Fluorescence Microscopy in Life Sciences

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.

The fundamental physical and chemical processes responsible for luminescence will be introduced and explained in this chapter. The classical division of luminescence into Fluorescence and Phosphorescence will be presented along the similarities and differences between these two processes. Starting with a simple introduction to light and electromagnetic waves and moving to atomic and molecular systems, the text will allow the reader to intuitively understand why physical systems absorb and emit light. This chapter represents the theoretical foundations on which the rest of the book is built upon, the platform from which all other chapters and processes explained therein can be adequately understood and apprehended.

This Chapter deals with the “molecular anatomy of dyes” as referred by Gurr [1], as well as with some chemical characteristics and properties. Obviously, structural aspects of dyes and fluorochromes are often shared and then they should be viewed as valid for both. In addition to shape and size, the molecular anatomy includes a collection of geometrical values such as planarity, bond lengths, dihedral and torsional angles, as well as the occurrence of substituents such as anionic, cationic, hydrophobic, hydrogen bonding, reactive, and metal complexing groups. Some of these physico-chemical parameters can be numerically formulated [2-4] (see section 3.2 and Chapter 15.2.1). The interdisciplinary character of color chemistry (i.e. staining and fluorescence methodology, modern microscopical techniques, industrial and medicinal applications, photosensitizing processes, etc.) is a fascinating research field of increasing importance.

This Chapter deals with the labeling of biomolecules with synthetic or natural fluorochromes. In spite of the development of enzyme and gold protocols for biomolecular labeling, the use of fluorescent tags keeps on being important procedures applied in biomedical research. Covalently attached fluorophores are the most general linking type and they can be either synthetic (FITC, TRITC) or natural (GFP and analogues, phycobiliproteins). Generally proteins are not fluorescent, but they can be labeled with suitable fluorophores. However, there are natural fluorescent proteins that are increasingly applied in biomedical studies. In addition to lipid labeling with synthetic dyes, there are also natural and synthetic fluorescent lipids that are more physiological for studies in vivo (i.e. pentaene derivatives). General aspects on the type and use of fluorescent labels can be found in [1-8]. Relevant issues for fluorescent labeling are the type of fluorophore, its reactive group and binding mode, and the biomolecule to be labeled. Increasing interest is now devoted to the development of new methods for fluorescent labeling [9], mainly using nanoparticles [10, 11].

Studies on the spectral (electronic) properties of dyes are often needed to know qualitative and quantitative aspects of their interactions with macromolecular substrates. This Chapter deals with the description and interpretation of absorption and emission spectra from some dyes and fluorochromes used in microscopical studies. General references on fluorescence spectroscopy can be found in [1-13]. For detailed information on microscopic photometry (i.e. ray paths, equipments, adjustments, and procedures), the book by Piller [14] should be consulted. At present, confocal spectral analysis generating 2D and 3D microscopical images of biological materials offers new possibilities for research applications, mainly in the localization of antitumor drugs within living cells [15]. Some key concepts and useful tips must be taken into account for a correct interpretation of spectra. Generally, emission spectra are corrected for the Raman scatter of the solvent and for harmonics. To record adequately far red emission, the detector sensitivity must be able to capture long wavelengths with fidelity.

The use of fluorescence microscopy requires knowledge of the elements making up the fluorescence microscope. In this chapter the basic elements combining to make a fluorescence microscope will be introduced and its role explained. Afterwards an introduction to the most common fluorescence microscopic techniques will be made followed by more advanced and developed approaches. These will include total-internal reflection and time-resolved microscopies, to name a few. The chapter will end with a brief introduction to flow cytometry, a highly related fluorescence technique in the biomedical field.

Fluorescence analysis has become a powerful tool in Biology and Medicine. Fluorescence microscopy and image technology are now widely used in many research fields. In this Chapter, most important issues are the description of cell and tissue structures as model systems, reactivity of biological substrates, binding modes of fluorochromes, fluorescence parameters, counterstaining, emission color, fading, autofluorescence, photo-enhancement, evaluation of fluorescent images, fluorescence of classical stains, etc. Some of these issues are also referred in other Chapters. In the case of reporting experimental observations and when otherwise is not stated, dyes and fluorochromes are applied simply dissolved in distilled water. Generally excitation and/or emission values are indicated directly on the figures.

Spectral changes of dyes related to pH and metal coordination have now increasing importance as analytical tools. Changes in the light absorption or emission that are characteristics of some dyes and reagents at different pH values or in the presence of metal ions, as well as their microscopical uses are described in this Chapter. Regarding histochemical and histophysiological detection, important metal cations are Mg, Ca, Fe, Zn, etc., with methodological procedures being spot tests, spectral studies and microscopic observations.

