Fluorescence Microscopy In Life Sciences E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Fluorescence Microscopy is a precise and widely employed technique in many research and clinical areas nowadays. Fluorescence Microscopy In Life Sciences introduces readers to both the fundamentals and the applications of fluorescence microscopy in the biomedical field as well as biological research. Readers will learn about physical and chemical mechanisms giving rise to the phenomenon of luminescence and fluorescence in a comprehensive way. Also, the different processes that modulate fluorescence efficiency and fluorescence features are explored and explained.
Key learning points covered in the book include:
Operation of fluorescence microscopy instruments as well as the different options available today for the scientist, from the classical to the most recent approaches
The wide range of biological detection possibilities that fluorescence microscopy offers in molecular biology, cell biology, histology and histopathology
Fluorescent chemical compounds
Breakthroughs in this field, such as non-linear microscopies and super-resolution techniques
Fluorescence Microscopy In Life Sciences is intended as a detailed guide for professionals, researchers and students (including graduates Ph.D. candidates) in life sciences, with special emphasis in the biomedical field. Researchers working in allied disciplines such as pharmacology, veterinary sciences and microbiology will also benefit from the information presented in this handbook.
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Veröffentlichungsjahr: 2017
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Fluorescence Microscopy in Life Sciences
Authored by:
Juan Carlos Stockert
&
Alfonso Blázquez-Castro
BENTHAM SCIENCE PUBLISHERS LTD.
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FOREWORD
Using fluorescence microscopy to study the components and processes of cells, tissues and organisms is not of course a novel investigatory approach, as it dates back to the early years of the twentieth century. However, the widespread use of this methodology had to wait for the commercial availability of fluorescence microscopes, in particular for commercialised confocal instruments. The parallel flowering of fluorescent probes for use with such microscopes, together with the continuing evolution of novel and powerful optical systems, further accelerated uptake of such methods within a wide variety of biomedical fields.
Consequently, many books — and even more chapters and reviews — have considered various specific aspects of fluorescence microscopy or, less often, provided more global overviews. Nevertheless, even in this crowded field, the present volume is ambitious, adopting, as it does, a rather different approach to most of its predecessors.
A key difference is its inclusive approach. Thus this book adopts a firmly didactic approach on the fundamentals and the applications of fluorescent microscopy. The aim is clearly to bring biologists and physicians up to speed with a basic knowledge of the physical chemistry, technical equipment, and practical manipulations underlying a functional understanding of fluorescence methods. However, the book also provides sufficient detail of practical applications, within an intelligible biomedical context, to allow readers whose backgrounds are chemistry or physics to understand the types of problem which are currently addressed by this approach. But more than that, to grasp possible future expansions of applications of fluorescence microscopy.
Another striking feature is the amount of critically selected information made accessible. Consider the physicochemical background. An account of colour theory – traditional, valence bond and molecular orbital – leads to a description of the molecular mechanisms of fluorescence. And so to the role of quenching, thereby answering the biologist’s plaintive enquiry, “But if that is how fluorescence arises, why are all dyes not fluorescent?” The separate discussion of fluorescent dyes and reactive fluorescent dyes (i.e., labels) will constitute a further useful clarification for many biomedical investigators. Perhaps, having read this account, readers will not be so likely to generate publications saying that a “rhodamine/Bodipy/fluorescein” dye was used – and imagine they are providing useful information. Indeed, there are explicit sections clarifying issues of nomenclature. Then there is extensive consideration given to the nuts and bolts of everyday practice – both regarding the instrumentation and the protocols used. Finally there are the many chapters addressing applications. These address the staining of all common biochemical components and organelles of cells. Again, the chosen examples of applications are broad based. Staining of both biopsy material and of living cells are separately discussed. Consequently, this book is just the place to send those, frequent, enquirers who ask, “Which dye should you use for … ?”
Another marked characteristic of this book, supporting and illustrating the features discussed above, are the numerous, informative and often visually attractive coloured figures. Presumably it is the use of an e-book format which makes such lavish illustration possible.
So do I think anything is missing? Well, there is no significant account of the burgeoning field of clinical diagnostic applications of fluorochromes – to detect or indicate the borders of tumours for instance. Nor of “smart probes” – those imaging compounds which exploit biochemical quirks to obtain selective staining, for instance of particular tumour cell lines. But no book can be all things to all people – and this book is certainly a remarkable fusion of information, explanation and beauty. In fact it’s hard to imagine who wouldn’t benefit from having this volume available for repeated consultation and, just as valuably, for mind and possibility-broadening browsing.
