvde with
microwave radiation at two temperatures and examined light microsrcopv, a) 70 C, 1800x, b) 80°C,
1800x.
Plate 8.
Rat kidney control fixed in formalin and
examined by electron microscopy.
Plate 9.
Rat kidney control fixed in glutaraldehyde
and examined by electron microscopy.
54
55
^8
J
Plate 10.
radiation
Rat ^idney fixed in saline with microwave
at 70 C and examined by electron microscopy.
40
Plate 11.
radiation
Rat £idney fixed in saline with microwave
at 80 C and examined by electron microscopy.
41
vi
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Page
Plate 12.
Rat kidney fixed in formalin with
microwave radiation at 70 C and examined by electron microscopy.
42
Plate 13.
Rat kidney fixgd in formalin with
microwave radiation at 80 C and examined by
electron microscopy.
43
Plate 14.
Rat kidney fixed inoglutaraldehyde
with microwave radiation at 70 C and examined
by electron microscopy.
44
Plate 15.
Rat kidney fixed inQglutaraldehyde
with microwave radiation at 80 C and examined
by electron microscopy.
45
vii
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INTRODUCTION
Statement of the Project
The aim of this project is to evaluate the use of
microwave radiation as a means of fixation for electron
microscopy.
Routine methods, though adequate,
have failed
to provide an all inclusive method of fixation without ser­
iously damaging the fragile cellular ultrastructure.
This
damage is of great concern to those involved in the rapidly
developing field of immunocytochemistry.
Moreover, the
time, money, and complexity of routine methods have signif­
icantly limited the use of electron microscopy in the diag­
nostic laboratory.
These limitations have led to many areas of research
in the goal of finding non-chemical methods of fixation.
Supported by numerous reports of success at the light micro­
scope level, microwave radiation may be the key to the com­
plete utilization of the electron microscope.
Therefore,
the goal of this project is to determine the feasibility
of microwave radiation as a means of fixation for the elec­
tron microscope.
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2
Literature Review
Since the days of Liewenhook (1662) scientists have
continually strived for ways to look at the world around
them.
This drive to look into the microscopic world has
led to the development of the most powerful instrument of
inspection, the electron microscope.
By using electrons
as the source of energy, researchers have been able to m a g ­
nify objects well over 40,000 times, allowing the individ­
ual the opportunity to examine a world unattainable by con­
ventional light microscopy.
However, along with the advan­
tages, there comes the disadvantage of magnifying the det ri­
mental effects characteristic of tissue preparation.
The
result is that since the development of the electron micro­
scope in the 1940's, researchers have sought ways to limit
the damage that fixation can incur in tissues.
Fixation, by definition,
is the method by which tissues
are preserved in a state that holds its components "fixed"
in situ so that they may be studied with the minimal amount
of alteration from the living state.
The main objectives of
fixation are to preserve the structure of cells with the
minimal amount of alteration from the living state with
regards to volume, morphologic detail, and spatial rela­
tionships of organelles and macromolecules, minimum loss of
tissue constituents and protection of specimens against the
subsequent treatments including dehydration, embedding,
staining, vacuum, and exposure to the electron beam (1).
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Within recent years, attention has shifted from tradi­
tional chemical methods to the products of modern technology
in search of methods that will better realize the goals of
fixation while minimizing any side effects.
Furthermore,
with the advent of new immunohistochemical studies,
the need
to find a non-chemical fixative has been accentuated in hopes
of minimizing chemical modifications of antigenic determin­
ants.
One possible method is microwave radiation; which if
proven reliable has the benefits of speed, ease, and low cost
without using chemicals.
Microwave Radiation
A complete explanation of microwave radiation is far
beyond the scope of this paper
(see Collins 2).
However,
a basic understanding of the properties of microwaves and
their effects are imperative for an understanding of how
one might utilize microwaves in the fixation process.
Microwave radiation is a form of electromagnetic
radiation which falls within the frequency of 300 m e g a ­
hertz to 300,000 megahertz
(3).
A hertz is one variation
or cycle per second and a megahertz 1,000,000 cycles per
second.
Its characteristics are based upon the theory of
electromagnetism and the understanding that microwave radi­
ation, like visible light moves in waves with frequency,
wavelength and period being its fundamental characteristics.
Microwave radiation exists naturally as a part of the
spectrum of radiant energy released by the sun.
It is now
possible to conduct and control man-made microwave energy
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by c o n d uc ti ng it from a t r a n s m i t t i n g source antennae,
re ce iv er antennae.
to a
The w av es tr an s fe r thr ou g h free space
or a m e d i u m as o s c i l l a t i n g electric and magnet ic
g
the speed of light, 3 X 10
me te rs per second.
fields at
Not only does m i c r o w a v e r a d i a t i o n travel at the same
speed as light, but
tics of light.
be reflected,
it also
has several other c h a r a c t e r i s ­
Like light, mi c r o w a v e s carry the a b i l i t y to
scattered,
r e fr ac te d and a bs orb ed by a medium.
It is by ab so rpt io n that m i c r o w a v e rad ia ti on can a c t iv el y
in fl uen ce bio logical systems.
However,
a bs o r p t i o n dep en ds
on the electromagnetic p ro p e r t i e s of the media.
important
variables are the d ia lec tri c
c o n d u c t i v i t y of the tissues
(3).
The most
constant and the
It has been sh own that
at
best only 1 0 % of the ra d i a t i o n is a b so r be d by b io l o g i c a l
tiss ue
(4).
It is im portant to rea liz e that w a te r compri ses
60-70% of a c e l l ’s volume.
10? is absorbed,
Therefore,
even t h o ug h only
bec au se of wat er 's hig h conductivity,
this
small p r o p or ti on of a b so rb ed r a d i a t i o n can have a drastic
effect on the mo l e c u l a r e n v i ro nm en t of water and the tiss ue
molecules
immersed in the water.
Fr om the ele ct ro ma gn et ic
r a d i a t i o n absorbe d by the
cell the amount of ener gy r e l e a s e d per p h o t o n can be c a l ­
c u l a t e d using E = hf
(E=6.625 x 10 “ ^
Joules second).
This
e q u a t i o n is known as Pl ank's e q u a t i o n after its ori gi na t or ,
Max Plank.
constant,
The energy
(E) equals the pr oduct of P l a n k ’s
h, times the f re q u e n c y
(f) o f the radiation.
e n e r g y a bso rb ed is r e l e a s e d into the mo l e c u l e as kinetic
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The
energy
system.
(thermal motions) that raises the temperature of the
If the absorbed energy is above 10 eV it can cause
ionization of the molecules.
