[Histonet] microwave processing tips?

Dick Paulson [Source Medical Products] dpconsult <@t> earthlink.net
Sat Dec 10 18:42:33 CST 2005


There are 2 books you should read to get a firm grasp on the state of
microwave technology in the laboratory.

“Microwaves for the Art of Microscopy”
by L.P. Kok and Mathilde E. Boon
Coulomb Press Leyden, 2003.

“Microwave Technology for Light Microscopy and Ultrastructural Studies”
by Anthony S-Y Leong, M.D.

I submit to you that “Microwaves for the Art of Microscopy” should be
included in the Reference Library of any lab investigating or using
microwave technology.  It is a unique collaboration between a physicist and
a pathologist.

I have included below the full Table of Contents for both books to peak your

You can get these books at:
Milestone Medical Products
Phone (866) 995-5300
$115.00 - “Microwaves for the Art of Microscopy”
$ 55.00 - “Microwave Technology for Light Microscopy and Ultrastructural

Dick Paulson
Source Medical Products
dpconsult <@t> earthlink.net


Microwaves for the Art of Microscopy by
L. P. Kok, Biomedical Engineering, University of Groningen
Mathilde E. Boon, Leiden Cytology and Pathology Laboratory, Leiden

370 Pages - 900 references

Mathilde E. Boon is the senior pathologist at, and the director of, the
Leiden Cytology and Pathology Laboratory. She was trained in the late
sixties as a cytologist at Ann Arbor (Michigan), then as a pathologist at
Leiden, The Netherlands. She has headed subsequently the Departments of
Cytology at the Leiden University and at Delft. She has authored more than
three hundred scientific papers on cytology, pathology, laboratory
techniques, and microwaves. 

L. P. Kok is a theoretical physicist and a full professor in Theoretical
Biomedical Engineering at the University of Groningen, The Netherlands. In
that city he also obtained his Ph.D. degree, in 1969. He worked for extended
periods in Italy, Israel, the US, and the USSR. He has authored more than
two hundred and fifty publications, starting off in the field of quantum
scattering theory of few-body systems. Books he (co)authored address
subjects in quantum mechanics, stereology, and the use of microwaves in
laboratory technique. 

Together they started to publish on the use of microwaves in the pathology
laboratory some twenty years ago. This work led to four books on this
subject, the most recent one being "Microwaves for the Art of Microscopy",
appearing in 2003. 

With their background in physics and pathology they did more innovative
work. In the nineties, they introduced neural-network scanning technology in
cervical screening. This application is still very successful, until now
some 700,000 patients were screened using this technique. 

In 1985, the pathologist Boon introduced microwaving and coagulant fixation
in her laboratory. These unconventional methods are, in a fine-tuned manner,
still used for all the incoming diagnostic material. They are the basics for
the workshop at the NSH Convention. There, Dr Kok will give a presentation
concerning the physics background of the use of microwaves in pathology. Dr
Boon will present the practical side of the application of microwaves for
fixation, (automated) histoprocessing, and other techniques. In addition,
she will conduct the discussion of artifacts with clinical examples.
Table of Contents


1.  Microwaves for beginners
•	Introduction to light and heat
•	Heat:  convection, conduction, and radiation
•	The electromagnetic spectrum
•	The dual nature of microwaves
•	New perspectives to the microscopist

2.  Microwaves are electromagnetic waves
•	Microwaves?  For microscopy?
•	Electromagnetic waves
•	Electromagnetic spectrum
•	Electromagnetic radiation
•	Microwaves
•	Terminology: (ir)radiation versus exposure and emission

3.  Microwaves and society
•	Origins of radar
•	Applications of microwaves
•	From radar, via cooking, to the laboratory
•	Hazards of microwaves
•	The 1982 ANSI standard and the 1988 IRPA standard
•	The body as an antenna
•	Hazards of microwave ovens

4.  Interaction between microwaves and matter
•	Introduction
•	The optics of microwaves: reflection and refraction
•	Concentration effects in the optics of microwaves
•	Molecular picture of dielectric media in electric fields
•	Macroscopic description of microwaves in media
•	Dielectric data and depth of penetration of microwaves
•	Depth of penetration into slightly conducting media
•	Penetration into highly conducting media
•	Antenna properties of exposed objects
•	Standing waves and standing-wave phenomena
•	“The microwave effect” and “The temperature effect”.  What is
•	Preferential stimulation; microwave stimulated diffusion
•	Microwave-assisted chemistry and chemical-reaction rates
•	Defrosting: runaway heating
•	Conclusion: lessons from the food industry