In this chapter, the fundamentals in regard to the use of transition metal complexes (TMCs) as luminescent probes will be presented. These metal complexes (MCs) between organic ligands and transition metal cations present many interesting properties that make them very suitable for a number of biomedical applications. First, a brief introduction to TMCs and their photophysics and photochemistry will be provided. Then, some particular applications of selected transition metals (TMs) in the field of fluorescence microscopy will be presented on a case-per-case basis. The relevant role played by lanthanide elements and their complexes in fluorescence microscopy, especially time-resolved, will be introduced. Some uses of actinide MCs will be presented as well.

In this chapter, the uses of most known fluorescent compounds for the microscopic visualization of lipids are described. Particular emphasis is given to the nature of substrates, fixing requirements, dye solubility, and examples of lipophilic stains. Probes for visualizing membranous or lipidic structures in live cells will be also described in Chapter 15.5. Hydrophobic azo and anthraquinone dyes are applied industrially to color fats, paints, plastic resins, rubbers, varnishes and waxes. Some of these dyes are employed to detect lipid droplets and fat-rich components of tissues. Several fat-soluble dyes, particularly bisazo Sudan dyes and Nile red have been routinely used in visualizing lipids [1-3]. Relevant natural and synthetic polyenes such as fluorescent antifungal antibiotics and unsaturated fatty acids will be also described.

Although the term carbohydrate is a general concept and applies to all the sugars and their derivatives, the name polysaccharides will be preferred as they are the common macromolecular substrates to be recognized in microscopy. Carbohydrates may be classified on account of their chemistry, histochemical responses in diagnostic pathology, or distribution and function in nature [1]. Some issues are central to this chapter: the use of bisazo dyes and fluorescent brighteners, chlorotriazines and PAS reaction, ionic binding and metachromatic reactions. Staining and fluorescence methods applied for histochemistry of polysaccharides have been reviewed [1-4], and some of them, in special glycoproteins (i.e. Congo red for amyloid), will be described in Chapter 13.11.3. More specific procedures involving the use of lectins can be found in Chapter 4.5.2.

On account of the effects on cell processes, binding of small ligands with nucleic acids (NAs) has important implications in biomedical sciences [1-4]. Several fluorescent ligands allow visualizing the microscopical site of interaction. Specific DNA sequences may also be visualized by intercalating and minor groove-binding drugs. Detailed descriptions on NAs structure and function can be found in the literature [5-10]. Microscopical assays for DNA damage are described in Chapter 16.2. Detection of NAs by FISH is considered in Chapter 17 (see also Chapter 4.4). Consequences of binding modes of small ligands with NAs have been reviewed [11- 14]. Structure-based design strategies have yielded new DNA-binding agents with clinical promise (i.e. hairpin polyamides and bis-intercalators [1, 3, 15]. Recognition of DNA sequences by fluorescent oligopeptides is a new approach. Examples are dansyloligopeptides from the lac repressor (containing sequences 19-32 and 53-71) that bind specifically to the lac operator DNA [16]. Computer-assisted molecular docking is increasingly applied for the design of molecules to target therapeutically relevant NAs structures. New analytical methods (i.e. electrospray ionization mass spectrometry [17]) allow assessment of the binding affinity of ligands to NAs.

At present, several histochemical methods for proteins using fluorescent dyes and reagents are available. Some of them utilize simple fluorochromes, whereas others involve preferential fluorogenic reactions with amino acids. Ionic, hydrophobic, and covalent interactions occur between protein groups and fluorochromes. In addition to these classical demonstrations of proteins, more selective methods such as immunofluorescence and detection of enzymatic activity are now widely used in microscopical studies. Also other macromolecules such as nucleic acids, polysaccharides, etc., can be visualized by these methods. Detection of macromolecules is also possible with haptens (i.e. dinitrophenol, biotin, digoxigenin) that are recognizable by suitable ligands (antibodies, streptavidin), or using labeled lectins, toxins, oligonucleotides, etc. (see Chapter 4). Fluorescent reactions for enzymatic activity and immunofluorescence are described in Chapters 14 and 17.

In recent years fluorescence techniques have become increasingly popular for the estimation and localization of enzymatic activities. Basic methodological concepts on enzyme histochemistry can be found in standard books and reviews [1 - 6]. Enzyme activity studies may be carried out to assess the localization of enzymes within cells and tissues, and to determine the effects of pH, ionic strength, temperature, presence of inhibitors, etc., on the rate of an enzyme-catalyzed reaction. In addition, enzymes can be applied as histochemical reagents to remove macromolecular substrates, examples being RNases, DNases, amylases, etc., or to identify substrates after their fluorescent labeling. Chromo- and fluorogenic reactions are usually employed to reveal enzymatic activity. A relevant issue to avoid diffusion artifacts or false localization is that the colored or fluorescent product of a given enzymatic reaction must have very scarce or null water solubility. However, insoluble or crystalline precipitates should be not confused with specific cell structures. Some examples will illustrate these points.