The energy per photon released by microwave radiation
-3
_
is 1.24 x 10
eV which is far below the 1.24 x 10' and 2.48
x 10^ eV released by a photon of X or Gamma radiation respec­
tively.
The importance of this is that microwave radiation,
unlike X or Gamma radiation (and other ionizing radiations,
see Table 1) is far below the energy level needed to cause
ionization of molecules.
Ionizing radiation may generate
destructive genetic mutations
(3).
The energy released by the microwave radiation does
have distinct effects on the cellular constituents, particu­
larly the proteins, nucleic acids and lipids.
Its effect is
indirect and mediated by its absorption by the water present
in the tissue.
The energy released by the microwave radia­
tion causes water molecules to vibrate at approximately
2,450 million times per second thereby rapidly raising the
tissue temperature.
Since microwaves travel at the speed
of light, the rise in temperature is extremely rapid.
Chemical Fixation
Tissue fixation can be induced by both physical and
chemical means, but chemical fixation has been studied the
most since it causes the least amount of cellular damage
at this time.
Chemical fixation, unlike physical methods
such as freezing, freeze drying or heating adequately pr e­
serves many cellular components.
Because of these ad-
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6
Table 1
ENERGIES OP ELECTROMAGNETIC RADIATION
O
1
Type of Radiation Wavelength Freauency Energy per Photon
Tnm)
(MHz)
(eV)
3.0 X 10 21* 1.24 X 107
1.0 X
X
5.0 X 10"1 6.0 X 1 0 23 2.48 X 106
Ultraviolet
1.5 X 1 0 1
Visible
3.9 X 1 0 2
2.0 X 1 0 17 8.27 X 101
0
7.7 X 1017 3.18 X 10
Infrared
7.8 X 1 0 2
3.8 X 1017 1.59 X 10°
Microwave
1.0 X 1 0 6
3.0 X 106
1.24 X 10"3
Radio Frequency
1.0 X 1 0 8
3.0 X 102
1.24 X 10-7
H
Gamma
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vantages,
chemical fixation (i.e. formaldehyde, glutar­
aldehyde, osmium tetroxide)
is the most extensively used
method of fixation.
There are three common factors which affect all methods
of fixation:
1)
rate of penetration,
2) pH of solution and
3) osmolarity
of
the fixative. These common factors must be
discussed before one attempts to discuss the individual che m­
ical reactions with cell components.
The rate
of
penetration by the fixative
upon five conditions:
of fixative,
1)
size
of block,
3) fixative concentration,
ation, and 5) temperature of fixative.
is dependent
2) diffusability
4) duration of fix­
The size of the
specimen has been reported to be the most common source of
failure in achieving good results
(1).
proper fixation for electron microscopy,
In order to achieve
the tissue block
must not be larger than 1 mm in thickness and ideally 0.5 mm.
Moreover,
it is of equal importance that the tissue blocks be
uniform in size, otherwise irregular fixation will occur which
results in a decrease in the quality and greater variability
of preservation.
Brunings and Preister
(4) reported the
difference between large and small blocks and demonstrated
artifacts such as extrusions of midgut epithelium of insects
in blocks which were thicker than 1 mm.
The second condition affecting the rate of fixation is
the rate of fixative diffusion.
This area has been studied
extensively and the results have shown a complex of variables
all of which can be altered thereby increasing the diffusion.
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Of these the best understood is the molecular weight of
the fixative.
However, recent studies by Dempster, et a l .
(5) have shown that the rate of diffusion is dependent
upon a coefficient of diffusability K where:
K = __________time________ ,
(depth of pe ne t r a t i o n ) “
K, the diffusability constant,
is a property of each
chemical independent of molecular weight.
Based on this
equation and numerous experiments it has been suggested
that the rate of diffusability for common fixatives in
decreasing order is:
(1)
Formaldehyde— Glutaraldehyde— Osmium Tetroxide
Fixation concentration is the third factor which pro ­
foundly influences the rate of diffusion.
Hyatt
(1) re­
ports that low concentrations of fixative require longer
duration of fixation than high concentrations.
Longer
fixation time may result in numerous detrimental side
effects including tissue autolysis.
It is important to
realize that the concentration of fixative can easily be
tailored for the needs and goals of thestudy.
The final two factors affecting the rate of fixation
is the reciprocal effect of temperature and duration of
fixation.
These factors are noteworthy for their flex­
ibility and relevancy to this study.
By increasing the
temperature one is able to increase the rate of penetra­
tion.
This decreases the duration of fixation by increas­
ing the availability of fixative and at the same time in­
creasing the reactivity of the cellular chemicals with the
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fixative.
However, one must contend with an increased
rate of autolytic destruction which is directly associ­
ated with increased temperatures.
It has been shown that
increasing the temperature with rapid fixatives (i.e. form­
aldehyde) results in better fixation because extremely
rapid fixation is capable of compensating for the increase
in autolytic activity
a slower fixative
(6).
On the other hand, heating of
(i.e. osmium tetroxide) results in ex­
cessive autolytic destruction (6).
Little is known about the effects of temperature on
the kinetics of the chemical reactions of fixation.
There­
fore, unless an optimal temperature for rapid molecular
fixation is determined the period between exposure to the
fixative and completion of the fixation process will permit
continued deterioration of tissue integrity (7).
The regulation of the ti s s u e ’s pH must be taken into
consideration for proper fixation.
The need for an ex­
ogenous buffer is necessitated by the inability of the
t i s s u e ’s buffering system to adequately handle the action
of the fixative (1).
In fact,
it has been shown by Claude
(8), that the application of unbuffered osmium tetroxide
for 48 hours resulted in a pH drop from 6.7 to 4.4.
The
effects of this unchecked pH are serious, especially in
light of the 7.0-7.4 physiologic range for animal tissues
(9).
Wrigglesworth and Packer
(10) showed that the pro-
tiens, which are the major cellular constituents, are de­
natured by low pH, resulting in abnormal protein struc­
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10
tures and subsequent deterioration of the structural pro­
teins.
Moreover, proteolytic and lipolytic enzymes are
activated at lower pH and are thereby increased in auto­
lysis of tissue.
In order to compensate for this drop in pH exogenous
buffers are required.
physiologic range.
These buffers keep the pH within
It is the narrowness of this range
that has limited the number of buffers in use.
The buffer
must be able to accommodate the hydroxyl and hydrogen ions
generated by the reaction between fixative and macromole­
cules through acid-base mechanisms.
Finally, osmolarity must be regulated for satisfac­
tory fixation.
To n i c i t y ’s effect on
easily understood,
tissue integrity Is
although regulation of tonicity is not
nearly as easy to achieve.