PREFACE
The current use of light for so many applications is without precedence in history. If the 20th century would probably be recalled as the “age of the electron”, then the 21st century will be, undoubtedly, the “age of light”. In the Life Sciences one of these light applications is at the very foundations to obtain knowledge: fluorescence microscopy. Fluorescence microscopy has offered, since its inception during the second half of the 20th century, a definitive enhancement in the signal-to-noise ratio in comparison to bright field microscopy. This increased contrast has done nothing but to grow as the years have passed by, accompanied by an unprecedented increase in resolution too. The improvement in resolving power has reached a level that empowers fluorescence microscopes to image nanoscopic objects with a detail that rivals some electron microscopy techniques. All of these without forgetting that fluorescence microscopy allows for real-time assessment of live samples, something at the reach of not so many analytical techniques.
Our goal with this book has been to present a straight, didactical work. We have strived to present all topics in a practical way, with as many real-world examples as possible. Also our aim has been to introduce concepts from an as intuitive as possible perspective, without neither being over simplistic nor eluding the theoretical foundations of the underlying processes. From our experience there has been a lack of a didactical work that presents fluorescence microscopy, as encompassing as the field is, to the broad audience in the Life Sciences. Therefore our intention has been to present a comprehensive yet user-friendly compendium of both basic and advanced topics in the field.
The book has been structured in three parts although it has not been explicitly divided into such. The first part (Chapters 1-7) presents the general concepts and processes to understand fluorescence microscopy, both from theoretical and applied points of view. The second part (Chapters 8-17) focuses on the main application areas of fluorescence microscopy. Our approach for this has been to present the principal biomolecules and organic molecules that make up living organisms and how fluorescence microscopy is employed in every case to obtain useful information. Finally in the third part (Chapters 18-20) we introduce to the reader some of the new application fields of fluorescence microscopy, many of which (e.g. luminescent nanoparticles or multiphoton microscopy) have evolved from interdisciplinary overlap with other scientific areas (solid-state physics and non-linear optics).
Fluorescence microscopy is a continuously evolving field, as we can testimony during this book writing process. As a consequence many techniques introduced in the book will transform over time. But our intention has been to make the fundamentals as clear and approachable as possible. Understanding the supporting concepts ensures the comprehension of the techniques, no matter their evolution and improvement. Finally there is, we must admit, an aesthetic side in fluorescence microscopy that makes this technique so appealing. The pure colours and intense contrasts commonly seen in fluorescence images put many of them on pair with art pieces. Truly an image speaks more than a thousand words when talking about fluorescence microscopy. In this sense, we coincide with Francis Bacon when he stated: “The beholding of the light is itself a more excellent and fairer thing than all the uses of it”.
DEDICATION
To my family, especially Federico and Alejandro (my heroes), and Gloria (my muse).
To my wife, Bego, for her patience and her support during this long road.
ACKNOWLEDGEMENTS
We thank the following colleagues and friends for encouraging support, fructiferous collaboration, and stimulating discussions:
María I. Abasolo, Ulises Acuña, Ümit Akbey, Marco A. Alvarez, José L. Bella, Gabriel Bertolesi, Thomas Breitenbach, Magdalena Cañete, Paul J. CaraDonna, Elisa Carrasco, Adriana Casas, Lucas L. Colombo, Pedro Del Castillo, Edgardo N. Durantini, Jürgen Elvert, Jesús Espada, Pedro Esponda, Juan M. Ferrer, Sergio Galaz-Leiva, Antonio Gómez, Jorge Herkovits, Richard W. Horobin, Amy M. Iler, Angeles Juarranz, Markus Kempf, John A. Kiernan, Claudia Lanari, José A. Lisanti, Daniel M. Lombardo, Isabel Lüthy, Maria Luiza S. Mello, Roberto Mezzanotte, Arturo Morales, Karen ní Mheallaigh, Santi Nonell, Claus Pelling, Peter R. Ogilby, Carolina Ollé, Cristina Ortega-Villasante, Viviana Rivarola, Antonio Romero, Steven E. Ruzin, Sergio H. Simonetta, Alberto J. Solari, Arnaldo T. Soltermann, Carlos Soñez, Cristina Soñez, Francisco Vicente Pedrós, Benedicto C. Vidal, Angeles Villanueva, and Clara I. Trigoso.
Finally, our apologies to many other people lacking explicit mention but also very interested in this e-book and always supporting our research work.
Our institutional acknowledgements go to the following institutions for their help and support:
JCS - Max-Planck-Institut für Biologie, Tübingen, Germany, Department of Biology, Faculty of Sciences, Autonomous University of Madrid (grant CTQ2013-48767-C3-3-R from the Ministerio de Economía y Competitividad, Spain), Institute of Research and Technology in Animal Reproduction, Faculty of Veterinary Sciences, University of Buenos Aires, and Institute of Environmental Sciences and Health, Buenos Aires, Argentina.