The visualization of cell structures by fluorescent vital probes to study cell organelles, cell viability and signaling processes has great importance in biomedical sciences. Main interest is devoted to the uptake, accumulation and prediction of localization of drugs and xenobiotics in cultured cells. Mechanistic aspects and predictive rules are studied using numerical parameters, which conform the quantitative structure-activity relationships (QSAR) for vital probes. The uptake of probes into cells is the first step for labeling. Several mechanisms of cell uptake and accumulation in cell organelles are known, the most relevant being passive diffusion and active endocytosis (i.e. adsorptive and fluid-phase, binding to receptors). In addition to theoretical predictions, direct cytotoxic effects, photodynamic action, and fading must be taken into account in the microscopical evaluation of uptake and localization of fluorescent probes.

In this chapter several fluorescent probes for the assessment of cell viability, types of cell death, identification of signals, cellular traffic and connections will be described. These biomedical fields have increasing interest [1-3]. The fluorescence histochemistry of enzymatic activity is currently applied in assays for cell viability, apoptotic processes, and detection of reactive oxygen species (ROS). Several probes of viability and signaling are also markers of oxidative stress and redox visualization. Numerous probes are now commercially available for studies on cell metabolism and toxicity, signal transduction, drug screening, molecular diagnosis, etc. Fluorescent labels and biosensors are applied to perform quali- and quantitative analysis of normal and pathological cell processes (i.e. sensing dyes for trans-membrane potential, ions, ROS, pH, etc). There exist also approaches to the direct real-time signal imaging of live-cells in vitro and in vivo, and for 2D and 3D analysis.

This chapter presents and summarizes two very important techniques in biomedical research, immunofluorescence and fluorescence in situ hybridization (FISH), which relay completely in the use of fluorescent methods to achieve their results. Although the particular molecules involved are different both methods make use of the same approach: the detection of a specific molecular sequence or moiety by a specific molecule (antibody or nucleic acid sequence) that has a fluorophore attached to it. This fluorophore permits the detection of the specific target. An improvement over fluorophore-tagged antibodies has been the introduction of fluorescent proteins, generically known as GFPs (green fluorescent proteins) or FPs (fluorescent proteins). Here we will elaborate a little bit further on their practical applications, as one can think of FPs as a “subtype” of immunofluorescence. Strictly speaking immunofluorescence and FISH provide no new concept to the principles of fluorescence. They rely on using a more or less classical fluorophore and the real breakthrough was the use of the exquisite specificity of antibodies and nucleobase complementarity to recognize certain substrates. However, immunofluorescence represents probably the most widespread employed technique in fluorescence microscopy. And FISH is only a step behind if at all in regards as its use as a diagnostic and research tool in genetics and medicine. Therefore we will introduce the basics of both techniques and some of their most promising recent applications in this chapter.

In this chapter, we will introduce the fundamentals and the main types of solid-state fluorophores in common use for fluorescence microscopy. The photophysics underlying the absorption and emission processes are universal and shared with other fluorophores already presented (e.g. organic molecules, see Chapters 2 and 3). However, fluorescent/luminescent solid state matrices present their own peculiarities, and it is convenient to introduce them in a generic way. As the reader will notice progressesing through the chapter, there is no common chemical basis for the presented solid-state fluorophores. The common theme is precisely solid state structures composed of a relatively large number of atoms (106-1012 atoms/structure) as compared to the low number of atoms (101-103 atoms/structure) of the fluorophores already presented in other chapters. The solid state is broadly divided into three categories according to the (increasing) electrical conductivity: insulators (dielectrics), semiconductors and conductors (metals). There are luminescent representatives within each of these categories. Therefore, we will begin the chapter by explaining the physical basis of electrical conductivity, which is directly related to the optical properties of the material, and then move forward to introduce the different subtypes of luminescent solid-state markers. To understand the electrical/optical properties of solids we will first present the band structure model.

This chapter deals with some of the most fascinating topics in fluorescence microscopy and optics, in general: non-linear optics. All the optical phenomena explained in the previous chapters, with some particular exceptions, have dealt with linear optics; this means that a single photon is involved in the optical process during and after being absorbed. However, it is possible to have a multiphoton optical response, one that depends on more than one photon interacting with the system “at the same time”. This leads to a whole new range of optical phenomena; many of them unobservable at the relatively low light intensities with characteristic of linear optics. Some of the non-linear optical phenomena provide very interesting and useful applications in fluorescence microscopy. In this chapter, we will introduce and explain the main non-linear optical processes employed in fluorescence microscopy, enumerate their advantages and drawbacks, and highlight their importance in the life sciences.

In this final chapter, the fundamentals and main super-resolution microscopy (SRM) techniques currently in use will be presented. Super-resolution allows imaging of structures that are not usually resolvable with optical frequencies in the visible range. There are different strategies to be able to do so and these will be introduced in this chapter. This field of SRM is relatively young yet it providies huge amount of information on biological process and structures. This is done with experimental setups that are much more benign to biological samples in comparison to other “superresolution” techniques like electron microscopy. In the chapter, the fundamentals and techniques will be presented in a simple and intuitive way. This is a field currently experiencing a huge development and new advancements are guaranteed in the upcoming years.