If tissues are subjected to a
hypotonic solution, laws of diffusion dictate that water
will move through the cell's semi-permeable membrane from
a greater concentration of solute to a lesser concentra­
tion.
This movement results in the abnormal and destruc­
tive swelling of tissues.
The opposite movement
is ex­
perienced in a hypertonic solution and the result is a
destructively shrunken cell which cannot be studied.
Therefore,
it is crucial for the buffer and the fixative
medium to be isotonic in relation to the tissues.
In
their comprehensive study, Bone and Ryan (11) report that
both the buffer and the fixative affect the osmolarity of
the solution.
Two possible methods can be used to regu-
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11
late osmolarity of the buffer.
These are the addition of
electrolytes and non-electrolytes to the fixative solution.
Common non-electrolytes used are polyvinylpyrrolidine
(PVP) and sucrose.
However, these non-electrolytes are
large enough to decrease the penetration rate of the fixa­
tive and increase the extraction of cellular components
(12, 13).
Furthermore,
some non-electrolytes such as PVP
interfere with the enzymatic activity of the cellular en­
zymes
(1).
It is these deleterious effects that have limited
the use of non-electrolytes in buffers.
Electrolytes,
such as sodium chloride and calcium
dichloride and others,
have been more successful in ade­
quately preventing osmolarity-evoked destruction.
Unlike
non-electrolytes, most electrolytes do not have the detri­
mental effects upon penetration rate and cellular extraction
synonymous with the larger non-electrolyte buffers.
With the three factors of rate of penetration, pH of
solution,
osmolarity of the fixative,
in mind,
it is now pos­
sible to turn the discussion towards the mechanism of fixation.
In the 1950's an assortment of fixatives were used which
proved to be inadequate.
Sabitini
(14),
in 1964, demonstrated
conclusively that glutaraldehyde was the most effective fixa­
tive for preserving cellular ultrastructure, while being flex­
ible enough to use for a broad spectrum of tissues.
More­
over, glutaraldehyde was shown to have a lower detrimental
effect on tissue proteins, both structural and enzymatic, and
therefore could be used for some enzymatic studies
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(14).
12
The specific intermolecular action of glutaraldehyde
is a complex and not completely understood.
Glutaraldehyde
is a straight chain five carbon dialdehyde.
It has a molecular weight of 100.12, a low viscosity and
reacts to an increased pH by polymerization (15).
The success of glutaraldehyde is directly related to
its ability to irreversibly crosslink proteins, thereby
stabilising cellular structure and deactivating its enzymes.
The reaction was first thought to be due to di ald ehydes '
ability to react with the free amino groups to form Schiff
bases:
1)
(16)
protein - NH^+ + CHO - (CH2 )3 " CH0---------protein
2)
-N =
CH -
(CHg) -
protein - M =
CH -
(CH^)^ -
CHO
CHO + +NH^_ protein
protein - N * CH - (CHp )3 - CH = N protein
However, this proposed mechanism of crosslinking was
not supported by experimental evidence that the glutaralde­
hyde fixed tissue can withstand acid treatment, which a
Schiff-base formation cannot
(18) and Hopwood
(17).
Moreover, both Richards
(19) have shown that the kinetics of this
reaction would not account for the fact that Schiff base
formation is reversible while the protein-glutaraldehyde
reaction is not
(17).
The recent theory of the reaction is complex.
What is
known is that the glutaraldehyde reacts primarily with the
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13
epsilon amino groups of lysine
(18).
Jansen,
et al.(20),
using chymotrypsin and zymogen, detailed the loss of enzyme
activity due to the reaction of epsilon amino groups with
glutaraldehyde.
It is now thought that alpha, beta unsatur­
ated aldehydes react with lysine to give a Michael type adduct.
A Michael addition is a conjugate nucleophilic ad­
dition of enolate ions to alpha, beta- unsaturated carbonyl
compounds.
The characteristics of this reaction are consis­
tent with the known glutaraldehyde reaction In that they are
irreversible,
stable to acid and have the appropriate pK (17).
Also, the resulting crosslink of amino groups with a five
carbon chain between two nitrogen atoms is consistent with
the known data detailing electron bridges observed at low
resolution studies on crosslinked lysozyme (1 8 ).
It is important to realize that these are theories of
intermolecular reactions between the dialdehyde, glutaral­
dehyde, and proteins.
They are not fact and are, therefore,
still open to debate.
No matter what the specific reaction
is the result is still the same:
the proteins are cross-
linked and polymerised and as a result,
the cell's structure
is stabilized and its enzymes deactivated.
The reaction of glutaraldehyde with nucleic acids is
not as complex nor as Important to glutaraldehydes action as
a fixative.
Furthermore, the reaction has not been studied
as extensively as it has with proteins.
Hopwood
(21), demon­
strated that the reaction of glutaraldehyde with RNA was
o
o
slight until 45 C and absent with DNA until 64 C. The reason
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14
for this non-reactivity Is related to the tertiary structure
of nucleic acids which are held together by hydrogen bonds.
It is only when the temperatures become high that these hydro­
gen bonds can be broken and the purine and pyrimidine bases
can be made available for reaction with the aldehyde as the
nucleic acids unwind.
It is known that routine glutaraldehyde fixation pro­
cedures utilize low temperatures and therefore,
it has been
theorized that there has to be another mechanism responsible
for the reaction of glutaraldehyde with nucleic acids.
It
has been suggested that the proteins associated with the
nucleic acids are polymerized through their reaction with
glutaraldehyde and form a complex meshwork which ensnares
the nucleic acids thereby "fixing” the nucleic acids without
specifically reacting with the nucleic acids.
Lipids,
the third major intracellular component, also
react with glutaraldehyde In the fixation process, however,
like the nucleic acids the reaction is not nearly as complete
as with theproteins.
There has been little examination of
the reaction, but Roozemond (22) with rat thalamus,
showed
that buffered glutaraldehyde is thought to be due to the
presence of free amino acids in the phospholipids which are
then bound by the aldehyde in the same manner as are the pro­
teins.
It Is important to realize that glutaraldehyde fixa­
tion of lipids is so incomplete that post-fixation with osmium
tetroxide is required, otherwise it is impossible to adequately
study structures made of lipids
(23).
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15
Presently the two most common chemical fixatives are
variations of pure glutaraldehyde:
Karnovsky's solution
and Miloneg's buffered glutaraldehyde.
Karnovsky
(24), in
1965, first reported a medium which used a mixture of for­
maldehyde and glutaraldehyde in an attempt z o broaden their
individual spectra of positive effects while minimizing
their limitations
(24).