ABC - Aarhus Institute of Advanced Studies (AIAS), Aarhus University, Aarhus, Denmark; Marie Skłodowska-Curie actions - Research Fellowship Programme (AIAS-COFUND programme 609033).
Introduction
Juan C. Stockert*Abstract
This book tries to be a guide for understanding fluorescence microscopy, and explaining its chemical and physical principles for workers in biomedical sciences, especially those with limited expertise in chemistry and physics. In contrast to early morphological studies, considerable background of physics and chemistry is at present necessary to make fluorescence microscopy a more fruitful technique. In this book we therefore attempt to simplify and make understandable the basis of fluorescence reactions and their biomedical applications. When possible, mechanistic approaches have been introduced regarding dye affinity and fluorescent selectivity. To make the text more didactic and amenable, cell and tissue pictures, diagrams, graphs and chemical structures are included. Obviously, overlapping of several issues concerning fluorochromes and fluorescence techniques will be found along chapters. As an example, consider binding of a fluorescent ligand to a biomolecule. This can be studied from the point of view of (1) ligand properties and binding, (2) substrate structure and affinity, (3) reaction methodology, mechanisms, instrumentation, etc. In turn, ligands can be simple molecules (fluorochromes, vital probes), macromolecules (phycobilins, fluorescent proteins), or multi-molecular complexes (labeled IgG, lectins, oligonucleotides), in which different fluorescent labels are used for visualizing a great variety of biological substrates.
1. FLUORESCENCE METHODOLOGY: GENERAL ASPECTS
At present, there is almost no biomedical field in which fluorescence methods are not applied. Research professionals have been increasingly interested in this methodology, and now new concepts, instruments and techniques have developed rapidly leading to constant progress in the design and application of fluorescence methods. The literature of fluorescence methodology is so extensive that only the most relevant references are included in each chapter. Likewise, no special emphasis is given to precise chemical description of biological substrates (i.e. lipids, polysaccharides, nucleic acids, proteins, etc.), because these are explained in detail in textbooks of biochemistry and cell biology.
On the other hand, the number of fluorescent images is overwhelming [1], and it must be recognized that even in an E-book, the number of pictures must be limited. It is unfortunate that many beautiful and interesting colored images remained out of this work. Many of them are, however, available at www.probes.com, www.lifetechnologies.com/bioprobes), and other commercial sources.
Classic books should be consulted for more detailed accounts in the fields of light, color and chemistry of dyes [2-9], microscopical and histochemical staining [10-17], and fluorescence methods [18-29]. In addition, exhaustive descriptions on specific properties and biological uses of dyes, fluorochromes, and fluorescent probes are available [1, 30-34]. Relevant critical reviews, and historical articles on the same subjects should also be consulted [35-45].
Here the terms, fluorescent dye and fluorochrome apply indistinctly to any fluorescent compound used for staining fixed cells or labeling live cells. The terms, luminophores and fluorophores are also used, meaning a chromophore (part of a molecule) that absorbs UV or visible light and emits light of a longer wavelength (i.e. fluorescence). Fluorescent probes are vital fluorochromes that localize selectively in some cell or tissue structures. Sometimes they are referred to as “fluoroprobes”. A fluorescent label indicates a fluorochrome that is covalently or otherwise strongly bound to a biomolecule. The generic term “staining” is often applied to denote the use of a fluorochrome in fluorescence microscopy, i.e. “acridine orange staining”.
Some misleading concepts should be taken into account and corrected regarding fluorescence microscopy. Often it is claimed that to observe fluorescence, ultraviolet (UV) excitation is required. This is true for only some fluorescent compounds, because others need blue or green excitation. Fluorochromes are sometimes abbreviated as “fluors”. “Immunofluorescence microscope” is a bad name, because there is no specific microscope to be used for this technique. Description of excitation filters by their applications instead wavelength (i.e. “Texas red excitation/emission filters”, “DAPI channel”, or “DAPI cube”) is another ambiguous and imprecise practice. High brightness is often taken as a synonym of strong affinity for a given substrate, but the emission intensity is only based on the fluorochrome chemistry and binding mode. Therefore, some prejudices and myths in the fluorescence methodology should be corrected.
2. COLOR AND ENERGY SPECTRUM
Color is the visual impression produced by light emitted by a luminous source or reflected from a material. Sometimes, a color is often subjectively described by comparing it with the color of a familiar object. A perceived color is largely dependent on the type of illumination and light intensity (e.g. brown is not a spectral color but a very dark orange). Clearly defined assessment of colored objects is possible through objective measurements and chromaticity diagrams.
