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The impact of histochemistry – a historical perspective
acta histochem. 102, 5±14 (2000) Ó Urban & Fischer Verlag
The impact of histochemistry ± a historical perspective Raymond Coleman Department of Anatomy and Cell Biology, Bruce Rappaport Faculty of Medicine, Technion ± Israel Institute of Technology, P. O. Box 96 49, Haifa 31096, Israel Received 5 October 1999 and in revised form 24 November 1999; accepted 27 November 1999
Summary Histochemistry has a remarkable long-term impact on cell and tissue biology, embryology, and pathology and remains at the forefront of research in these disciplines. Histochemical techniques, and particularly immunohistochemical and in situ hybridization techniques, are now more widely used than ever and, in conjunction with new developments in microscopical imaging and analysis, will continue to have an important position in the life sciences and medicine. However, histochemistry is often mistakenly perceived as an archaic discipline, and its contributions to cell and molecular biology are not always given the credit it deserves. Key words: histochemistry ± enzyme histochemistry ± immunohistochemistry ± in situ hybridization ± living cell histochemistry
Historical impact of histochemistry The new millennium is a good opportunity to evaluate the impact and contributions of specific disciplines in medical and biological sciences. The exact impact can only be recognized and appreciated when it is evaluated in a longterm or historical context. The long-term impact of histochemistry is remarkable in many respects. Histochemical techniques introduced in the beginning Correspondence to: Prof. R. Coleman, phone.: +9 72-4-8 29 53 95, fax: +9 72-4-8 29 53 92, e-mail: [email protected] http://www.urbanfischer.de/journals/actahist
0065±1281/00/102/1±005 $ 12.00/0
6
R. Coleman
of the 20th century or even before are still commonly used (Pearse, 1985). There are only few other disciplines in experimental biology or medicine that can make a similar claim. Some of these ªhistoricalº contributions of histochemistry had such an enormous impact that they changed our concepts in biology and medicine. For example, the silver impregnation staining of neurons as developed by Camillo Golgi and Ramon y Cajal is still used at present and few histochemists or neurobiologists today can improve the quality of their preparations. The Feulgen staining of DNA, the Nissl staining for neurons, and techniques for localizing extracellular reticular and elastic fibers all have been introduced decades ago, but are still widely used. Periodic acid-Schiff (PAS) staining continues to provide useful information and is used routinely in histological and pathological departments until today. Perls' Prussian blue method to demonstrate iron, was introduced in 1867, and is the method of choice today. Von Kossa's method to demonstrate the presence of calcium was developed in 1901 and is still widely used to stain bone sections almost a century after its introduction. Classical histochemical techniques to demonstrate lipids or metachromatic compounds in tissue are still of great value. The list of histochemical techniques that have been developed a long time ago, but are still in use, is extensive. In many cases, we have no longer access to the original publications and rarely refer to them. However, it does not mean that a technique lacks scientific merits only because it is old, or even very old. Unfortunately this viewpoint is not always shared by referees of manuscripts today. The impact of histochemistry throughout the last century has been enormous although this is not reflected by commonly accepted methods to determine ªimpactº. The widely used Journal Impact Factors and Citation Indices only measure short-term impact in 3 years maximally after a publication has appeared and do not measure long-term impact, which can only be assessed historically. Journal Impact Factors are greatly misused and have only limited relevance as a measure of scientific impact in slow moving and more traditional medical disciplines (Coleman, 1999). Histochemistry and cytochemistry are methodological approaches in biology and medicine that allow precise analysis of the chemistry of cells and tissues in relation to structural organization (Padykula, 1983). Pearse reviewed the history of histochemistry in his classical histochemistry books (Pearse, 1951, 1960, 1980) and put forward the viewpoint that progress in histochemistry was continuous and that the aims and principles remained essentially the same. This viewpoint is still valid, although the contributions of histochemistry to our understanding of cell biology and molecular biology are not always fully appreciated. Until the mid-1950s, histochemistry and cytochemistry were largely perceived as ªstainingº techniques, and many experimental biologists continue to hold this perception today. In the last half a century, histochemistry has contributed considerably to concepts of the dynamic processes in cells and tissues and to diagnostic pathology and continues to do so.
The impact of histochemistry ± a historical perspective
7
Enzyme histochemistry The golden decades for histochemistry were in the 1950s and 1960s, when its popularity and esteem were greatest, especially due to the development of new techniques in enzyme histochemistry and cytochemistry. According to Pearse (1960), in 1953 there were histochemical methods for localization of the activity of 18 enzymes. In the late 1960s, the number of techniques had risen to approximately 75 and today the figure is in excess of 100 (Lojda et al., 1979; Chayen and Bitensky, 1991; Van Noorden and Frederiks, 1992 a). The development and application of metal salt techniques, azo dye, tetrazolium dye and indigo dye methods for the localization of enzyme activity were a source of great excitement in cell biology. Furthermore, the introduction and popularization in the early 1960s of fixatives based on glutaraldehyde and the ability to localize enzymatic activities in fixed tissues at the ultrastructural level also had enormous impact. The mid-1960s were also marked by increasing availability of transmission electron microscopes and associated tissue preparation techniques. Enzyme localization techniques were instrumental in elucidating the functions of cell organelles such as lysosomes, peroxisomes and Golgi apparatus. The 1970s saw further refinements in ultracytochemical localization techniques, and the introduction of cerium as replacement for lead improved capture reactions (Van Noorden and Frederiks, 1992 b). Enzyme histochemistry has remained one of the tools for the pathologist, despite the fact that many of the techniques were developed a long time ago. Histopathological diagnosis of muscle biopsies, for example, is still to a large extent dependent on fiber typing with the use of enzyme histochemistry that is performed on frozen sections (Johnson, 1988).