5.  From Domestic microwave ovens to laboratory microwave processors
•	Introduction
•	Operation and precautions
•	The microwave oven as a resonant cavity.  Hot spots
•	Power-level control of microwave ovens
•	Power and efficiency of microwave ovens
•	Shielding and detection of unwanted microwaves
•	Microwave-transparent materials, susceptors, and active packaging
•	Domestic microwave oven or laboratory processor?  A legal question

6.  Microwave processors, temperature control, and robustness of procedures
•	Temperature measurement in microwave environment
•	Historical ways of measuring temperature in microwave cavities
•	Infrared thermometry
•	Variation of temperature of exposed objects
•	A uniform microwave heating pattern is a fiction
•	General characteristics of microwave processors
•	The rise, usage, and fall of the water load

7.  Transport properties
•	Introduction
•	Viscosity of liquids
•	Diffusion in liquids
•	Heat capacity
•	Thermal conductivity and transport of heat
•	Electric conductivity
•	External versus internal heating: the beef experiment
•	The power of vacuum and low-pressure histology
•	High-altitude microscopy and superheating

8.  History of the use of microwaves for microscopy
•	Introduction
•	The first two decades of microwave publications
•	Microwave publications ordered by subject
§	History of microwave fixation and stabilization
§	History of microwave histoprocessing
§	History of microwave-stimulated staining
§	History of microwaves and immunostaining
§	History of microwaves and enzyme incubations
§	History of microwaves and ELISA
§	History of microwaves and botany
§	History of microwaves in entomology
§	History of microwaves and antigen retrieval
§	History of microwaves and electron microscopy
§	Miscellany

9.  Grossing
 	The gross room
§	Grossing large operation specimens
§	Microwave hardening of large operation specimen
§	Hardening of human brains
§	Hardening of small human embryos
§	Grossing small operation specimens
§	Grossing biopsies
§	Surgical margins of microwave-treated operation specimen
§	From the grossing room to the pathology report
§	In situ stabilization brains in live rats by Løberg and Torvik

10.  Microwave-Stimulated fixation with formalin
§	Introduction
§	Formaldehyde fixation and microwave exposure
§	DNA and RNA in formalin-fixed tissue
Formaldehyde-glutaraldehyde mixture for histochemistry
§	Microwave-stimulated fixation for in situ hybridization
§	Microwave-stimulated formalin fixation of perfused laboratory
§	Microwave exposure with low formalin concentrations for the
detection of dopamine

11.  Microwave stimulation of coagulant formalin-free fixatives
§	Coagulant fixatives
§	Ethyl alcohol – polyethylene glycol fixatives: Kryofix and BoonFix
§	Selecting a fixative for DNA and RNA preservation
§	Experiences with Kryofix
§	Comparing microwave coagulation with microwave-stimulated Kryofix
fixation: the egg experiment
§	Microwave-stimulated Kryofix fixation
§	Flow of specimen in the Kryofix laboratory

12.  Microwave stabilization of fresh tissue by microwave heating
§	Introduction
§	Microwave stabilization without postfixation
§	Hybrid method: microwave stabilization with postfixation
§	Microwave stabilization of lungs
§	Perfusion of laboratory animals
§	Combining microwave stabilization and paraffin embedding
§	Combining microwave stabilization and frozen sectioning
§	Comparison of different microwave stabilization and fixation methods
§	Microwave stabilization of tissue and cells for immunocytology
§	Microwave stabilization of myocard to delineate necrosis

13.  Microwaves for paraffin histoprocessing
•	Introduction
•	The fixative factor
§	Formalin as fixative for paraffin sections
§	Coagulant fixatives for paraffin sections
Reagents used for microwave histoprocessing
§	Dehydration fluid
§	The intermedium
•	Paraffin wax
Differences between microwave and conventional histoprocessing
•	Vacuum histoprocessing in the Milestone LAVIS microwave processor
§	Exploiting low boiling temperatures under vacuum
§	Troubleshooting in the first-generation microwave vacuum
Introduction of a vacuum-drying step in 1997
•	The Milestone MicroMED URM vacuum Histoprocessor
•	The Milestone RHS-1 vacuum processor
•	Vacuum histoprocessing using JFC solution in the Milestone
•	Vacuum histoprocessing using ethyl alcohol/isopropanol solution in
the Milestone processors
•	Microwave histoprocessing in the Pelco processors
•	The Tissue-Tek continuous rapid processor of Sakura
•	The story of microwave histoprocessing using domestic ovens
•	The story of the H2500 and the H2800 microwave processor
•	The future of microwave histoprocessing and automation

14.  Combining microwave and freeze techniques
•	Introduction
•	Combining cryostat-sectioning and microwave-stimulated fixation in
surgical pathology
•	Combining cryostat-sectioning and microwave-stimulated fixation for
•	Microwave-stimulated fixations of cryosections for histochemistry
•	Microwave cryostat procedure for large specimen

