Microwave Processing Techniques for Microscopy
From Energy Beam Sciences, Inc., Agawam, MA
How does a microwave oven work?
How does a laboratory microwave processor
differ from a kitchen microwave oven?
Safety Features and Temperature Control
What are some applications where a microwave processor
would be useful?
How is a laboratory microwave oven useful in electron microscopy?
Energy Beam Sciences' Microwave
Processors
Microwaves are electromagnetic waves. Electromagnetic
waves can be classified by their frequencies, and include radio
waves,
television signals, radar beams, infrared waves, visible and ultraviolet
light,
X-rays and gamma rays. Electromagnetic waves which have a frequency
between 300 MHz and 300 GHz are classified as microwaves. These
two frequencies correspond to wavelengths of 1 m and 1 mm, respectively.
All domestic microwave ovens and laboratory microwave processors
operate at 2.45 GHz (corresponding to a wavelength of 12.2 cm,
or just over 4-3/4").
Microwave technology evolved out of the development of radar (Radio
Detection And Ranging). Because microwave pulses can be very short,
they can be used for distance and time measurement. The simplest
form of radar measures the time for an echo to return from a certain
direction. Microwaves penetrate fog and clouds, travel in straight
lines, and give distinct shadows and reflections.
The original magnetron was invented by Albert Hull at the GE Research
Laboratory in 1916. The microwave oven was invented in 1945 by
Percy Spencer of Raytheon, who received a U.S. patent in 1950.
The first commercial microwave oven appeared on the market in 1947.
In the early 1950's, 50-100 units were sold per month, at a price
of about $4000.00 each. In 1967, Amana introduced a counter-top
model with a retail price of less than $500.00. Ten years later,
in 1977, 3% of U.S. households owned a microwave oven. In 1987,
12.8 million microwave ovens were sold. According to some sources,
in 1992, 90% of U.S. households owned a microwave oven, and the
number worldwide now exceeds 100 million.
Microwave ovens provide an effective way of heating many nonconductive
materials. Microwaves penetrate the material; whether or not heat
is generated is determined by the specific dielectric properties
of the material itself. In most materials, the microwave-power
absorption is proportional to the water content of the material.
The frequency of commercial microwave ovens (2.45 GHz) was selected
so that a standard portion of food would be heated uniformly. Because
the heat does not have to be conducted thermally through the food,
but is generated inside the materials, microwaving reduces the
time needed for heating the food to a uniform temperature.
Microwaves cause heating within a material by exciting molecules
to rotate. This rotation produces energy in the form of heat. Unlike
conventional heating, this effect occurs simultaneously throughout
the whole material being microwaved. This has important implications
for microscopy, because the basis of much specimen preparation
is the effective diffusion of fluids in and out of tissue blocks
or sections. Heat increases the rate of diffusion, and microwave
(internal) heating can enhance it even more effectively.
As an example, two 2 x 2 x 2 cm3 cubes of beef (striated muscle)
were dehydrated. One cube was heated externally at 70 C in 100%
ethyl alcohol for 5 minutes, the other kept at that temperature
by microwave exposure. In the case of external heating, only the
outer part of the cube was slightly dehydrated (hard and grey),
but the microwaved cube was completely dehydrated (hard and grey
all the way through), illustrating the more effective diffusion
of alcohol into the interior of the material.
These same properties of microwave heating will dictate the choice
of which processing fluids to use. Different substances subjected
to the same amount of microwave energy heat up at different rates.
For example, 100 ml of water needs 2.2 times more heat to warm
up than 100 ml of alcohol. The materials that heat up fastest are
comprised of non-symmetrical polar molecules, which are easily
rotated by microwave energy. This can have important implications
for the microscopist. For example, xylene has been the clearing
agent of choice for most conventional histology because of its
fast diffusion rate, despite the fact that it is flammable, causes
dermatitis and can shrink tissue. With microwave processing, however,
isopropanol penetrates faster than xylene; and isopropanol is much
less harmful, and causes less shrinkage of specimens.