Frozen sections Frozen sections are now widely used in diagnostic pathology (Chayen and Bitensky, 1991). The first ªcryostatº (microtome for frozen sections) was developed in 1938 and cryostats became readily available in the 1950's (Pearse, 1960). In practice, modern cryostats and freezing techniques are still similar to what they were some 50 years ago and the technology has improved only marginally over the years. Commonly used methods for cryoprotection and freezing are still rather primitive and uncontrolled and produce often poor and/or unpredictable results. Modern cryostats have been improved with respect to automation, digital temperature control and disposable knives, but on the whole the morphology of frozen sections is not much better than that of frozen sections obtained half a century ago. Freezing artifacts in frozen sections typically result from poor freezing rather than sectioning with the cryostat. The development of computer controlled techniques for tissue freezing should result in improved morphology and more precise localization of molecules in frozen sections.
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Ultracryomicrotomy for ultrastructural cytochemical localization purposes is still, unfortunately, not very popular, owing to the very demanding technical skills that are needed to obtain good results, despite the fact that it has contributed very significantly to our understanding of, in particular, intracellular trafficking processes.
Radioautography Radioautography is a cytochemical procedure to determine sites containing radioactivity in tissue sections (Rogers, 1979). Techniques in radioautography were at their peak of popularity in the 1960s and 1970s and helped to elucidate many fundamental dynamic processes in cell biology including endocytosis, exocytosis, protein synthesis and storage, and functioning of the Golgi apparatus. Our concepts of cell differentiation, migration, and proliferation were markedly influenced by information derived from radioautographical studies. The use of electron microscopical radioautography to localize and quantify enzyme activities at the ultrastructural level was possible in the late 1960s (Saltpeter, 1967). In terms of technology and methodology, relatively few advances have been made in radioautography in the last 40 years. Use of radioautography and its contribution to ªhistochemistryº have markedly declined in recent years partly due to the increased safety restrictions with respect to the use of radioactive chemicals and partly due to the availability of alternative immunohistochemical and cytochemical methods.
APUD Concept The 1960s saw the introduction and development of the APUD (Amine Precursor Uptake and Decarboxylase) concept by Pearse (1969). It was firmly based on histochemical observations. This concept, which showed that many polypeptide hormone-secreting cells have similar features, is one of the major contributions of histochemistry and cytochemistry to cell biology over the last century. The APUD concept provided a strong stimulus for cell biological research and debate (especially on the origin of APUD cells) and contributed tremendously to our understanding in endocrine pathology. In recent years, the concept of the diffuse neuroendocrine system has replaced the APUD concept, but the terms ªAPUDº and ªapudomaº remain part of the pathological nomenclature (Pearse, 1977; Van Bogaert, 1982).
Immunohistochemistry The history of immunohistochemistry began in 1941, when Coons and his colleagues labelled an antibody with a fluorescent dye and used it to identify an antigen in tissue sections (Coons et al., 1941; Polak and Van Noorden, 1997).
The impact of histochemistry ± a historical perspective
9
Since the 1970s, the use of immunohistochemical techniques has taken off exponentially (Brandtzaeg, 1998; Stoward et al., 1998; Coleman, 2000) in line with the development of specific molecular markers. For example, molecular markers that are used to determine sequences or cascades of events involved in apoptosis has lead to a reevaluation of many of our concepts concerning embryologic organ development, cellular differentiation, programmed cell death and disease processes. Furthermore, the possibility to distinguish functionally different cell types with similar morphology on the basis of immunohistochemical detection of surface markers has totally altered our understanding of immunology. There are few areas of modern experimental biology or diagnostic medicine that have not been greatly affected by applications of immunohistochemical methodologies. The overall impact has been enormous, but one of the paradoxes is that many of the users of immunohistochemical techniques in routine diagnostics or experimental research, do not associate themselves or their work with histochemistry, they do not publish in histochemical journals and lack any affiliation with histochemical societies. Since the introduction in the mid-1980s of the microwave oven as a laboratory tool in histopathology, microwave techniques have had an enormous impact on immunohistochemistry, and are widely used now for antigen retrieval in archival formalin-fixed paraffin-embedded material (Kok and Boon, 1992; Gu, 1994).
Avidin-biotin techniques The discovery of the avidin-biotin interaction in the early 1970s was undoubtedly one of the major discoveries that pushed developments in histochemistry and one that has had enormous impact in many fields of biological sciences (Wilchek and Bayer, 1990). The avidin-biotin complex is the strongest bond that is known to exist between a protein and a ligand (Wilchek et al., 1997). Its use was originally categorized as ªaffinity histochemistryº, but avidin-biotin systems have since been applied to many areas in molecular biology, cytogenetics, immunoassays, affinity chromatography, cell selection, in vivo diagnostics, drug targeting etc. Since its discovery, the avidin-biotin market has generated estimated annual commercial sales exceeding US$ 400 million and more than 800 associated patents. The development of the avidin-biotin techniques has revolutionized immunochemistry. Immuno-diagnostic kits are widely available and used extensively in departments of diagnostic pathology and cell biology. Almost every diagnostic pathology laboratory uses routinely over 100 specific antibodies for the detection of antigens. Most of these kits are based on the principle of avidin-biotin or streptavidin-biotin binding. Despite the widespread use of avidin-biotin or streptavidin-biotin complexes in so many areas in the life sciences, the biological significance of this strong binding is still not known (Wilchek et al., 1997).