15.  Microwaves and staining
•	Introduction
•	Theory and practice of staining in the microwave processor
•	Microwave Warthin-Starry method
•	Microwave Southgate mucicarmine method
•	Microwave Alcian-blue method
•	Microwave Fontana-Masson method
•	Microwave Grimelius silver method
•	Microwave Grocott methenamine silver-nitrate method
•	Microwave Jones periodic acid methenamine silver method
•	Microwave Jones-Marres method
•	Microwave Perls iron stain 
•	Microwave rhodamine method 
•	Microwave Bosma-Steiner method
•	Microwave Elastica Van Gieson method
•	Microwave Azan staining method
•	Microwave Romanowsky-Giemsa method for plastic-embedding bone-marrow
•	Microwave PAS staining method for glycol methacrylate sections
•	Microwave Hanker-Giammara silver staining method of
cytochemical-reation products
•	Microwave AgNOR method
•	Microwave staining method for nerve and muscle biopsies
•	Microwave Nissl method
•	Microwave Klüver-Barrera method
•	Microwave Bodian silver method
•	Microwave King silver method
•	Microwave Rio-Hortega method
•	Microwave Häggqvist method for human brain

16.  Microwaves for decalcification
•	Introduction
•	Decalcification in the Milestone processors
•	Decalcification in the Pelco 3440 microwave processor
•	Microwave-stimulated decalcification using nitric acid
•	Microwave-stimulated decalcification using formic acid
•	Microwave exposure for the study of bone canaliculi

17.  Microwave exposure in immunostaining
•	Introduction
•	Temperature controlled microwave-enhanced incubations for
immunostaining in the Milestone processors
•	Immunostaining in the Pelco microwave processors

18.  Microwaves and cell fixation for TEM and SEM
•	Introduction
•	Microwave-stimulated fixation of single cells
•	Microwave stabilization of single cells
•	Microwave-stimulated aldehyde fixation of cell monolayers
•	Microwave-stimulated aldehyde fixation of cell pellets
•	Comparison of microwave stabilization and fixation
•	Cytochemistry and immunocytochemistry at EM level in microwaved
•	Microwaving for immunoelectron microscopy of cultured cells
•	Microwave stabilization for scanning electron microscopy
•	Microwave-stimulated fixation of cells for scanning electron
•	Microwave method for electron microscopy in aspiration cytology

19.  Microwaves and tissue fixation for TEM and SEM
•	Introduction
•	Working with the Milestone REM Processor for EM
•	Microwave-stimulated fixation for TEM using the Pelco 3440
•	Microwave procedure for TEM fixation with membrane protection
•	Microwave-enhanced fixation for TEM of the parathyroid
•	Microwave procedure for TEM fixation of brain slices
•	Microwave stabilization for SEM
•	Microwave-stimulated fixation for SEM
•	Microwaving for electron microscopy in Boston

20.  Microwave exposure in immunoelectron microscopy
•	Introduction
•	Temperature control in the immunogold technique
•	Immunogold labeling of antigens on mesothelial cells using microwave
•	Lysosmal localization of acid phosphatase in prostate epithelium
using microwave exposure
•	Microwaving and preembedding in immunoelectron microscopy

21.  Microwaves and embryology
•	Introduction
•	Combining microwave-stimulated formalin fixation and frozen
•	Combining microwave stabilization and frozen sectioning
•	Combining microwave stabilization and paraffin embedding
•	Microwave-stimulated formalin fixation of embryos in fertilized fish
•	Microwave treatment of chimeric mouse embryos
•	Microwave-stimulated Kryofix fixation of cuticulized embryos

22.  Microwave exposure and epoxy-resin embedding for TEM
•	Introduction
•	Working with the Milestone REM Processor for EM embedding
•	Working with the Pelco processors for EM embedding
•	Microwave embedding for TEM using the Pelco processors
•	Temperature control in the BioRad H2500 in the dehydrations steps
•	Microwave embedding for TEM

23.  Microwaves and botany
•	Introduction
•	The AgNOR story in botany
•	Microwave-enhanced immunostainings in botanic sections
•	Microwave exposure for rapid killing and fixing of plant tissue
•	Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of
plant tissue
•	Microwave-stimulated glutaraldehyde fixation of yeast cells
•	Microwave-stimulated fixation for the preservation of soluble
•	Microwave-stimulated staining of plant virus inclusion bodies
•	Microwave-stimulated staining of plastic sections
•	Processing plant tissues for ultrastructural studies
•	Processing plant tissue in the Pelco 3450 Processor