The problem in establishing routine laboratory procedures using
microwave technology has been the inadequacy of kitchen microwave
ovens for laboratory use. Kitchen microwave ovens are rated by
their maximum output power levels (e.g. 700 watts), and the only
way of varying the amount of microwave energy entering the oven
cavity is by switching the magnetron on and off over a period of
time. All kitchen microwave ovens have preset cycle times, usually
between 15 and 30 seconds. Therefore, if an oven with an output
of 700 W and a cycle time of 30 seconds is required to operate
at half its power (350 W), the magnetron is on for the first 15
seconds and off for the subsequent 15 seconds. In laboratory use,
this often results in a cycle of heating and cooling that produces
suboptimal and inconsistent results. At even lower power levels,
the problem is exacerbated by the fact that the magnetron needs
a second or so to warm up and begin emitting microwaves. For example,
if 150 W of power is required, the magnetron should only emit microwaves
for the first 6 seconds of each 30 second cycle. But, in this case,
it makes no difference whether total time has been set for 6, 15
or 30 seconds. In all of these cases, the magnetron will actually
emit microwaves for 6 seconds only. In the same example, time settings
of 2:08 and 2:30 minutes give the same amount of exposure, whereas
2:36 minutes will give much more.
Further control of the temperature of microwaved materials can
be achieved through use of a temperature probe which is connected
to power-level control. After a cycle of exposure, temperature
is checked against a preset value. When this value is reached,
the exposure pattern is adapted to maintain this temperature. However,
this control is still very imprecise with kitchen microwave ovens.
Often, temperatures can only be set in multiples of 5 C or over
a limited range of temperatures. More important, when after a cycle,
the desired temperature has almost, but not quite, been reached,
the next cycle may overshoot the preset temperature. The lack of
fine control becomes especially dramatic in the case of small laboratory
samples, which can easily overheat and become damaged. Conversely,
in the case of relatively microwave-transparent materials (like
paraffin), this pattern does not usually suffice to maintain the
desired temperature.
A further way of controlling temperature is
through the use of a "dummy load"- a vessel of tap
water placed in the back of the oven which functions as a heat
sink, and thereby reduces
the power absorbed by other specimens in the oven. In general,
the rate of temperature rise slows in proportion to the size of
the dummy load, but the shape of the container, its location, and
the initial temperature of the water, all have an effect.
The H2800 is a microwave processor designed specifically for laboratory
use. It differs from kitchen microwave ovens in respect to its
safety features, and in the degree of user control it provides.
In a microscopy laboratory, solvents and toxins are heated, producing
fumes. In any kitchen microwave oven, there is a risk that these
fumes could be inhaled, or that they could enter the electrical
control system, where high voltage switching occurs, with subsequent
risk of ignition. The Energy Beam Sciences H2800 Microwave Processor
is outfitted with a high-powered extraction system which removes
air from the cavity at a rate exceeding 100 cfm (cubic feet per
minute), which is then vented into a fume hood or other exhaust
system.
Moreover, this extractor is interlocked with the microwave control
system to prevent operation of the instrument should the fan fail,
or the venting become obstructed. A range of specially-designed
plasticware is available to avoid the use of metal and glass containers
in the H2800.
The H2800 incorporates a unique, custom-made
and very sensitive temperature probe, which is accurate to +/-1/2
C. Other features
include a rotator and a sophisticated wave stirrer, both designed
to minimize temperature variations within the cavity. An adjustable-speed
air pump agitation system is provided to produce even distribution
of temperature within a container of stain or other reagent. Cycle
time can be selected by the user. Since a shorter cycle time results
in more precise temperature control, a cycle time of 2 seconds
is recommended. The key component of the instrument is a built-in
microprocessor which allows almost perfect realization of an ideal
temperature curve. Two modes of timer control are available: one, "total
time", in which the total process time is selected, and the
timer begins counting down as soon as the "run" control
is activated, and a second, "time at temperature", in
which the timer is activated only after a preset desired temperature
is reached.
With the H2800, either an actual temperature, or a power setting
can be selected by the user, depending on the microwave procedure
being followed. Reliable temperature control within 1/2 C can be
achieved. In the power control mode, relative settings from 1%
to 100% can be chosen, and both the percentage power and the temperature
are continuously displayed during operation.
Fixation: Microwave Stabilization of Unfixed
Tissue:
Fixation of Tardigrades (Echiniscus viridissimus)
Microwave-Stimulated Fixation with Fixatives:
Histoprocessing: Microwave Histoprocessing
Using Paraffin
Combining Microwave and Freezing Techniques:
Special stains
Antigen retrieval
Specimen preparation for transmission electron microscopy
Fixation: Microwave Stabilization of
Unfixed Tissue:
The great advantage of microwave stabilization
is that there are no chemicals involved which would extract important
components
from the tissue. Researchers have found that up to 40% of protein
can be lost after formaldehyde fixation. However, other researchers
have found significant disadvantages in this method (shrinkage,
sponginess of tissue, and breakdown of red blood cells). Kok and
Boon recommend a "hybrid" method, in which chemical postfixation
is done after the initial microwave stabilization. In this case,
the "poaching" effect of microwave stabilization seems
to create channels through the tissue, permitting subsequent enhanced
diffusion of fixatives into the cell.