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Lectin histochemistry Lectins are proteins and glycoproteins with specific affinities for carbohydrates. They are also called agglutinins. The major advances in carbohydrate histochemistry ensuing from the discovery of lectins possessing affinity for specific terminal sugar groups have had enormous impact in cell biology and histopathology (Brooks et al., 1996). The use of a battery of these agglutinins conjugated to horseradish peroxidase or a fluorescent label permits localization of specific sugars in cells and tissues (Danguy, 1990; Spicer, 1993). In the last decade lectin histochemistry has emerged as a powerful tool to identify and localize carbohydrate moieties. Today, lectins are widely used as histochemical probes to localize aberrant glycosylation in cancer, metabolic diseases and other pathological conditions. Carbohydrate immunohistochemistry has also played a major role in distinguishing endocrine cells in the pituitary gland, islets of Langerhans and the diffuse endocrine cells of the intestinal and respiratory tracts.
Fluorescence microscopy The impact of immunohistochemistry in the life sciences was possible not only by the developments in signal amplification techniques, but also by improvements in imaging techniques. Fluorescence microscopy has always been a major visualization technique in immunohistochemistry because of its sensitivity (Ploem and Tanke, 1987; Herman, 1997). The last 2 decades have seen major improvements in fluorescence microscopy due to the introduction of confocal scanning laser techniques, that allowed higher spatial resolution of fluorescent signals by optical sectioning, and digital imaging that allowed higher sensitivity and 3D reconstruction (Sheppard and Shotton, 1997). The possibility to combine signals from different antibodies in one microscopical image has also provided major advances in immunohistochemistry. The spectacular images obtained with fluorescence confocal scanning laser microscopy are widely used on front covers of research journals in biology and medicine and in the advertising material for molecular probes, more than those of any other visualization technique.
Colloidal gold and ultrastructural cytochemistry Colloidal gold was first introduced as cytochemical marker for transmission electron microscopy (TEM) in 1971 (Faulk and Taylor, 1971) and for scanning electron microscopy (SEM) in 1975 (Horisberger et al., 1975). Colloidal gold labeling offers high-resolution localization of specific antigens and their quantification at the subcellular level and has contributed enormously to the understanding of intracellular processes (Beesley, 1989; Hodges and Carr,
The impact of histochemistry ± a historical perspective
11
1990). Double-labeling techniques in TEM using different sizes of gold particles has allowed subcellular localization of more than one antigen in a single preparation.
In situ enzyme histochemistry The major development in the field of enzyme histochemistry in recent years has been the replacement of catalytic histochemistry by immunohistochemistry as the preferred means of visualizing enzymes (Stoward et al., 1998). However, there are fundamental differences between the two approaches, because the former demonstrates the functional activity of an enzyme, whereas the latter demonstrates the protein of an enzyme be it active or inactive (Van Noorden and Jonges, 1995). Furthermore, the last 2 decades have seen the development of histochemical techniques to determine in situ kinetic behavior of enzymes (Van Noorden and Jonges 1995; Stoward et al., 1998). It is easy now to measure reaction rates of enzymes in tissue sections by monitoring the formation of the final reaction product throughout the incubation. Such powerful tools may provide significant impact in histochemistry in the future as they enable quantitative determinations of metabolic pathways in situ in conjunction with advanced imaging of tissues and cells.
In situ hybridization In situ hybridization (ISH) techniques and, in particular, fluorescence ISH (FISH) have proved to be invaluable molecular tools for visualizing nucleic acids in their cellular environment. The techniques were introduced in the early 1980s. ISH has had a tremendous impact in the last 10 years in cytogenetics and cancer pathology (DNA-ISH). Furthermore, precise localization of mRNA enables the elucidation of when and where specific genes are transcribed and specific proteins are produced (Leitch et al., 1994; Stoward et al., 1998). The applicability of ISH is still limited by restricted detection sensitivity (Speel, 1999). In the last 10 years, techniques have been developed to improve nucleic acid detection in situ by amplification of target nucleic acid sequences before ISH by ªin situ PCRº, which is a marriage of ISH and reverse transcriptase-polymerase chain reaction (PCR) (Stoward et al., 1998; Long 1998). More recently, alternative methods for improving detection sensitivity have been developed involving tyramide signal amplification using the catalyzed reporter deposition (CARD) technique (Speel et al., 1998, 1999). Several methods for in situ hybridization at the ultrastructural level have been reported as well permitting the detailed localization of nucleic acid sequences (Le Guellec, 1998). The new in situ techniques are emerging as extremely powerful tools for basic and diagnostic research in histochemistry, cell biology and pathology.