24.  Microwaves and entomology
•	Introduction
•	Microwave exposure to extrude hidden cuticular parts for SEM

25.  High-temperature heat treatment for immunhistochemistry and molecular
•	Introduction
•	Chemical composition and pH of the retrieval solution
•	Heat treatment in domestic microwave ovens
•	The ER story
•	The MiB-1 story
•	The p53 story
•	Working the Milestone T/T Mega for high-temperature heat treatment
•	Microwave superheating in the Milestone Pressure Reactor
•	Combining microwave heat treatment with signal amplification
•	Multiple immunostainings: microwave heat treatment for blocking of
cross-reactivity, false-positive staining and antigen retrieval
•	Multiple immunoenzyme staining of paraffin tissue sections
•	DNA and RNA retrieval in formalin-fixed tissue and sections by
microwave heat treatment
•	Microwave heating for RNA-ISH in formalin-fixed tissues
•	Antigen retrieval for immunoelectron microscopy
•	Antigen retrieval in plastic sections

26.  Miscellany
•	Introduction
•	Mordanting in the microwave oven
•	Bleaching melanin in the microwave oven
•	Drying and attaching sections using the microwave oven
•	Treating plastic and glass equipment (sterilizing?)
•	Preparation of bacteriological-culture media
•	Microwave-stimulated Ce-Pb conversion
•	Cryoquenching and microwaving
•	Treating cryosections in the microwave oven
•	Microwave digestion for forensic pathology
•	Destaining slides in the microwave oven
•	Deparaffinizing sections in the microwave oven
•	Various
•	Microwaves and wine

The formalin-free microwave laboratory


Microwave Technology
for Light Microscopy and Ultrastructural Studies
by Anthony S-Y Leong, M.D.
Dr. Anthony S-Y Leong is one of the world's best-known pioneers in the use
of microwave technology for histopathology. This book is a compendium of the
knowledge he has gathered in 20 years of activity. It describes the variety
of purposes to which microwaves can be applied, providing detailed
information on the techniques and procedures that he and his colleagues have
This book is an excellent primer for any pathologist or histologist who is
considering the transition to microwaves. It is also good reading for anyone
who has already made that transition and now wants to get the most out of
their new technology.

Table of Contents:
1. Introduction
•	Effects of low dose microwave and radio frequency radiation on
mammalian tissues 
•	Microwaves - physical properties 
•	Background to applications in histopathology 
2. Tissue Fixation
•	Microwave fixation of large specimens 
•	Microwave fixation of tisue blocks 
•	Motable differences in the properties of microwave -fixed and
formalin-fixed sections 
•	Incorporation of microwave -fixation with conventional tissue
•	Formaldehyde toxicity 
•	Principles of tissue fixation 
•	A classification of Microwave fixation 
•	Microwave -stimulated aqueous formaldehyde fixation 
•	Microwave fixation and microwave stimulated fixation of whole organs

•	Microwaves for fixation in neurochemical analysis 
•	Optimal temperatures for Microwave fixation and Microwave stimulated
•	Protocol for Microwave fixation for light microscopy 
•	Are organisms destroyed during Microwave fixation? 
•	Microwave fixation for electron microscopy 
•	Ultrastructural enzyme and antigen preservation in Microwave -fixed
•	Protocol for Microwave stimulated fixation of specimens for electron
•	Microwave stimulated fixation for accelerated processing of fine
needle biopsy specimens for ultrastructural diagnosis 
•	Protocol for rapid Microwave stimulated fixation and processing of
fine needle aspiration biopsies 
•	Microwave fixation for cytological preparations 
3. Microwave Accelerated Demineralisation
4. Cryostat Sections
5. Histochemical and Immunohistological Staining
•	Light microscopy 
•	Microwave -stimulated staining of reticulin in plastic sections with
ammoniacal silver nitrate (Leong and Pulbrook, 1989) 
•	A new Microwave -stimulated stain for melanocytic lesions (Leong &
Gillham, 1989) 
•	Electron microscopy 
•	Immunohistological staining 
•	General comments 
6. Antigen Retrieval
•	Introduction 
•	Microwave antigen retrieval 
•	Antigen retrieval for cytological preparations 
•	Protocol for immunolabelling of cytological preparations 
•	Antigen retrieval for electron microscopy 
•	Proposed mechanism of formalin fixation and antigen retrieval 
7. Applications in Molecular Analyses
8. Rapid Tissue Processing
•	Conventional Tissue Processing 
•	Microwave -Stimulated Tissue Processing 
•	Impact of Microwave -Stimulated Tissue Processing 
•	Protocol for Microwave -Stimulated Tissue Processing 
•	Rapid Processing for Electron Microscopy 
9. Chemical and Industrial Applications
10. Conclusions

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