Cyan, yellow and magenta are the three primary colors. All the remaining solid (absorption) colors can be obtained by mixing the primary ones. Techniques for color restitution in trichromy are shown in Fig. (1.1). Therefore, colors derived from light absorption or emission correspond to different physical processes. When a given color (wavelength) is absorbed by a material illuminated with white light, then the complementary color is observed (blue, green, red, respectively). This feature, as well as the distribution of radiation energy over a large scale, is illustrated in the next chapter (see Fig. 2.2).
Fig. (1.1)) Diagram of primary colors (absorption and emission), and trichromic RGB synthesis.Depending on the energy (wavelength) of the light that is absorbed by a compound, several types of interactions occur between photons, electrons, atoms or the whole molecule, and can have different effects (i.e. heating, luminescence). According to the wavelength of photons their interaction with molecules results in electronic excitation (UV-visible), atomic vibration and rotation (near infrared, NIR), or movement of whole molecules (far infrared, FIR) (see Chapter 2.4). Luminescence consists in light emission from the excited matter. Photoluminescence is caused by light absorption that induces fluorescence and phosphorescence. When a chemical reaction takes place, the energy it generates can be released as light (chemoluminescence). In the case of enzymatic catalysis of chemiluminescent reactions, the process is known as bioluminescence. In contrast to the color of the world that surrounds us (due to the differential absorption of white light), luminescence color (e.g. fluorescence) has the true color (wavelength) of the emitted light.
3. BRIEF HISTORY OF STAINING AND FLUORESCENCE METHODS
First uses of dyes for general or histochemical staining reactions are listed in Fig. (1.2). It is only an approximate survey, because of doubts about the priority of some discoveries. As Lillie [31] validly stated: One can never be certain how long any particular individual will remain credited with being the “first” to do something. Unfortunately, current fashion is to quote only the most recent works, leaving the original sources in oblivion. Therefore, historical and cultural aspects of science and technology are often ignored in recent publications.
Fig. (1.2)) Brief history of microscopical and histochemical staining (see [31, 34]).3.1. Natural Dyes
The natural compound indigo (indigo blue, vat blue 1, CI 73000), known and applied as textile dye for 5000 years, is produced by fermentation of the glycoside “indican” from leaves of Indigofera tinctoria. Dimerization of the resulting indoxyl produces the insoluble blue dye, indigo. After alkaline reduction, indigo becomes soluble and colorless (indigo white, leuco-indigo). It is then applied to the fabric, and the blue color reappears by air oxidation. Now, the current commercial dye is synthetic. In the textile industry indigo is used mainly to dye blue jeans and other denim products.
Interestingly, although in the free form indigo undergoes photodegradation (fading), the dye is protected in some environments. In addition to faded indigo, Mayan artwork from the 6th century found in the Yucatan peninsula also contained the dye (produced from the leaves of the añil plant, Indigofera suffruticosa) included within channels of the clay palygorskite. This inclusion compound (Maya blue) conserved a bright blue color because of high chemical- and photo-stability of indigo when protected within the clay lattice [46, 47].
Tyrian purple (6,6’-dibromoindigo, the most precious indigoid dye, made in pre-Roman times by the Phoenicians), is obtained from the marine snails Bolinus (Murex) brandaris, Hexaplex trunculus, Purpura pansa, Nucella lapillus,etc. (Muricidae, Neogastropoda: Mollusca). The dye was greatly prized in antiquity because the color became brighter with weathering and sunlight. On account of their high cost, purple-dyed textiles became status symbols (i.e. royalty, prelates). Exploitation of Tyrian purple is known from as early as the 13th century B.C., and has attracted the interest of historians, artists, dyers, archeologists, chemists, biologists, and pharmacologists [48, 49]. Tyrian purple derivatives (as indirubin compounds) show important antiproliferative activity [50]. Histochemical use of indigogenic substrates is described in Chapter 14.
It is interesting to mention that murexide (ammonium purpurate) is a deep purple dye known since late the 18th century, which increased the fascination for the purple (“the color of power”). Its name (from Murex mollusks) evokes the Tyrian purple of the ancients used for the dresses, and was first prepared by heating uric acid with nitric acid, and then treating the dried solid with ammonia. Murexide has been used for textile dyeing, as product from the reaction to reveal uric acid, and for Ca2+ detection in tissues [10].
The first microscopic staining reaction using the natural dye saffron (crocin, CI 75100; from stigma of Crocus sativus; see Fig. 3.7) is attributed to Leeuwenhoek in 1714 [51]. Other natural dyes were also applied to stain different cell and tissue components, notably cochineal (carmine, CI 75470) and hematoxylin (CI 75290). The latter compound is extracted from one of the trees with red wood [52], found by Spanish explorers in Yucatan, Mexico, in 1502. The generic name (Hematoxylon; Haematoxylum according to Linnaeus, 1753) derives from “blood-colored wood”. Historical aspects of hematoxylin and its oxidized product were described [34, 53] (Fig. 1.3) (see also Chapter 3.4.3).