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Quantitative histochemistry The combination of histochemical localization and accurate quantitation has been fraught with complications in the past, usually because of problems linked with the many variables introduced by specimen processing and/or imaging. Quantitation of staining intensities in defined areas of sections is possible when the final colored reaction product is stable and has sufficient contrast (Stoward and Ploem, 1982). In the 1970s, this became feasible using integrating microdensitometers such as the Zeiss Scanning Microscope Photometer or the Vickers M85 Scanning Microdensitometer (Chayen and Bitensky, 1991). In the last 2 decades, such instruments have been largely replaced by image analysis systems as a result of the widespread introduction of CCD cameras linked with personal computers and user-friendly software. Cytophotometric and integrated densitometric quantitation has become more and more easy to use. However, the pitfalls of quantitative measurements, especially with respect to ªstaining intensityº levels of immunohistochemically stained sections or cells, are not always appreciated. The introduction of spectral imaging in the last few years has provided greatly improved quantitative and analytical tools for histochemistry and histopathological diagnosis and are undoubtedly important in future developments (Malik and Rothmann, 1999). Quantitation of fluorescence signals in fluorescence microscopy is still problematic owing to signal depletion (photobleaching or quenching effects) and this is an area where technology is still seeking suitable answers.
Live cell histochemistry The great paradox in histochemistry and cytochemistry of the last century is that most of our concepts were developed from the analysis of dead biological material, whereas the molecular and structural events in cells are dynamic processes (Van Noorden, 1999). Vital and supra-vital staining is a topic in histochemistry that urgently needs to be developed. Relatively few techniques are available yet. In the past, vital dyes have been useful, for example, for the understanding of dynamic processes such as phagocytosis. The last few years have been the scene of a remarkable return of vital labeling, in particular with the discovery of new fluorescent probes and the introduction of new imaging techniques to study dynamic processes that occur in living cells. A 2-day meeting held in conjunction with the Microscopy and Microanalysis '99 conference, entitled ªOptical microscopy in the next millenniumº, was recently held in Portland, Oregon (July 31±August 1, 1999). Some 30 papers presented a wide range of applications of the new methodologies in optical microscopy, many of which could be described as leading edge dynamic histochemistry involving real-time imaging, in particular using multiphoton microscopy and spectroscopy. Themes included applications of new molecular probes, espe-
The impact of histochemistry ± a historical perspective
13
cially for protein trafficking. Green Fluorescent Protein (GFP) of jelly-fish origin is so extensively used today for live fluorescence imaging and possesses such remarkable properties (for example it is the best intracellular pH indicator), that it must be considered to be a prime candidate for the nomination to become ªmolecule of the yearº. In conclusion, it can be stated that histochemistry and cytochemistry have had an enormous long-term impact in cell biology and molecular biology during the last century, and will continue to contribute to our understanding of dynamic cellular and pathological processes, despite the fact that these disciplines do not always achieve the recognition they really deserve.
References Beesley JE (1989) Colloidal gold: a new perspective for cytochemical marking. Bios, Oxford Brandtzaeg P (1998) The increasing power of immunohistochemistry and immunocytochemistry. J Immunol Meth 216: 49±67 Brooks SA, Leathem AJC, and Schumacher U (1996) Lectin histochemistry. A concise practical handbook. Bios, Oxford Chayen J, and Bitensky L (1991) Practical Histochemistry. 2nd ed. Wiley Publishers, Chichester and New York Coleman R (1999) Impact factors: use and abuse in biomedical research. Anat Rec 257: 54±57 Coleman R (2000) Histochemistry in the new millenium ± a time to change our terminology. Acta histochem (in press) Coons AH, Creech HJ, and Jones RN (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47: 200±202 Danguy A (1990) Lectins emerging in histochemistry. Eur Microsc Anal 6: 15 Faulk WP, and Taylor GM (1971) An immunocolloid method for the electron microscope. Immunohistochemistry 8: 1081±1083 Gu J (1994) Microwave immunocytochemistry. In: Gu J and Hacker GW (Eds) Modern methods in analytical morphology. Plenum Press, New York, pp 67±80 Herman B (1997) Fluorescence microscopy. 2nd ed. Bios, Oxford Hodges GM, and Carr KE (1990) Colloidal gold immunocytochemistry using SEM. Eur Microsc Anal 6: 17±20 Horisberger M, Rosset J, and Bauer H (1975) Colloidal gold granules as markers for cell surface receptors in the scanning electron microscope. Experientia 31: 1147±1149 Johnson MA (1988) Muscle histochemistry: new perspectives for the 1990s. Proc Royal Microsc Soc 23: 33±36 Kok LP, and Boon ME (1992) Microwave cookbook for microscopists: art and science of visualization. 3rd ed. Coulomb Press Leyden, Leiden Le Guellec D (1998) Ultrastructural in situ hybridization: a review of technical aspects. Biol Cell 90: 297±306 Leitch AR, Schwarzacher T, Jackson D, and Leitch IJ (1994) In situ hybridization. Bios, Oxford Lojda Z, Gossrau R, and Schiebler TH (1979) Enzyme histochemical methods. A laboratory manual. Springer Verlag, New York Long AA (1998) In situ polymerase chain reaction: foundation of the technology and today's options. Eur J Histochem 42: 101±109 Malik Z, and Rothmann C (1999) Spectral imaging of histological and cytological specimens. Acta histochem 101: 30