The reason for the superiority of
the mixture over using each fixative alone can be attributed
to the symbiotic effect of the two fixatives.
a monoaldehyde,
Formaldehyde,
is a small compound able to penetrate a
tissue faster, thereby stabilizing the cellular constituents
before autolysis can distort
cellular architecture.
Also,
the lack of permanence and limited range associated with
formaldehyde is alleviated by the presence of glutaraldehyde.
This diaidehyde penetrates more slowly due to its larger
size, however,
its previously discussed ability to permanent­
ly crosslink proteins is the source of stability and per­
manence
(1) .
By determining the proper concentrations of each fixative
the microscopist can take advantage of their respective posi­
tive characteristics while minimizing their drawbacks.
The
original mixture suggested by Karnovsky was a 52 glutaraldehyde
buffer (pH 7.2) containing 0.05% calcium dichloride
formulation, reports Hayat
(24).
This
(1), is extremely hypertonic with
an osmolarity of 2010 osmols, which results in severe cellular
distortion.
This has been corrected and now the most widely
used solution is 1-3% glutaraldehyde and 0.5%-2% formaldehyde
with a lower osmolarity.
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16
The Miloneg's solution, on the other hand, utilizes a
pure 2 % glutaraldehyde with a phosphate buffer (25).
The
use of this system is based primarily on the phosphate's abil­
ity to keep the tissue within the proper pH range without
severely affecting the osmolarity.
By keeping the solution
within these restricted parameters one is able to optimize the
effects of glutaraldehyde fixation.
The result is that although chemical fixation is the most
frequently used method of fixation for electron microscopy,
it
still has its drawbacks, primarily due to its dependence on
chemicals.
The detrimental effects of varying tissue pH and
osmolarity have previously been discussed at
length.
But along
with these, there are other causes of tissue destruction in
chemically fixed tissue.
One variable introduced by routine chemical fixation is
the effect of the time delay upon structural preservation.
Standard glutaraldehyde fixation takes two hours; during this
time delay allows for a significant amount of autclytic activi­
ty especially if conditions such as pH are not optimal.
Pre­
sently, there is no way to determine exactly the effects of pro­
longed fixation,
but it is reasonable to assume they are signifi­
cant .
In the discussion of fixation mechanisms it becomes obvious
that although glutaraldehyde is the most effective chemical fix­
ative, it is also limited as to the cellular constituents it can
fix effectively.
fixing proteins,
While extremely effective in crosslinking and
glutaraldehyde is net nearly as effective in
immobilizing carbohydrates and lipids.
Lipid fixation is so
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17
poor that post-fixation with osmium tetroxide is often neces­
sary.
Therefore, the tissue is placed in a second medium which,
like the first, must be adjusted with respect to osmolarity,
pH, temperature and concentration.
This multiplies the number
of variables that will influence the quality of tissue preser­
vation .
A final disadvantage of chemical fixation is the effect
the chemicals have on various special techniques which are
imperative for complete utilization ^f the electron microscope.
Of the special techniques limited by glutaraldehyde fixed
tissue are its deleterious effects on antigen,
hormone and
enzyme reactivity in such fields as immunchistochemistry and
hormone receptors.
It is thought that the glutaraldehyde
changes the secondary and tertiary conformations of the pro­
teins
(antigens, recepters and enzymes),
thereby destroying
their ability to react with the reagents of the specific stain­
ing techniques.
In conclusion,
it becomes obvious that chemical fixation,
despite its position as the preeminent method of fixation, does
not meet the requirements of the demanding, rapidly changing
field of electron microscopy.
Therefore,
the goal of this
study grows out of the need tc find a method of fixation which
removes the negative effects introduced by chemicals.
Physical Fixation
Physical fixation methods, on the other hand, have so
far proven to be seriously limited for electron microscopic
use.
The two primary methods of physical fixation are
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18
heating and freezing.
Freeze techniques,
using special media
and temperatures below -20°C are presently used In surgical
pathology.
This method introduces low temperatures and slow
freezing resulting in the coagulation of tissue molecules,
stabilizing the tissue and causing it to be hard enough for
sectioning.
However, along with the coagulation of molecules
is the production of ice crystals which cause severe damage
at the light microscope level.
This mthod offers adequate
fixation for light microscope examination,
however it is com­
mon knowledge that these standard freeze techniques are inad­
equate for electron microscopy.
Other methods of freeze fixation are presently in use
in research electron microscopy.
Tokuyasu
(2b) has been
successful using a combination of chemical and freeze fixa­
tion.
The procedure consists of a I hour fixation in a
less than 2 % concentration of glutaraldehyde fixative at 4JC.
This step stabilizes the tissues,
but does not cause anti­
genic damage or complete fixation.
Thin sections are then
o
cut at 300-900 Angstroms at a temperature of -7 0 to -90 C.
using a special cry oattachment.
This step completes the
fixation process by freeze methods thereby preserving the
antigenic sites while causing complete fixation.
A more recent development
is a method known as freeze
substitution.
Here the tissues are fixed in liquid helium
o
at 4 Kelvin and then transferred through liquid nitrogen
into liquid acetone at temperatures around 0°C.
The
acetone Infiltrates and stabilizes the tissues by replacing
the water present in the tissues
(27).
This technique has
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19
seen extensive use in the immunocytochemical research
laboratories.
Despite the tremendous utilization and results
gathered by these techniques in research laboratories, they
are not used in diagnostic laboratories.
These methods are
limited mainly by the tremendous cost for routine use.
Special equipment such as cryoattachments, liquid helium
o
and nitrogen, and freezers capable of 4 K all are too ex­
pensive for the clinical laboratory.
Moreover,
the time
consumption and difficulty of the technique make its routine
usage unfeasible.
The use of heat as a method of fixation,
not met with great success.
likewise has
Theoretically it is possible
because high heat coagulates and stabilizes proteins.
But,
the time lapse between routine heating and complete protein
stabilization is long and allows enough time for severe
destruction of the tissue by proteolytic enzymes and fat
oxidation.
Microwave Fixation
As discussed previously, microwave radiation is cap­
able of vibrating molecules at speeds high enough to cause
a rapid increase in kinetic energy.
This property of al­
most instantaneously increasing temperature to a high level
is the basis of* microwave radiation as a method of fixation.
Proteins, the major cellular constituent,
natured and coagulated, thereby fixed when the
of the system reaches 50-60°C.
can be de­
temperature
However, traditional heating
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20
methods take several minutes before these temperatures
are reached.
As Bell
(28) vividly details, the enzymatic
activity is increased significantly to the point of
severely damaging the tissues when the temperature of the
system is increased slowly.
If one v/ere to limit the time
lag during the rise in temperature to only a few seconds,
(i.e. microwave radiation)
it might then be possible to de ­
nature and coagulate the proteins without subjecting the
tissues to the prolonged autolytic activity of the un­
harnessed enzymes.