A vigorous trade soon developed related to preparing hematoxylin for use in dyeing fabrics in Europe. In the mid 1800s, amateur microscopists applied hematoxylin to stain nuclei. Today, the aluminum complex of hematoxylin remains the most popular nuclear stain in histology and histopathology. It is interesting to note that the name Brazil comes from the term “brasa” (live coal) due to the occurrence in that country of a tree with red wood (brazilwood, Caesalpinia echinata), from which the hematoxylin-related dye brazilin (CI 75280) was obtained. This dye also occurs in other leguminous plants from Sumatra (C. sappau) and Central America (C. brasiliensis, C. crista).
Fig. (1.3)) Hematoxylin. A, B: The logwood Haematoxylon (Caesalpinia) campechianum, Leguminosae (Campeche’s, wood). C: Oxidation process yielding the dye product, oxidized hematoxylin (hematein) showing the numbering of atoms.3.2. Synthetic Dyes
The first synthetic dye, mauveine (mauve, aniline purple, Perkin´s violet, CI 50245), was obtained by the eighteen-year-old English chemist W.H. Perkin in 1856 using potassium dichromate and sulfuric acid on crude aniline. Its industrial application was very successful for dyeing silk and cotton fibers with higher brightness and fastness than natural dyes then in use. Mauveine is commonly mentioned in books on color chemistry on account of its great historical interest [54], but at present no application as a textile or histological dye is known. After examination, a sample of the original dye (λab: 548 nm) showed an exceptional high tinctorial value [30]. It is structurally related to safranine O (CI 50240) and thus red fluorescence would be expected to occur for mauveine.
Before Perkin’s synthesis other dyes were also produced (reviewed by Lillie [31]). As early as about 1300, French purple (orcein, orseille; likely the first synthetic dye!) was synthesized by air oxidation of orcinol from the orchil Rocella tinctoria and other lichens in the presence of ammonia (from fermented urine). Indigocarmine was obtained in 1740 by treating indigo with sulfuric acid. Another artificial dye was murexide. Picric acid was prepared by Woulfe in 1771 [30] and produced commercially one hundred years later. Rosolic acid was synthesized by Runge in 1834, and produced commercially thirty years later. In contrast, the time interval between mauveine synthesis (1856) and industrial production (1857) by Perkin was only one year. Almost all dyes are now derived from raw materials ̶ mainly benzene, toluene, naphthalene, anthracene ̶ obtained from coal tar or petroleum. These aromatic hydrocarbons provide the molecular framework for the final structure of a dye, which is a substance that can confer color.
3.3. Fluorescent Compounds
On the other hand, fluorescence methods also have an interesting history (Fig. 1.4) (for details see [42, 45]).
Fig. (1.4)) Brief history of fluorescence methodology.In 1565 the Spanish botanist and physician Nicolás Bautista Monardes (1508-1588) noted that a water extract of a Mexican wood exhibited an odd blue shimmer. Kircher (1601-1680) also referred to the blue tinge of water contained in a cup made from this Mexican wood. The medicinal wood, known in Europe in that time as Lignum Nephriticum from the Mexican tree Eysenhardtia polystachya (Leguminosae) had been used to treat kidney and bladder diseases; Aztec healers had already noticed the “blue” shine of infusions of slices of this wood.
Recent studies have revealed that at least one flavonoid derivative of this tree (the glycosyl dihydrochalcone, “coatline B”) is rapidly converted by oxidation into the water-soluble fluorophore “matlaline”, with an intense blue emission (λem: 465 nm) only at alkaline pH, with fluorescence quantum yield ΦF: 1 (Fig. 1.5) [55]. According to Boyle’s (1627-1691) description, this pH dependence (blue shine or not at all at alkaline or acid pH, respectively) is possibly the first observation on a fluorescent pH indicator [42]. The same fluorophore seems to form also from infusions of similar trees such as E. officinalis and the Philippine Pterocarpus indicus.
Fig. (1.5))A: Blue fluorescence of an aqueous infusion of Lignum Nephriticum under daylight illumination (reprinted and adapted with permission from [45], Copyright 2011, American Chemical Society). B: Chemical structure of matlaline in its acid form. C, D: Resonant forms of the ionized fluorophore at alkaline pH, showing the conjugated double bonds (blue) and charges, similar to oxonols, coumarins and xanthenes (see Figs. 3.6 and 3.18). E: Chemical structure of the glycosyl residue at R.Almost three hundred years later, the red emission of green leaf extracts (chlorophyll) and the blue emission of quinine were described by Brewster and Herschel, respectively. George G. Stokes and Adolf von Baeyer greatly contributed to establish the importance of fluorescence methods. Their potential was already understood in 1877, when Baeyer demonstrated a link between the headwaters of Danube (which flows into the Black Sea) and Rhine (which flows into the North Sea) [45, 56]. He suggested that 10 liters of concentrated uranin solution (disodium salt of fluorescein) be thrown into the Danube (near Immendingen). Fifty hours later, the characteristic green fluorescence of the dye was found in the river Aache (12 km to the south), then in Lake Constanz and finally in the Rhine. At present, fluorometric methods are used for tracking water pollution in rivers and lakes [57].