Dr. Anthony S.-Y. Leong has published a method of processing 30,000
surgical biopsies per year, incorporating a microwave stabilization
step. This method involves microwaving 20 blocks of tissue in cassettes,
placed in a beaker of 500 ml normal saline on a rotator for 5 minutes
at 68 C. The stabilized blocks are then transferred to a tissue
processor for dehydration, clearing and paraffin embedding. According
to Leong, the absence of noxious formalin fumes in the processing
room is an important advantage of the procedure, and the elimination
of formalin in both the fixation process and in the processing
has improved the quality of antigen preservation in the tissue
sections.
Fixation of Tardigrades (Echiniscus viridissimus)
From Ruth and Bill Dewel, Appalachian State University, College
of Arts and Sciences, Department of Biology, Boone, North Carolina,
28608.
Tardigrades (Echiniscus viridissimus) were placed in 2ml of preheated
prefix (see below) and immediately exposed to microwaves. The instrument
(an Energy Beam Sciences H2500 Microwave Processor) was set for
maximum power with a 40 degree C endpoint measured by the temperature
probe set in water in a beaker. The specimens were then rinsed
in 0.2M s-Collidine buffer for 20 minutes and postfixed (see below)
using the same method as above.
Prefix:
4% paraformaldehyde
1.5% glutaraldehyde
0.1M cacodylate buffer
0.025% Calcium chloride
Postfix:
2% Osmium tetroxide
0.2M s-Collidine buffer
0.07% Calcium chlorid Microwave-Stimulated Fixation with Fixatives:
Microwave exposure can be used to enhance diffusion of fixation
reagents into the tissue, and to accelerate the chemical process
by which the fixative cross-links with the protein of the tissue.
The most common histological fixative, formalin, is a solution
containing methylene glycol and a little fomaldehyde. Normal formalin
fixation takes place in three steps: first, the methylene glycol
quickly penetrates the tissue (formalin will penetrate a 5 mm block
in 4 hours); second, some methylene glycol is slowly converted
to formaldehyde by dehydration; third, formaldehyde binds very
slowly to the proteins in the tissue by cross-linking. All three
of these steps can be accelerated by microwave exposure.
However, simply microwaving tissue in formalin
produces disappointing results, because the outside of the tissue
fixes so rapidly and
well that it effectively prevents further diffusion of fixative
into the central part of the biopsy. For that reason, a "hybrid" procedure
is recommended. First, tissue blocks are soaked in formalin for
4 hours at room temperature (longer, if blocks thicker than 5 mm
are used); next, the blocks are microwaved for 1.5 minutes at 55
C. Some researchers use shorter soaking times in diluted formalin
solutions. Excellent immunostaining has been achieved using this
hybrid method of fixation.
However, excellent fume extraction (such as the system supplied
with the H2800) is a necessity when using formalin, fumes are still
highly unpleasant, and great care must be taken when handling the
heated formalin. For these reasons, we recommend the use of Leong's
method, or the substitution of an ethyl alcohol-based fixative,
Kryofix..
The absence of formalin is clearly a great advantage for the clinical
personnel and laboratory technicians. However, the microscopist
will be confronted with a slightly different morphology than that
of formalin-treated tissue. There are some disadvantages (more
pronounced shrinkage, for example) and some advantages, particularly
in the field of immunopathology, where Kok and Boon have found
superior positive staining. As with formalin, better results are
achieved by soaking tissue in Kryofix. prior to microwaving; however,
the soaking times are much shorter than for formalin.
Histoprocessing: Microwave Histoprocessing
Using Paraffin
Paraffin wax has been used as an embedding medium in histoprocessing
for over 100 years. It is a good embedding medium for routine histology
because it can thoroughly permeate the tissue in its liquid form
(when warm), and it solidifies (when cooled) with little damage
to the tissue. However, before tissue can be embedded, it must
be subjected to the following procedures:
Completion of fixation;
Removal of formalin from the tissue (if fixed in formalin);
Gentle and complete dehydration;
Removal of the dehydration fluid with an intermedium (clearing
agent) miscible both with the dehydration agent and paraffin
wax;
Impregnation of the tissue with melted paraffin wax. Most histopathology labs now use automated tissue processing machines
which use 12 containers and require 6-20 hours for processing.