Like proteins, nucleic acids react to high tempera­
tures in a similar manner.
Temperatures above 50°C re­
sult in denaturation of the nucleic acids thereby fixing
them.
Unlike proteins, however,
if the increase and sub­
sequent decrease in temperature are slow and gradual enough
it is possible to renature the nucleic acids
(28).
In
this case, the additional important characteristic of
microwave radiation is it does not have the energy content
capable of causing mutagenic ionization which could destroy
the structure of the nucleic acids
(28).
Therefore,
it is
reasonable to assume that microwave radiation could serve
as a possible method for fixation of nucleic acids.
Lipids, likewise react to high temperatures in some
way.
However, the exact effect of the rapid increase in
temperature on these lipids has not been determined.
The use of microwave radiation for tissue fixation has
already been shown to be effective at the light microscope
level (29).
Login (30), reports that tissue heated to 60°C
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21
In a saline medium in the microwave oven were superior
structurally to all other microwave and traditionally
fixed tissue.
The result
is that by using microwave fix­
ation the time for fixation was reduced, cheaper solutions
were used (saline vs. formaldehyde), toxic fumes were
eliminated all without damaging the cellular architecture
more than what is found in standard procedures.
Other reports have appeared detailing the advantages
of microwave fixation of fetal tissues
tured cells
(31), tissue cul­
(3 2 ), and even the elution of antibodies from
sensitized red blood cells
(33).
However, only one study
has been reported concerning the use of microwave radiation
for electron microscope study
(3*0.
In their study, Chew,
et al (3*0 examined the preservation of ultrastructure in
microwave fixed rat tissue
(kidney and liver).
The result
of their work is the proposal that the preservation of
ultrastructure was equal to the standard fixation in 2.5^
glutaraldehyde for two hours while also causing no diffi­
culty in sectioning or staining.
As stated previously, the feasibility of microwave
radiation depends upon the speed at which it stabilizes
the cellular constituents.
If it does not achieve this,
microwave radiation will be no better than the discarded
methods of conventional heating.
Another cause for con­
cern is the random, rapid heating of both structural and
enzymatic proteins.
Whereas denaturation of enzymatic
proteins is beneficial to ultrastructural preservation,
the denaturation of structural proteins without the benefit
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22
of crosslinking agents might cause severe structural damage.
Nucleic acids also might suffer from denaturation without
crosslinking resulting in destruction instead of preserva­
tion.
Yet, despite these possible drawbacks previous re­
search has only been positive.
It is now important to de­
termine the feasibility of fixing human tissues with micro­
wave radiation.
Rationale
The aim of this investigation is to evaluate the use­
fulness and reliability of microwave fixation of human
tissues for electron microscope study.
Theoretically and
practically, microwave radiation is a successful method
of fixation for light microscopy
(30-32).
However,
very
scanty evidence is available as to the effect of high in­
tensity microwave radiation on tissues examined at the
ultrastructural level.
This study will compare convention­
al formalin and modified Miloneg's glutaraldehyde fixatives
to a series of trials where the fixation will be done by
microwave radiation using chemical fixatives or saline.
Furthermore, the temperature to which the tissue will be
heated will also be systematically varied to determine the
optimal temperature of fixation.
Ultimately, the goal will
be to determine the fastest and most efficient method of
fixation which maintains the quality of preservation needed
for diagnostic use.
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MATERIALS AND METHODS
Login (30) showed that the optimal temperature for high
o
quality fixation for electron microscopy ranged from 60-65 C.
Using these temperatures as a base, a two stage project was
proposed that first used 3 fixative mediums
(saline, glutar­
aldehyde, formalin) at six separate temperatures (50, 55, 60,
o
65, 70, 80 C) under optimal conditions in hope of isolating
the proper temperature and mediums.
The temperature-medium
combinations that gave adequate quality under optimal con­
ditions would then be tested on surgical specimens.
is defines as:
Optimal
amount of time between excision of tissue
and complete fixation less than 15 minutes.
The kidneys of a 125 gram albino rat were excised and
placed immediately in 0.09? saline at room temperature.
Tissue blocks at 1 mm^ were cut and randomly placed in either
saline, formalin or glutaraldehyde and heated to temperatures
_ o
ranging from 50-80 C.
Control tissues placed in 2% glutar­
aldehyde, and in formalin were fixed overnight.
The samples were individually heated to the proscribed
temperatures using a Kennore model 565 Microwave oven oper­
ating at 2450 MHz and 600 watts.
The temperature of the
medium was monitored by a heat sensor which was previously
shown to be accurate before the experiment.
The microwave fixed tissues and controls were processed
by routine osmium tetroxide post-fixation, dehydration with
23
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ethanol and propylene oxide and epon embedding (Appendix 1).
Thick sections were cut at 1 micron were stained with toluidine blue and basic fuschin (Appendix 2).
ated by light microscopy.
These were evalu­
Those tissues which exhibited ade­
quate fixation were thin sectioned at 8rnm.
Sections were
stained with lead citrate and uranyl acetate (Appendix 3)
and were viewed with a Philips 300 electron microscope.
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RESULT?
The thick sections,
seen in Plate 1, show excellent
preservation of the renal anatomy using normal glutaralde­
hyde and formalin fixation methods.
The glomeruli are well
preserved with no evidence of cellular distortion or dis­
organization.
The tubules,
likewise, are preserved with
good cellular integrity and continuity.
The tissues fixed with the microwave oven using all
three media
(saline, formalin and glutaraldehyde) have a
great variation in preservation
fixed at 50°, 55 °t
(see Plates 2-4).
Those
60 ° 65° all had inadequate preservation
for further study.
The glomeruli were obliterated with
little recognizable structure.
The tubules are disorganized
with severe nuclear and cytoplasmic clumping within the
cells.
The lumina are filled with displaced epithelial cells
Overall, these temperatures did not produce adequate pre­
servation regardless of the medium used.
When the
temperature of fixation was raised to 70°
and 80°C the preservation of structure in all media was im­
proved
(see Plates 5-7).
The glomeruli were well organized
and the tubules were intact with prominent nuclear staining.
These sections were equal to if not better than the control
sections.
From these results tissues fixed in all media at
o
n o
70 and 80 C were chosen for further processing and study by
electron microscopy.
25
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26
Plate 1.
Rat kidnev controls fixed in two standard fixatives
and examined by light microscopy, a) formalin, 1800x, b)
glutaraldehyde, 1800x.
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27
Plate 2.