In contrast, some developments in fluorescence microscopy have only a short history. Immunofluorescence and epifluorescence, for example, are about 75 and 50 years old, respectively [58, 59], and now rapid developments occur in the case of new methods such as super-resolution fluorescence microscopy (see Chapter 20).
4. TYPES OF DYES AND FLUOROCHROMES: NOMENCLATURE
For practical purposes, the terms dyes, colorants and stains will be used here as synonyms and in a wide sense. Synthetic dyes were first used in the textile industry, and can be classified according to methods of application to fabrics. Today, dye classification is mainly based in the chemical structure of chromophores, although names and characteristics derived from industrial dyeing also appear in descriptions of dye properties. Dyes and fluorochromes can be classified according several criteria (Fig. 1.6).
Fig. (1.6)) General classification of dyes according to different criteria.Anionic and cationic dyes are defined by the occurrence of negative or positive groups in the molecule, amphoteric dyes having both groups. Non-ionic, hydrophobic dyes have no charged groups. Solvent dyes are colored, nonpolar compounds that dissolve in lipids. Reactive dyes remain bound to the substrates by covalent linkages. Direct dyes are anionic or amphoteric compounds originally designed for staining cellulosic materials (see Chapter 11.2). Mordant dyes combines with metal ions to form metallic complexes (see Chapter 8.2). Vat dyes generally correspond to anthraquinone or indigoid dyes that are applied as leuco-compounds, and color is regenerated by oxidation. In general, names of dyes have initial capitals only for words that are proper nouns, as in Congo red, Nile blue, Texas red, etc.
On the other hand, trivial names or acronyms are often used to refer to more complex chemical denominations of dyes or well known staining procedures. Thus it is practice to employ the current name of H&E for the staining sequence aluminum ions (alum)-oxidized hematoxylin followed by eosin Y, the staining method most commonly used in histology and histopathology. Other examples are Bodipy (dipyrromethene-boron difluoride), DAPI (4’,6-diamidino-2-phenyl indole), DiOC1(3) (3,3’-dimethyl-oxacarbocyanine), etc.
Regarding the chemical composition of natural and synthetic dyes, they belong to a great variety of chemical groups such as anthraquinone, azine, azo, di- and triphenylmethane, indigo, oxazine, phthalocyanine, polyene, thiazine, xanthene, etc. Several of these compounds are fluorescent, mainly those containing chemical groups such as acridine, aryloxazole, benzimidazole, benzoxazole, benzothiazole, bimane, carbocyanine, coumarin, dipyrrole-boron, flavone, indole, naphthalimide, phenanthridine, porphyrin, stilbene. More precise descriptions will be found in Chapters 3 and 4.
Unfortunately, dye nomenclature is variable and ambiguous. Some dyes have many synonyms and they can be easily confused. Therefore, in addition to the usual name, the Colour Index (CI) provides a precise way to identify dyes, providing a unique number for each chemical structure. Unfortunately, many fluorescent compounds are not included in the CI. In cases of contradictions in chemical numbering of chromophore rings, that of the Merck Index is here used [60]. Numerous names and acronyms of fluorochromes are cryptic and difficult to understand (e.g., Alexa, Cy3, Cy5, FM 4-64, PKH 26, PKH 67). Some trivial names are unfortunate (indigocarmine is not red but blue, fluorone black is not black but red) or misleading (true blue, fast blue). Likewise, postfixes are generally unintelligible and therefore they are often omitted. This is a bad practice because postfixes define the type of dye. They often refer to the shade, solubility or other characteristics of the compounds (Y: yellowish; B: bluish; S: soluble).
Although at present numerous fluorescent probes are available, chemical structures are not always revealed by their manufacturers and vendors. Thus Molecular Probes and Ursa BioScience have produced fluorophores, some of which are their own, with non-revealed chemical structures. This secrecy hinders understanding of their properties and reaction mechanisms with biological substrates. Often fluorochromes are sold in kits with attractive or fantastic names, designed to promote their consumption: examples are AttoPhos®, GelGreen®, NanoOrange® protein reagent, OliGreen® ssDNA reagent, PicoGreen® dsDNA reagent, ProQ Diamond®, etc. Alexa fluorochromes have become fashionable and mainly correspond to coumarins, rhodamines and carbocyanines (all sulfonated). Advantages are mentioned for Alexa fluorochromes: high brightness and photostability, good solubility in water, insensitivity to pH changes, and multiple emission colors [1]. An attractive goal for usage is to design a gallery of dyes covering the full spectrum of colors.