When microwaving is used in histoprocessing, these procedures
are not only accelerated, but fundamentally changed:
Completion of fixation is achieved prior to histoprocessing
in the microwave oven.
Dehydration is achieved in one step, instead of the 2-6 steps
used in conventional procedures. The use of a graded series
of alcohols is not necessary in the microwave method.
Isopropanol can be substituted for xylene as a clearing agent,
and one bath is sufficient.
Higher temperatures are required for the impregnation of the
paraffin wax. Therefore, microwave histoprocessing becomes a three-step process:
One dehydration step
One clearing step
One paraffin wax step (at two temperatures) Using
the H2800, processing schedules for thin (>1 mm), medium-thin
(1-2 mm) and thick (2-5 mm) blocks of tissue take a total time
of 25 minutes, 60 minutes and 165 minutes, respectively. This technique
allows the histology lab to routinely process tissue for same-day
diagnosis. Using four special TeflonŽ histoprocessing racks, which
each accommodate 24 standard cassettes, up to 96 cassettes can
be processed at a time using this method. The cassettes themselves
are never handled during this process. The racks holding the cassettes
are transferred from a tray containing the ethyl alcohol to a tray
containing isopropanol, then to a tray containing liquid paraffin.
One laboratory which processes 300 biopsies a day saved more than
$10,000 in reagents over the first year it employed this technique.
Combining Microwave and Freezing Techniques:
The principal advantage in freezing techniques in surgical pathology
is the speed of preparation of a tissue block that can be cut.
In addition, no fixative is required. The hardening of tissue,
necessary for cutting thin sections, is a one-step process, and
cutting can be carried out in minutes. The principal disadvantage
of the cryostat technique is the relative poor quality of the ultimate
light microscope images. Major improvement can be achieved by subsequent
microwave-stimulated fixation.
In the method recommended by Kok and Boon, frozen sections are
cut by a cryostat and mounted on slides. Next, the sections are
quickly covered with a few drops of Kryofix.. The slide is then
quickly transferred, in the horizontal position, to a polystyrene
platform in the H2800, and microwaved for 20 seconds at 450 W.
Finally, the slides are stained with Hematoxylin-Eosin. This procedure
takes less time than conventional frozen section techniques, and
produces substantially improved microscopic images.
Microwave Staining:
Staining of tissue is based on two factors: diffusion of the dye
into the cells, and binding of the dye to the substrate. Diffusion
is a physical process, and can be enormously accelerated by microwave
exposure. Binding of stains to cell substrates is a physical-chemical
process, and the role of microwave exposure depends on various
factors. Generally, staining methods that normally take minutes
can be done in a microwave oven in seconds; those that take hours,
in minutes; and those that take days or even weeks can be completed
in a matter of hours using microwave techniques.
There are two basic methods for staining in the microwave oven.
Whenever possible, we recommend that staining be performed in a
plastic coplin jar or staining rack, with the temperature probe
of the H2800 inserted into the fluid in the jar. This allows use
of the probe for very accurate temperature control. The optimum
temperature for most non-metallic stains is around 60 C, and for
metallic stains approximately 95 C. Stirring the solution by air-bubble
agitation in the H2800 is usually advantageous, facilitating more
even staining from top to bottom of the slide. A second method
involves covering the slide with a few drops of staining solution,
placing the slide on a platform in the microwave oven, and microwaving
for 20-30 seconds.
Kok and Boon present numerous methods for microwave staining of
histologic sections in their Microwave Cookbook for Microscopists.
These methods include techniques for both paraffin and plastic
sections.
For more on-line information on staining, please
see the complete listing of Technical Papers from
Energy Beam Sciences, Inc.
Antigen Retrieval:
Microwave techniques for antigen retrieval have become increasingly
popular in the years since Dr. Shi first pioneered this technique.
Still, the use of kitchen microwaves for antigen retrieval has
produced notoriously inconsistent results, with poor reproducibility.
For optimum results, the slides should be placed in plastic racks,
and a temperature probe used to measure and regulate the temperature.