Rat kidney fixed in saline with microwave radiation
at vagious temperatures and examined by light microscopy,
a) 50 C, 1800x, b) 55°C, 1800x, c) 60°C, 1800x, d) 65 C, 1800x.
I
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Plate 3. Rat kidney fixed in formalin with microwave radiation
at various temperatures and examined by light microscopy,
a) 50 C, 1800x, b) 55 C, 1800x, c) 60°C, 1800x, d) 65°C, 1800x.
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31
Plate 4.
Rat kidney fixed in glutaraldehyde with microwave
va
radiation at varigus
temperatures and examined by light
microscopy, a) 50 C, 1800x, b) 55°C, 1800x, c) 60°C, 1800x,
microgcopy,
d) 65 C , 1800x.
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Plate 5.
Rat kidney fixed in saline at two temperatures
with microwave radiation and examined by light microscopy,
a) 70 C , 1800x, b) 80°C, ISOOx.
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34
Plate 6.
Rat kidney fixed in formalin with microwave radi­
ation at two temperatures and examined bv light microscopy,
a) 70 C , 1800x, b) 80°C, 1800x.
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35
Plate 7.
Rat kidney tixed in glutaraldehyde with microwave
radiation at two temperatures and examined by light m icros­
copy, a) 70 C , 1800x, b) 80°C, 1800x.
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36
The selected tissues were thin sectioned at
The controls shown in plates 8 and 9 demonstrate good
preservation of kidney ultrastructure.
Both glomeruli and
tubules show excellent organization and continuity with no
damage to structural integrity.
fy all cell types
It is possible to identi­
(endothelial, epithelial and mesangial)
and the nucleus and cytoplasm of each have excellent clari­
ty.
ing.
There is no appreciable disruption or chromatin clump­
The pedicles are seen as extensions of continuous
epithelial cells and the basement membranes are clearly discernable.
Tubules were comparably preserved
(not shown).
Plates 10 and 11 are representative of microwave fixed
tissue at 7 0° and 80°C in saline.
Glomeruli and tubules
are distorted with obvious destruction of their anatomical
continuity and integrity.
The glomeruli are not preserved
and it is difficult to understand the structure and identify
its characteristic
cells.
The podocytes have lost their
cellular attachment and are partially degraded.
The base­
ment membrane has lost its integrity and continuity.
The
tubular epithelial cells contain altered mitochondria, dis­
torted borders with loss of intercellular adhesion and
clumping of both the nuclear and cytoplasmic matrix.
Overall
the preservation is totally inadequate.
The microwave-formalin fixed tissues at 70° and 80^C
likewise show an unacceptable amount of ultrastructural
damage with nearly the same negative features as seen In the
tissues fixed in saline.
As can be seen in Plates 12 and 13
the preservation though slightly better is still inadequate.
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37
Finally, Plates 14 and 15 illustrate that tissues
microwave fixed in glutaraldehyde at 70^ and 80°C are also
inadequately preserved despite its better preservation of
glomerular podocytes.
Once again, the overall continuity
and integrity of both the glomeruli and tubules along with
the intracellular damage, particularly the nuclear damage,
is too great to use the tissues for diagnostic
study.
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r
38
Plate 8 . Rat kidney control fixed in formalin and examined
by electron m icroscopy,10,260x.
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39
Plate 9. Rat kidney control fixed in glutaraldehyde and ex­
amined by electron microscopy, 10,260x.
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40
PlateQ 10.
Rat kidney fixed in saline with microwave radiation
at 70 C and examined by electron microscopy, 4500x.
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41
Plate ll.QRat kidney fixed in saline with microwave radia­
tion at80 C and examined by electron microscopy, 4500x.
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■I
42
Plate 12.
Rat kidney fixed in formalin with microwave
radiation at 70 C and examined by electron microscopy, 4500x.
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43
Plate 13.
Rat kidney fixed in formalin with microwave radiation
at 80°C and examined by electron microscopy, 4500x.
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f
44
Plate 14.
Rat Jeidney fixed in glutaraldehyde with microwave
radiation at 70 C and examined by electron microscopy, 4500x.
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45
Plate 15.
Rat jeidney fixed in glutaraldehyde with microwave
radiation at 80 C and examined by electron microscopy,4500x.
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DISCUSSION
Data Analysis
This study was carried out to investigate the feasibil­
ity of using microwave fixation for electron microscopy in the
surgical pathology laboratory.
duce prior claims of success
Its design was chosen to repro­
(34) and to test the hypothetical
advantages of non-chemical fixatives.
The two stage program
was used in order to eliminate the number of trials by elimin­
ating those trials that were inadequate under ideal conditions.
The different temperatures were used in hopes of isolating the
optimal temperature for fixation.
The different media were
used to determine if saline could replace formalin and/or
glutaraldehyde in the hope that this would eliminate the need
for expensive, damaging,
toxic chemicals.
The results illustrate that none of the temperaturemedium combinations resulted in an acceptable preservation
of ultrastructure.
Since these results were obtained with
rat tissues obtained promptly,
there was no justification
for studying microwave fixation of surgical pathology spec­
imens which have varying amounts of delay prior to fixation.
Exactly why microwave fixation resulted in such poor
preservation in light of
certain.
Chew et a l . (34) results is un­
The light microscope study was consistent with
other investigations
(30- 3 2 ) and originally led to expecta­
tions of good results at the electron microscope level.
46
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47
However,
it is obvious that the demands of electron micros­
copy are much more rigorous than light microscopy.
As stated previously,
the quality of fixation is dir­
ectly related to the fixative's ability to stabilize cellu­
lar proteins, particularly enzymatic proreins.
Microwave
heating does this by increasing the system's vibrational
energy to the level where the heat produced unravels the
complex protein structure.
This process,
ation, allows the proteins to crosslink,
known as denaturleading to fixation.
There are two ways in which the microwave heating
mechanism might result
hibited.
in the structural alterations ex­
First, the time elapsed between introduction of
the heat and when denaturation and fixation are complete
might be long enough to allow oxidation of lipids and in­
creased activity of proteolytic enzymes to destroy the
tissue.
Since the heating is almost
instantaneous and
temperatures of 70° and 80°C are reached in the matter of
seconds,
it is unlikely that the increase in proteolytic ac­
tivity associated with the rise in temperature will have a
prominent effect in this short period of time.
Alternatively, the energy increase may be too random
and severe resulting in the non-selective denaturation of
both globular
(enzymes) and fibrous
(structural) proteins.
On the one hand, denaturation of enzymatic proteins needs
to be complete.
The rapid rise in temperatures to levels
of 70° and 80°C are excellent for deactivating the enzymatic
proteins thereby inactivating their autolytic activity.