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Padykula HA (1983) Histochemistry and cytochemistry. In: Weiss L (Ed) Histology, cell and tissue biology. 5th ed. Elsevier Biomedical, New York and Amsterdam, pp 88±108 Pearse AGE (1953) Histochemistry, theoretical and applied. 1st ed. Churchill Livingstone, Edinburgh Pearse AGE (1960) Histochemistry, theoretical and applied. 2nd ed. Churchill Livingstone, Edinburgh Pearse AGE (1969) The cytochemistry and ultrastructure of polypeptide-hormone producing cells of the APUD series and the embryologic, physiologic, and pathologic implications of the concept. J Histochem Cytochem 17: 303±313 Pearse AGE (1977) The diffuse neuroendocrine system and the APUD concept: Related endocrine peptides in brain, intestine, pituitary, placenta, and anuran cutaneous glands. Med Biol 55: 115±125 Pearse AGE (1980) Histochemistry, theoretical and applied, Vol. 1. 4th ed. Churchill Livingstone, Edinburgh Pearse AGE (1985) Histochemistry, theoretical and applied, Vol. 2. 4th ed. Churchill Livingstone, Edinburgh Ploem JS, and Tanke HJ (1987) Introduction to fluorescence microscopy. Bios, Oxford Polak JM, and Van Noorden S (1997) Introduction to immunocytochemistry. 2nd ed. Bios, Oxford Rogers AW (1979) Techniques of autoradiography. Elsevier, Amsterdam Saltpeter M (1967) Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle. J Cell Biol 32: 379±389 Sheppard CJR, and Shotton DM (1997) Confocal laser scanning microscopy. Bios, Oxford Speel EJ, Saremaslani P, Roth J, Hopman AH, and Komminoth P (1998) Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides. Histochem Cell Biol 110: 571±577 Speel EJ, Hopman AH, and Komminoth P (1999) Amplification methods to increase the sensitivity of in situ hybridization: play card(s). J Histochem Cytochem 47: 281±288 Spicer SS (1993) Advantages of histochemistry for the study of cell biology. Histochem J 25: 531±547 Stoward PJ, and Ploem JS (1982) The histochemical basis of quantitative histology. J Microsc 128: 49±56 Stoward PJ, Nakae Y, and Van Noorden CJF (1998) The everchanging advances in enzyme histochemistry, 1986±1996. Eur J Histochem 42: 35±40 Stoward PJ, and Pearse AGE (1991) Histochemistry, theoretical and applied, Vol 3. 4th ed. Churchill Livingstone, Edinburgh Van Bogaert L-J (1982) The diffuse endocrine system and derived tumors. Histological and histochemical characteristics. Acta histochem 70: 122±129 Van Noorden CJF, and Frederiks WM (1992 a) Enzyme histochemistry: a laboratory manual of current methods. Bios, Oxford Van Noorden CJF, and Frederiks WM (1992 b) Versatility of cerium for light and electron microscopical histochemistry. Proc Royal Microsc Soc 27: 185±189 Van Noorden CJF, and Jonges GN (1995) Analysis of enzyme reactions in situ. Histochem J 27: 101±118 Van Noorden CJF (1999) Microscopy of living cells and tissues. Acta histochem 101: 233±237 Wilchek M, and Bayer EA (1990) Applications of avidin-biotin technology: literature survey. Meth Enzymol 184: 14±45 Wilchek M, Kulomaa M, and Bayer EA (1997) Avidin-biotin ± past, present and future. Acta histochem 99: 349±350
The impact of histochemistry ± a historical perspective Raymond Coleman Department of Anatomy and Cell Biology, Bruce Rappaport Faculty of Medicine, Technion ± Israel Institute of Technology, P. O. Box 96 49, Haifa 31096, Israel Received 5 October 1999 and in revised form 24 November 1999; accepted 27 November 1999
Summary Histochemistry has a remarkable long-term impact on cell and tissue biology, embryology, and pathology and remains at the forefront of research in these disciplines. Histochemical techniques, and particularly immunohistochemical and in situ hybridization techniques, are now more widely used than ever and, in conjunction with new developments in microscopical imaging and analysis, will continue to have an important position in the life sciences and medicine. However, histochemistry is often mistakenly perceived as an archaic discipline, and its contributions to cell and molecular biology are not always given the credit it deserves. Key words: histochemistry ± enzyme histochemistry ± immunohistochemistry ± in situ hybridization ± living cell histochemistry
Historical impact of histochemistry The new millennium is a good opportunity to evaluate the impact and contributions of specific disciplines in medical and biological sciences. The exact impact can only be recognized and appreciated when it is evaluated in a longterm or historical context. The long-term impact of histochemistry is remarkable in many respects. Histochemical techniques introduced in the beginning Correspondence to: Prof. R. Coleman, phone.: +9 72-4-8 29 53 95, fax: +9 72-4-8 29 53 92, e-mail: [email protected] http://www.urbanfischer.de/journals/actahist
0065±1281/00/102/1±005 $ 12.00/0
6
R. Coleman
of the 20th century or even before are still commonly used (Pearse, 1985). There are only few other disciplines in experimental biology or medicine that can make a similar claim. Some of these ªhistoricalº contributions of histochemistry had such an enormous impact that they changed our concepts in biology and medicine. For example, the silver impregnation staining of neurons as developed by Camillo Golgi and Ramon y Cajal is still used at present and few histochemists or neurobiologists today can improve the quality of their preparations. The Feulgen staining of DNA, the Nissl staining for neurons, and techniques for localizing extracellular reticular and elastic fibers all have been introduced decades ago, but are still widely used. Periodic acid-Schiff (PAS) staining continues to provide useful information and is used routinely in histological and pathological departments until today. Perls' Prussian blue method to demonstrate iron, was introduced in 1867, and is the method of choice today. Von Kossa's method to demonstrate the presence of calcium was developed in 1901 and is still widely used to stain bone sections almost a century after its introduction. Classical histochemical techniques to demonstrate lipids or metachromatic compounds in tissue are still of great value. The list of histochemical techniques that have been developed a long time ago, but are still in use, is extensive. In many cases, we have no longer access to the original publications and rarely refer to them. However, it does not mean that a