Proper fume extraction is necessary when heavy metals (lead or
zinc) are present in the antigen retrieval solutions.
Microwave Tissue Fixation for EM:
The pioneering work in this area has been done by Gary Login and
Ann Dvorak at Beth Israel Hospital, in Boston, MA, U.S.A.. They
present, in series of publications, a comprehensive approach to
microwaving for electron microscopy, comprised of five aspects:
A standardization method for testing microwave oven performance.
Postfixation and processing of microwave-fixed specimens for
EM.
Ultrafast microwave fixation.
Evaluation of specimen morphology following microwave exposure
in different solutions.
Postembedding immunoelectron microscopy of microwave-fixed
specimens. Detailed procedures for each are provided in Chapter 19 of the
Microwave Cookbook for Microscopists.
Microwave Exposure in Immunoelectron Microscopy:
This seems to us to be one of the most promising areas for further
exploration. Several studies indicate that excellent results can
be obtained when gold-labelling is performed in the microwave oven
under controlled conditions. For successful labelling, accurate
temperature control such as that provided by a laboratory microwave
like the H2800, is essential (it is desirable to keep the temperature
in the antigen-antibody system at around physiologic temperature).
In one technique cited by Kok and Boon, free floating 405 Vibratome.
sections are used for preembedding imummunoelectron microscopy.
This procedure was performed in an H2800.
Microwave Exposure and Epoxy-Resin Embedding for Transmission
EM:
Thin sectioning of epoxy-resin embedded material is the most widely
used method for TEM investigations of biological material. As in
histoprocessing techniques, the cells or tissues are first fixed,
then dehydrated and embedded. (The epoxy resins are miscible with
alcohol, so no clearing agent is required). Again as with histoprocessing,
conventional methods are very time-consuming, requiring up to three
days. This is particularly true in cases where the resin has to
penetrate barriers such as thick cell walls. Such long processing
times give rise to embedding artifacts and a low overall contrast
(mainly a result of extraction of subcellular compounds). Therefore,
it seems worthwhile to try to shorten these steps through the use
of microwave-stimulated diffusion of the reagents through the thick
barriers.
Microwave techniques have successfully been demonstrated for dehydration
for epoxy-resin embedding through a series of graded ethyl alcohols.
Microwaving can shorten dehydration times, with no noticeable disadvantages.
For the embedding in plastic itself, the processing time can also
be shortened, but the quality of the resulting blocks using kitchen
microwaves has often proved unpredictable. Beverly Giammara and
Jacob Hanker have been studying these techniques, with good results
using EPON/Araldite mixtures and silicone rubber molds. A dummy
load may be used to absorb excess microwave energy. Giammara has
been able to achieve consistent polymerization in approximately
30 minutes when blocks of the same size and fixation were placed
carefully in the same locations within the microwave cavity. Using
the H2800 Microwave Processor, rather than a kitchen microwave
oven, the procedures that Giammara has developed can be further
improved through careful temperature control. Preliminary experiments
have been encouraging, even with LR White resin.
Rapid Processing of Tissues for Transmission Electron Microscopy
Technique courtesy of: Ed Calomeni Dept. of Pathology Medical
College of Ohio Toledo, Ohio 43699
Note: Set the microwave at 60% power, and use a 300ml water load
for all microwave steps.
Fix in 3% glutaraldehyde for 5 minutes. Microwave
for 30 seconds at 50°C. Note: Tissue should be trimmed to 2mm
or smaller. If the tissue was not received already in formalin,
increase
the
glutaraldehyde fixation time to 15 minutes.
Rinse 2X with sodium cacodylate for 30 seconds each.
Post-fix in 1% OsO4 for 14-1/2 minutes. Microwave for 30 seconds.
Rinse in s-collidine 2X for 30 seconds each.
Tertiary fix in saturated Uranyl Acetate for 15 minutes. Microwave
for 30 seconds.
Dehydrate in a graded series of ethanols (30, 50, 70, 90, 95,
32X 100%) for 30 seconds each in the microwave.
Clear with 100% acetone 4X for 30 seconds each in the microwave.
Infiltrate with a 1:1 mixture of acetone: Spurr's resin (rapid
cure) for 1 hour, microwaving every 10 minutes.
Place tissue in filled BEEM capsules, and
place in convection oven at 70°C.
Increase temperature 2-1/2°C every 10 minutes. At the end of
1-1/2 hours, remove blocks and section. Do not exceed 100°C. |