If enzymes were the only proteins denatured by high tempera-
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48
cures the results would have been excellent quality of
fixation.
However,
enzymatic proteins,
the heat increase affects not only the
but also the structural proteins.
Fibrous proteins,
on the other hand, are proteins
noted to have an axial secondary configuration which makes
them ideal for their structural responsibilities.
Located
both intra and intercellularly, examples include collagen,
alpha keratin and elastin.
It is probable that the rapid
heating of these proteins results
in the denaturation of
not only their quananery and tertiary structures,
their secondary structures.
but also
Whereas the denaturation of
enzymatic proteins is a positive factor in fixation,
the
denaturation of the structural proteins secondary struc­
tures results in an unravelling and disassociation of their
basic chemical structure without the crosslinking action
of chemical fixatives.
As a result, the elastin and collagen
fibers integral for intercellular adhesion and basement
membrane integrity may be totally disrupted and destroyed.
Moreover, the organelle destruction exhibited in the trials
may be due to the disruption of the membrane proteins.
Therefore,
it can be theorized that the majority of ultra-
structural damage seen in the 70° and 80°C trials can be
attributed to the denaturation of the secondary structures
of the fibrous proteins.
However,
it is unlikely that the tremendous destruc­
tion of tissue seen at 70° and 80°C can be attributed solely
to the denaturation of fibrous proteins.
The other cellular
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4S
constituents,
fats and nucleic acids, also showed evidence
of heat destruction, though not as pronounced as the pro­
teins denaturation.
The nuclear chromatin clumping and destruction results
from the destruction of the nucleic acid structure by high
Q
temperatures.
Temperatures above 35 C are enough to rup­
ture over 50% of the hydrogen bonds, which hold the double
helical structure intact.
In doing this,
there is complete
denaturation and destruction of nucleic acids.
there is evidence of partial cleavage
Below 35°C
(less than 50 % )
of
the hydrogen bonds and also denaturation of the hydrophic
forces between stacked pyrimidine and purine bases.
though the rupture of the structure is incomplete,
Al­
there is
enough energy to cause the clumping and destruction seen
in the 70° and 80°C trials.
At lower temperatures
(50°, 55°, 60°, 65°C) the tis­
sue destruction can be attributed to non-deactivation of
the enzymatic proteins.
As previously discussed,
temperatures over 65°C
until
reached, the levels of enzymatic
activity actually increases proportionally to the tempera­
ture increase.
Therefore,
fix the proteins,
the lower temperatures do not
but actually increase their activity.
Their unleashed activity can easily cause the tremendous
destruction seen at light microscopy, through increased
autolytic and biodegradative processes.
The fact that
70°C fixed tissue,
independent of medi­
um, resulted in the best fixation accentuates the diphasic
pattern of destruction exhibited by these tissues.
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At this
50
temperature the enzymatic proteins had been best stabil­
ized with the minimal amount of structural protein dena­
turation,
fat oxidation and nucleic acid destruction which
are artifacts of higher temperatures.
However, it is uno
o
likely that a temperature between 70 and 80 C would re­
sult in better preservation because the difference between
the preservation in these two temperatures is very subtle.
The result of using microwave radiation is poor pre­
servation for electron microscopy no matter what the tem­
perature-medium combination used.
Though the damage may
be minimized to the extent that the tissues can be studied
with the light microscope,
the demands of electron micros­
copy are far too rigorous to accept the poor preservation
and severe distortion induced by microwave radiation.
Therefore, unless there is devised a way to selectively
heat the proteins,
lipids, and nucleic acids so as to mini­
mize the side effects of microwave radiation,
it will not
be a successful tool for fixation of tissues for electron
microscopy.
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51
Overall Conclusions
Prom this study it can be stated that microwave radi­
ation cannot be used as a fixative for electron microscopy.
The effects of the high, rapid, random heating,
character­
istic of microwave radiation, destroyed the delicate ultra­
structure leaving thetissue inadequate for diagnostic inter­
pretation.
This study can be used to remind those who wish
to investigate other methods of heat fixation that the ef­
fects of high heat are still too severe for the delicate
structures.
Unless there is developed a technique to limit
the negative effects inflicted by the heat while retaining
its fixative capabilities,
heat will never be useful as a
fixative for electron microscopy.
However, this does not exclude microwave radiation
from the diagnostic
laboratory.
This study lends credence
to prior results of good fixation for light microscopy.
Al­
so, with a better understanding of how microwave radiation
is used in light and electron microscopy,
to use microwaves
for other steps in the preparation of tis­
sues for electron microscopy;
ising.
it may be possible
staining being the most prem­
Therefore, although microwave radiation is an inad­
equate means of fixation for electron microscopy,
its useful­
ness might be realized in other areas of routine preparation.
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REFERENCES
Hayat M.A.: Fixation for Electron M i c r o s c o p y , Academic
Press, Mew York, N .Y . , 1 9 8 1 .
2.
Collins R.E.: Foundations for Microwave Engineering,
McGraw Co., New York, N.Y., 1966.
3.
Lin J.: Microwave Auditory Effects and Applic a t i o n s ,
Charles C~. Thomas C o . , Springfield II., 1978.
4.
Metalsky I.: Non-Ionizing Radiation.
In Industrial
Hygiene Foundation , L.V. Cralley and G. D. Clayton, Eds.,
McGraw Co., Mew York, N.Y., 1968, pp. 140-179.
5.
Brunings E.A. and dePriester W.: Effect of Mode Fixa­
tion on the Formation of Extrusions In the Midgut Epi­
thelium of Calliphora.
Autobiology 4_, 487 (1971 ).
6.
Depmster W.T.: Rates of Penetration of Fixing Fluids.
AM. J. Anat. 107, 59 (I960).
7.
Hayat M.A.: Principles and Techniques of Electron Microscopy: Biological A D D lications, University Press, Balti­
more^ ~MD . , 1981“
8.
Paris R.B., Kelley J., Drury S., Sauer K . : The Hill Re­
action of Chlcroplasts Isolated from Glutaraldehydefixed SDinach Leaves.
Proc. Natl. Acad. Sci. 55, 1056
(1 9 6 6 ).
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9.
Claude A.: Fixation of Nuclear Structure by Unbuffered
Solutions of Osmium Tetroxide In Slightly Acid Distilled
Water.
Proc. Inter. Congr. Electr. Microsc. 5th ed. 2,
1-14 (1962).
10.
Butler T.C., Waddell W.J., Poole D.T.: Intracellular pH
based on the Distribution of Weak Electrodes.
Fed.
Proc. 26, 1327 (1967).
11 .
Wrigglesworth J.M., Packer L.: pH Dependent Conforma­
tional Changes In Submitochondrial Particles.