technique lacks scientific merits only because it is old, or even very old. Unfortunately this viewpoint is not always shared by referees of manuscripts today. The impact of histochemistry throughout the last century has been enormous although this is not reflected by commonly accepted methods to determine ªimpactº. The widely used Journal Impact Factors and Citation Indices only measure short-term impact in 3 years maximally after a publication has appeared and do not measure long-term impact, which can only be assessed historically. Journal Impact Factors are greatly misused and have only limited relevance as a measure of scientific impact in slow moving and more traditional medical disciplines (Coleman, 1999). Histochemistry and cytochemistry are methodological approaches in biology and medicine that allow precise analysis of the chemistry of cells and tissues in relation to structural organization (Padykula, 1983). Pearse reviewed the history of histochemistry in his classical histochemistry books (Pearse, 1951, 1960, 1980) and put forward the viewpoint that progress in histochemistry was continuous and that the aims and principles remained essentially the same. This viewpoint is still valid, although the contributions of histochemistry to our understanding of cell biology and molecular biology are not always fully appreciated. Until the mid-1950s, histochemistry and cytochemistry were largely perceived as ªstainingº techniques, and many experimental biologists continue to hold this perception today. In the last half a century, histochemistry has contributed considerably to concepts of the dynamic processes in cells and tissues and to diagnostic pathology and continues to do so.
The impact of histochemistry ± a historical perspective
7
Enzyme histochemistry The golden decades for histochemistry were in the 1950s and 1960s, when its popularity and esteem were greatest, especially due to the development of new techniques in enzyme histochemistry and cytochemistry. According to Pearse (1960), in 1953 there were histochemical methods for localization of the activity of 18 enzymes. In the late 1960s, the number of techniques had risen to approximately 75 and today the figure is in excess of 100 (Lojda et al., 1979; Chayen and Bitensky, 1991; Van Noorden and Frederiks, 1992 a). The development and application of metal salt techniques, azo dye, tetrazolium dye and indigo dye methods for the localization of enzyme activity were a source of great excitement in cell biology. Furthermore, the introduction and popularization in the early 1960s of fixatives based on glutaraldehyde and the ability to localize enzymatic activities in fixed tissues at the ultrastructural level also had enormous impact. The mid-1960s were also marked by increasing availability of transmission electron microscopes and associated tissue preparation techniques. Enzyme localization techniques were instrumental in elucidating the functions of cell organelles such as lysosomes, peroxisomes and Golgi apparatus. The 1970s saw further refinements in ultracytochemical localization techniques, and the introduction of cerium as replacement for lead improved capture reactions (Van Noorden and Frederiks, 1992 b). Enzyme histochemistry has remained one of the tools for the pathologist, despite the fact that many of the techniques were developed a long time ago. Histopathological diagnosis of muscle biopsies, for example, is still to a large extent dependent on fiber typing with the use of enzyme histochemistry that is performed on frozen sections (Johnson, 1988).
Frozen sections Frozen sections are now widely used in diagnostic pathology (Chayen and Bitensky, 1991). The first ªcryostatº (microtome for frozen sections) was developed in 1938 and cryostats became readily available in the 1950's (Pearse, 1960). In practice, modern cryostats and freezing techniques are still similar to what they were some 50 years ago and the technology has improved only marginally over the years. Commonly used methods for cryoprotection and freezing are still rather primitive and uncontrolled and produce often poor and/or unpredictable results. Modern cryostats have been improved with respect to automation, digital temperature control and disposable knives, but on the whole the morphology of frozen sections is not much better than that of frozen sections obtained half a century ago. Freezing artifacts in frozen sections typically result from poor freezing rather than sectioning with the cryostat. The development of computer controlled techniques for tissue freezing should result in improved morphology and more precise localization of molecules in frozen sections.
8
R. Coleman
Ultracryomicrotomy for ultrastructural cytochemical localization purposes is still, unfortunately, not very popular, owing to the very demanding technical skills that are needed to obtain good results, despite the fact that it has contributed very significantly to our understanding of, in particular, intracellular trafficking processes.
Radioautography Radioautography is a cytochemical procedure to determine sites containing radioactivity in tissue sections (Rogers, 1979). Techniques in radioautography were at their peak of popularity in the 1960s and 1970s and helped to elucidate many fundamental dynamic processes in cell biology including endocytosis, exocytosis, protein synthesis and storage, and functioning of the Golgi apparatus. Our concepts of cell differentiation, migration, and proliferation were markedly influenced by information derived from radioautographical studies. The use of electron microscopical radioautography to localize and quantify enzyme activities at the ultrastructural level was possible in the late 1960s (Saltpeter, 1967). In terms of technology and methodology, relatively few advances have been made in radioautography in the last 40 years. Use of radioautography and its contribution to ªhistochemistryº have markedly declined in recent years partly due to the increased safety restrictions with respect to the use of radioactive chemicals and partly due to the availability of alternative immunohistochemical and cytochemical methods.