5. MICROSCOPIC STAINING AND FLUORESCENCE
Without having differences in the refractive index or absorption characteristics between different cell structures, the need of staining to distinguish them becomes evident. Although staining and fluorescence reactions will be presented in detail in other Chapters, some examples are illustrated here. Ortho- and metachromatic reactions are very common in microscopy, and Fig. (1.7) shows representative bright-field images after staining with thiazine and xanthene dyes. In addition, fluorescence reactions induced in plastic semi-thin sections can be observed in Fig. (1.8). In the case of acridine orange, the differential orthochromatic (green) and metachromatic (red) fluorescence reactions of specific structures in live and dead (fixed) cells are shown in Fig. (1.9) (see Chapter 15.1).
Fig. (1.7)) Bright-field metachromatic reactions in Epon semithin sections of mouse large intestine stained by toluidine blue (A) and pyronine Y (B) (both dyes at 10-4 M, pH 5). Observe the violet (A) and orange (B) metachromatic reactions of mucin. Nuclei appear in orthochromatic blue and pink-red, respectively. Fig. (1.8)) Examples of fluorescence reactions from semithin Epon sections of mouse large intestine (A, B), kidney (C), and uterus (D) stained with 10-6 M DAPI (A), saturated morin in distilled water (B), 10-4 M berberine (C), and 0.2 mg/mL orcein (D). In C and D, dye solutions were in borate buffer pH 9.2. Mucin from goblet cells, chromatin, and reticular and elastic fibers are shown in A and B, C, and D, respectively. Scale bars: 30 µm. Fig. (1.9)) Acridine orange staining of dead and living cells. A: Ehrlich ascites tumor cells fixed in methanol, showing metachromasia of cytoplasmic RNA (arrows). B: Living Pam212 keratinocytes in culture with metachromatic lysosomes (arrows). Acridine orange was applied as follows: 50 µg/mL, 1 min (A), and 5 µg/mL, 15 min (B).It is well known that fluorescence methods offer advantages of enhanced sensitivity and contrast over transmitted light absorption methods. Fluorescence microscopy is no exception: fluorescent cell and tissue structures have higher visibility than those observed by bright-field microscopy and stand out against a black background. But not all bright structures contrasting on a dark background are necessarily fluorescent ones. Thus, this type of image pattern may be observed in dark-field illumination, and linear or circular polarization microscopy (Fig. 1.10) [61]. In this context, an intriguing artifact was described in the histological technique. If paraffin is not adequately removed from sections, bright birefringent chromatin spots are seen within nuclei under polarization microscopy [62].
Fig. (1.10)) Fluorescence-like images: dark-field and polarization microscopy. A: Dark-field image of a plant anaphase after silver staining showing bright prenucleolar bodies. B: HeLa cells treated with a sunflower oil emulsion showing birefringent lipid droplets in the cytoplasm. C: Starch granules of the arrowroot rhizome (Maranta arundinacea) showing typical Maltese crosses, polarization microscopy. D: Same image as C, but observed trough a λ-plate.6. ADVANTAGES AND APPLICATIONS OF FLUORESCENCE METHODOLOGY
At present, luminescent molecules have many advantages and are very important research tools in biomedical sciences. Regarding sensitivity, conventional detectors (e.g. the eye) record the emitted light with intensity at least three orders of magnitude lower than the reflected light after specific absorption by a colored body. Therefore, the major advantage of fluorescence methods is their greater sensitivity (103 to 105 times higher than that achieved by light absorption). Very high fluorescence quantum yields exist in some natural fluorophores such as phycobiliproteins and those from the green fluorescent protein family, which show bright fluorescence (see Chapter 4.6).
On account of its high sensitivity, fluorescence is an extremely powerful tool for several analytical applications, including detection of biomolecules, ligand-receptor interactions, enzymatic activity, etc. [22, 56]. A survey of fluorescence methods that have important applications in biomedical fields include:
Microscopical procedures in microbiology, genetics, histology, histochemistry, cell and molecular biology, developmental biology, toxicology, pathology (fluorescent vital probes, viability assays, fluorescent indicators, FISH, immunofluorescence).Immunological and image methods for medical diagnosis.Analytical (qualitative and quantitative) techniques in chemistry and biochemistry (electrophoresis, chromatography).Population analysis of cells (flow cytofluorometry).A few examples will illustrate these issues. The diagnosis of some infectious diseases is now greatly improved by using fluorescent microscopic methods. The detection of Mycobacterium tuberculosis remains a diagnostic challenge in many resource-poor countries. Classical auramine O fluorescence is a useful method for mycobacteria [63, 64] (Fig. 1.11A). Application of light-emitting diodes (LEDs, see Chapter 6.1) as excitation sources in fluorescence microscopy has resulted in rapid, efficient and low cost methods for mycobacteria screening in clinical specimens [65, 66]. Auramine O also allows easily recognizing kinetoplast DNA in trypanosomes (Fig. 1.11B).