Arch.
Biochem. B i c p h y s . 1 3 3 » 194 (196 8 ).
12.
Bone Q., Ryan K.P.: Osmolarity of Osmium Tetroxide and
Glutaraldehyde Fixatives.
Histochem. J. 2, 1 (i9 6 0 ).
52
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13.
Wood R.L., Luft J.H.: The Influence of the Buffer System
on Fixation with Osmium Tetroxide.
J. Cell Biol. 19, 83A
(1974) .
14.
Hagstrom L. Bahr G.F.: Penetration Rates of Osmium Tefcrox
ide with Different; Fixation Vehicles.
Histochemistry 2,
(I960).
15.
Sabitini D., Bebsch K., 3arnett R.: New Means of Fixation
for Electron Mxcroscooy and Histochemistry.
A n a t . Record
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16.
Korn A.H., Feairheller S.H., Filaohione E.M.: Glutaraldehyde: Mature of the Reagent.
J. Mol. Biol. £5, 525529 (1972).
17.
Peters T., Richards F.:
46, 253 (1 9 7 2 ).
18.
Richards F.M., Knowles J.R.: Glutaraldehyde as a Protein
Crosslinking Reagent.
J. Mol. Biol. _37 231-233 (1968).
19.
Annual Review of Biochemistry.
Hcpwood D.: Review of
Glutaraldehyde as a
Histochem. J. 4, 267-303 (1972).
Fixative.
20.
Jansen E. Tomimnastee Y., Olsen F . : Crosslinking of
Alpha Chymotrypsin and Other Proteins by Reaction with
Glutaraldehvde. Arch. Biochem. Bioohys. 144, 394-400
(1971).
21.
Hopwood D.: The Reactions of Glutaraldehyde with Nucle­
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22.
Roozemond R.B.: The Effects of Fixation ’
w ith Formalde­
hyde and Glutaraldehyde on the Composition of Phospho­
lipids Extractable from Rat Thalamus.
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Cytcchem. 5, 123-128 (1972).
23.
Wood J.: The Effects of Glutaraldehyde and Osmium Tet.roxide on Froteins and Lioids of Myelin and Mitochondria.
3iochem. 5ioph.ys. Acta. 329, 118-127 (1973 ).
24.
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High Osmolarity for use In Electron Micro s c c o y . J.
Cell Biel. 27, 137 (1965).
25.
Miloneg G.: Advantages of a Phosphate Buffer for CsO^
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26.
Tokuyasu K.T.: A Study of Positive Staining of ultrathin
Frozen Sections.
J. Ultrastr. Research 63, 287-307
(1978).
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Personal Communication.
May 1 9 8 5 .
27.
Papermaster D.:
28.
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Physiology and B i o c h e m i s t r y . 8th e d . Williams and
Wilkens Co., Baltimore, M D . , 1972.
29.
Schneider D., Pelt B., Goldman H . : On the Use of Micro­
wave Radiation Energy for Brain Tissue Fixation.
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Neurochem. 38_, 749-752 (1 9 8 2 ).
30.
Login G.: Microwave Fixation versus Formalin Fixation
of Surgical and Autopsy Tissue.
Am. J. Med. Technol.
44, 435-437 (1978).
31.
Petere J., Scharden J.: Microwave Fixation of Fetal
Specimens.
Stain Technology 55_, 71-75 (1980).
33.
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tural Study of Microwave Fixation of Tissues.
Cell
Biol. Intern. Reports 7, 135-139 (1 9 8 3 ).
54
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A P P E N D IX
I
ROUTINE FIXATION, POST FIXATION, DEHYDRATION
AND EMBEDDING PROCESS EMPLOYED AT THE
WEST HAVEN VA MEDICAL CENTER
Fixation:
1.
2% Glutaraldehyde for 2 hrs. or overnight.
2.
Rinse tissues in Q.05M sodium cacodylate for 5
minutes at 25°C.
3.
Post Fix in 1% Osmium Tetroxide for 1 hour at 4°c.
4.
Rinse in sodium cacodylate for 5 minutes at 25 C .
Dehydrat i o n :
1 . 50$ ethanol
for 5 minutes at room
temperature.
2 . 7 0 $ ethanol
for 5 minutes at room
temperature.
3 . 9 5 % ethanol
for 5 minutes at room
temperature.
4.
Two rinses, 15 minutes each at room temperature,
100$ ethanol.
i
5.
100$ ethanolrpropylene oxide
at room temperature.
6.
Propylene Oxide for 15 minutes at room temperature
7.
Propylene oxide:Epon
8.
Epon until embedding.
(1:1) for 15 minutes
(1:1) 1 hour or longer.
EMBEDDING:
1.
Embed in epon for 48 hours at 5c°-60°C.
55
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A P P E N D IX
II
ROUTINE TOLUIDINE BLUE AND BASIC FUSCHIN
STAINS USED AT THE WEST HAVE
VA MEDICAL CENTER
Toluidine Blue:
1.
Place thick section on slide in a drop of* distilled
water.
2.
Dry on hot plate to permanently Tix section to slide
3.
Place a Lev/ drops of toluidine blue stain so to cove
the sections.
Heat until steam is visible, but do not evaporate
s tain.
5.
Wash slide under running tap water, thenfinal rinse
with distilled water.
Basic Fuschin:
1.
Make sure slide is completely clear of* toluidine
blue by adequately rinsing with distilled water.
2.
Drop a few drops of basic fuschin on slide so to
cover the sections.
3.
Heat for 15 seconds.
4.
Wash slide under running tap water,
rinse with distilled water.
5.
Dry,
then final
coverslip and label.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
APPENDIX III
URANYL ACETATE AND LEAD CITRATE STAINING
PROCEDURES USED AT THE WEST
HAVEN VA MEDICAL
CENTER
Uranyl Acetate:
1.
Centrifuge the uranyl acetate In 25% ethanol
m i n utes.
2.
Place drop of uranyl acetate on a piece of parafil
in a petri dish.
3.
Put grids with sections down on top of uranyl ace­
tate drop.
Stain for 15 minutes.
4.
Wash by immersing the grids in 3 changes
et h anol.
5.
Dry on #50
of
for 5
25%
filter paper.
Lead Citrate:
1.
2.
3.
Centrifuge the lead citrate for 5 minutes before
staining.
Place drop
in a Petri
of lead citrate on a piece of parafilm
dish containing a few pellets of NaOH.
Place grids with sections down on top of lead cit­
rate drop.
Stain for 4 minutes.
Wash in a stream cf 0.02M NaOH and Immerse in 3
changes of distilled water.
5.
Dry on #50
filter paper.
57
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