APUD Concept The 1960s saw the introduction and development of the APUD (Amine Precursor Uptake and Decarboxylase) concept by Pearse (1969). It was firmly based on histochemical observations. This concept, which showed that many polypeptide hormone-secreting cells have similar features, is one of the major contributions of histochemistry and cytochemistry to cell biology over the last century. The APUD concept provided a strong stimulus for cell biological research and debate (especially on the origin of APUD cells) and contributed tremendously to our understanding in endocrine pathology. In recent years, the concept of the diffuse neuroendocrine system has replaced the APUD concept, but the terms ªAPUDº and ªapudomaº remain part of the pathological nomenclature (Pearse, 1977; Van Bogaert, 1982).
Immunohistochemistry The history of immunohistochemistry began in 1941, when Coons and his colleagues labelled an antibody with a fluorescent dye and used it to identify an antigen in tissue sections (Coons et al., 1941; Polak and Van Noorden, 1997).
The impact of histochemistry ± a historical perspective
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Since the 1970s, the use of immunohistochemical techniques has taken off exponentially (Brandtzaeg, 1998; Stoward et al., 1998; Coleman, 2000) in line with the development of specific molecular markers. For example, molecular markers that are used to determine sequences or cascades of events involved in apoptosis has lead to a reevaluation of many of our concepts concerning embryologic organ development, cellular differentiation, programmed cell death and disease processes. Furthermore, the possibility to distinguish functionally different cell types with similar morphology on the basis of immunohistochemical detection of surface markers has totally altered our understanding of immunology. There are few areas of modern experimental biology or diagnostic medicine that have not been greatly affected by applications of immunohistochemical methodologies. The overall impact has been enormous, but one of the paradoxes is that many of the users of immunohistochemical techniques in routine diagnostics or experimental research, do not associate themselves or their work with histochemistry, they do not publish in histochemical journals and lack any affiliation with histochemical societies. Since the introduction in the mid-1980s of the microwave oven as a laboratory tool in histopathology, microwave techniques have had an enormous impact on immunohistochemistry, and are widely used now for antigen retrieval in archival formalin-fixed paraffin-embedded material (Kok and Boon, 1992; Gu, 1994).
Avidin-biotin techniques The discovery of the avidin-biotin interaction in the early 1970s was undoubtedly one of the major discoveries that pushed developments in histochemistry and one that has had enormous impact in many fields of biological sciences (Wilchek and Bayer, 1990). The avidin-biotin complex is the strongest bond that is known to exist between a protein and a ligand (Wilchek et al., 1997). Its use was originally categorized as ªaffinity histochemistryº, but avidin-biotin systems have since been applied to many areas in molecular biology, cytogenetics, immunoassays, affinity chromatography, cell selection, in vivo diagnostics, drug targeting etc. Since its discovery, the avidin-biotin market has generated estimated annual commercial sales exceeding US$ 400 million and more than 800 associated patents. The development of the avidin-biotin techniques has revolutionized immunochemistry. Immuno-diagnostic kits are widely available and used extensively in departments of diagnostic pathology and cell biology. Almost every diagnostic pathology laboratory uses routinely over 100 specific antibodies for the detection of antigens. Most of these kits are based on the principle of avidin-biotin or streptavidin-biotin binding. Despite the widespread use of avidin-biotin or streptavidin-biotin complexes in so many areas in the life sciences, the biological significance of this strong binding is still not known (Wilchek et al., 1997).
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Lectin histochemistry Lectins are proteins and glycoproteins with specific affinities for carbohydrates. They are also called agglutinins. The major advances in carbohydrate histochemistry ensuing from the discovery of lectins possessing affinity for specific terminal sugar groups have had enormous impact in cell biology and histopathology (Brooks et al., 1996). The use of a battery of these agglutinins conjugated to horseradish peroxidase or a fluorescent label permits localization of specific sugars in cells and tissues (Danguy, 1990; Spicer, 1993). In the last decade lectin histochemistry has emerged as a powerful tool to identify and localize carbohydrate moieties. Today, lectins are widely used as histochemical probes to localize aberrant glycosylation in cancer, metabolic diseases and other pathological conditions. Carbohydrate immunohistochemistry has also played a major role in distinguishing endocrine cells in the pituitary gland, islets of Langerhans and the diffuse endocrine cells of the intestinal and respiratory tracts.
Fluorescence microscopy The impact of immunohistochemistry in the life sciences was possible not only by the developments in signal amplification techniques, but also by improvements in imaging techniques. Fluorescence microscopy has always been a major visualization technique in immunohistochemistry because of its sensitivity (Ploem and Tanke, 1987; Herman, 1997). The last 2 decades have seen major improvements in fluorescence microscopy due to the introduction of confocal scanning laser techniques, that allowed higher spatial resolution of fluorescent signals by optical sectioning, and digital imaging that allowed higher sensitivity and 3D reconstruction (Sheppard and Shotton, 1997). The possibility to combine signals from different antibodies in one microscopical image has also provided major advances in immunohistochemistry. The spectacular images obtained with fluorescence confocal scanning laser microscopy are widely used on front covers of research journals in biology and medicine and in the advertising material for molecular probes, more than those of any other visualization technique.