Likewise, acridine orange is also used for selective detection of mycobacteria, to which it imparts red-orange fluorescence [67, 68]. In addition, acridine orange is currently used for recognizing Plasmodium falciparum in human blood smears [69], providing a simpler and more rapid procedure for malaria diagnosis than traditional Giemsa staining. Comparable results can be achieved by means of DAPI staining. Examples of other diagnostic applications are the immunofluorescent detection of the CD-138 antigen in plasma cells from multiple myeloma, and the antigen pp65 from cytomegalovirus (pathogenic in fetuses and immuno-suppressed patients) in neutrophil leucocytes.
Fluorescent methods are also involved in the development of new fields in biomedical research. Examples are emerging areas such as systems biology (genomics, proteomics, lipidomics, glycomics), nanobiology (nanotoxicology, nanomedicine), redox biology, design of biosensors, cellular signaling, diagnostic studies, imaging technology, etc. Applications of fluorescence methodology in whole organisms are especially attractive, by using relatively innocuous probes and labels [38]. This is the case for the uptake and visualization of fluorescent photosensitizing dyes in living organisms such as the nematode Caenorhabditis elegans [70], one of the simplest and best known animal models (Fig. 1.12).
Fig. (1.11)) Fluorescence of auramine O: uses in microbiology and parasitology. A: Visualization of Mycobacterium tuberculosis by auramine O fluorescence. B: Fluorescence of kinetoplast DNA (arrows) from Trypanosoma cruzi epimastigotes stained with auramine O. Fig. (1.12))Caenorhabditis elegans observed under bright-field (A, C) and fluorescence microscopy (B, D). Adult worms (SS104 strain) treated for one hour either with 10 µM rose Bengal (B) or 10 µM acridine orange (D). High fluorescence of the rostral (B) and caudal (D) digestive tract is seen (arrow). Pharynx and eggs are indicated (courtesy of J.I. Bianchi and S.H. Simonetta).Fluorescence methods are very useful in morphological studies on bone development in whole organisms [71], an example being the use of calcein for visualizing skeletal calcification in living zebrafish embryos (Fig. 1.13).
Fig. (1.13)) Skeletal development in zebrafish embryo by calcein labeling. Ventral view of a zebrafish (Danio rerio) embryo at 5 day postfertilization showing normal bone development after treatment of living animals for 3 min with 0.2% calcein, followed by washing and observation by bright-field (A) and fluorescence microscopy (B). Calcified structures in the head emit green fluorescence (courtesy of M.A. Alvarez).Diagnosis of tumors in vivo can be aided by clinical tests using fluorescent antineoplastic drugs used e.g. in photodynamic therapy. Although the dream of a “magic bullet” carrying a photo-active drug directed to the neoplastic tissue has not yet been achieved [72], fluorescent tumor imaging shows that some suitable drugs are selectively accumulated into neoplastic tissue after intravenous injection (Fig. 1.14); this is an interesting prospect for diagnosis and therapy of cancer [73, 74].
In addition to applications in microscopic staining, fluorochromes and dyes are employed (1) in industry as pigments and lakes for dyeing paper, textiles, leather, and plastics, (2) as additives to food, cosmetics and cleaning products, (3) in pharmacy for coloring medicaments, and (4) in medicine as diagnostic and therapeutic agents [30, 34, 39, 75, 76]. Treatment of whole organisms with photoactive dyes can induce either useful therapeutic actions [73] or adverse toxic effects [77, 78].
Fig. (1.14)) Fluorescent localization of tumors by the photosensitizers, zinc(II)-phthalocyanine (ZnPc) and meso-tetra(4-N-methylpyridyl)porphine (TMPyP). C57BL/6 mice bearing a subcutaneous B78H1 amelanotic melanoma were subjected to intratumoral or intravenous injection of ZnPc in DPPC liposomes, and TMPyP in PBS (0.5 mg/kg and 4.1 mg/kg body weight, respectively). Fluorescence was recorded with an Aequoria MDSTM imaging system (Hamamatsu) and converted to pseudocolor images (ImageJ, LUT: fire).CONFLICT OF INTEREST
The author (editor) declares no conflict of interest, financial or otherwise.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Ministerio de Economía y Competitividad (CTQ2013-48767-C3-3-R), Spain.