Colloidal gold and ultrastructural cytochemistry Colloidal gold was first introduced as cytochemical marker for transmission electron microscopy (TEM) in 1971 (Faulk and Taylor, 1971) and for scanning electron microscopy (SEM) in 1975 (Horisberger et al., 1975). Colloidal gold labeling offers high-resolution localization of specific antigens and their quantification at the subcellular level and has contributed enormously to the understanding of intracellular processes (Beesley, 1989; Hodges and Carr,
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1990). Double-labeling techniques in TEM using different sizes of gold particles has allowed subcellular localization of more than one antigen in a single preparation.
In situ enzyme histochemistry The major development in the field of enzyme histochemistry in recent years has been the replacement of catalytic histochemistry by immunohistochemistry as the preferred means of visualizing enzymes (Stoward et al., 1998). However, there are fundamental differences between the two approaches, because the former demonstrates the functional activity of an enzyme, whereas the latter demonstrates the protein of an enzyme be it active or inactive (Van Noorden and Jonges, 1995). Furthermore, the last 2 decades have seen the development of histochemical techniques to determine in situ kinetic behavior of enzymes (Van Noorden and Jonges 1995; Stoward et al., 1998). It is easy now to measure reaction rates of enzymes in tissue sections by monitoring the formation of the final reaction product throughout the incubation. Such powerful tools may provide significant impact in histochemistry in the future as they enable quantitative determinations of metabolic pathways in situ in conjunction with advanced imaging of tissues and cells.
In situ hybridization In situ hybridization (ISH) techniques and, in particular, fluorescence ISH (FISH) have proved to be invaluable molecular tools for visualizing nucleic acids in their cellular environment. The techniques were introduced in the early 1980s. ISH has had a tremendous impact in the last 10 years in cytogenetics and cancer pathology (DNA-ISH). Furthermore, precise localization of mRNA enables the elucidation of when and where specific genes are transcribed and specific proteins are produced (Leitch et al., 1994; Stoward et al., 1998). The applicability of ISH is still limited by restricted detection sensitivity (Speel, 1999). In the last 10 years, techniques have been developed to improve nucleic acid detection in situ by amplification of target nucleic acid sequences before ISH by ªin situ PCRº, which is a marriage of ISH and reverse transcriptase-polymerase chain reaction (PCR) (Stoward et al., 1998; Long 1998). More recently, alternative methods for improving detection sensitivity have been developed involving tyramide signal amplification using the catalyzed reporter deposition (CARD) technique (Speel et al., 1998, 1999). Several methods for in situ hybridization at the ultrastructural level have been reported as well permitting the detailed localization of nucleic acid sequences (Le Guellec, 1998). The new in situ techniques are emerging as extremely powerful tools for basic and diagnostic research in histochemistry, cell biology and pathology.
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Quantitative histochemistry The combination of histochemical localization and accurate quantitation has been fraught with complications in the past, usually because of problems linked with the many variables introduced by specimen processing and/or imaging. Quantitation of staining intensities in defined areas of sections is possible when the final colored reaction product is stable and has sufficient contrast (Stoward and Ploem, 1982). In the 1970s, this became feasible using integrating microdensitometers such as the Zeiss Scanning Microscope Photometer or the Vickers M85 Scanning Microdensitometer (Chayen and Bitensky, 1991). In the last 2 decades, such instruments have been largely replaced by image analysis systems as a result of the widespread introduction of CCD cameras linked with personal computers and user-friendly software. Cytophotometric and integrated densitometric quantitation has become more and more easy to use. However, the pitfalls of quantitative measurements, especially with respect to ªstaining intensityº levels of immunohistochemically stained sections or cells, are not always appreciated. The introduction of spectral imaging in the last few years has provided greatly improved quantitative and analytical tools for histochemistry and histopathological diagnosis and are undoubtedly important in future developments (Malik and Rothmann, 1999). Quantitation of fluorescence signals in fluorescence microscopy is still problematic owing to signal depletion (photobleaching or quenching effects) and this is an area where technology is still seeking suitable answers.
Live cell histochemistry The great paradox in histochemistry and cytochemistry of the last century is that most of our concepts were developed from the analysis of dead biological material, whereas the molecular and structural events in cells are dynamic processes (Van Noorden, 1999). Vital and supra-vital staining is a topic in histochemistry that urgently needs to be developed. Relatively few techniques are available yet. In the past, vital dyes have been useful, for example, for the understanding of dynamic processes such as phagocytosis. The last few years have been the scene of a remarkable return of vital labeling, in particular with the discovery of new fluorescent probes and the introduction of new imaging techniques to study dynamic processes that occur in living cells. A 2-day meeting held in conjunction with the Microscopy and Microanalysis '99 conference, entitled ªOptical microscopy in the next millenniumº, was recently held in Portland, Oregon (July 31±August 1, 1999). Some 30 papers presented a wide range of applications of the new methodologies in optical microscopy, many of which could be described as leading edge dynamic histochemistry involving real-time imaging, in particular using multiphoton microscopy and spectroscopy. Themes included applications of new molecular probes, espe-
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cially for protein trafficking. Green Fluorescent Protein (GFP) of jelly-fish origin is so extensively used today for live fluorescence imaging and possesses such remarkable properties (for example it is the best intracellular pH indicator), that it must be considered to be a prime candidate for the nomination to become ªmolecule of the yearº. In conclusion, it can be stated that histochemistry and cytochemistry have had an enormous long-term impact in cell biology and molecular biology during the last century, and will continue to contribute to our understanding of dynamic cellular and pathological processes, despite the fact that these disciplines do not always achieve the recognition they really deserve.
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