There are many biological structures and processes which cannot be imaged or probed by using intrinsic fluorescence due to endogenous fluorophores.
For example, DNA does not exhibit any fluorescence. In these cases, labeling of these biological structures with exogenous fluorophores is needed for bioimaging. Some fluorophores can stain (disperse) throughout the cell, while others localize in a particular organelle. In some cases, a fluorophore can be chemically conjugated to derive the feature of targeting a specific biological site, a particular type of cancer cell, or an individual organelle in a cell. Thus, the specificity and sensitivity of a fluorescence probe can be used to derive valuable structural, biochemical, and biophysical information on cells and tissues (Harper, 2001).
In this section, selection criteria for exogenous fluorophores for bioimaging are discussed and examples of fluorophores used for targeting various organelles are presented. A more comprehensive discussion of currently available fluorophores for bioimaging can be found in the book Principles of Fluorescence Spectroscopy (Lakowicz, 1999), in the Handbook of Fluorescent Probes and Research Products (Haugland, 2002) and from the website of a company, Molecular Probes, which commercializes fluorophores (see Section 8.9). The fluorophores (fluorochromes), useful for flow cytometry, are also discussed in Chapter 11.
For bioimaging, the basic requirements for an ideal fluorophore are as follows:
• Dispersability (solubility) of the fluorophore in the biological medium to be probed
• Specific association with a target molecule, organelle, or cell
• High quantum efficiency of emission
• Environmental stability
• Absence of photobleaching
Photobleaching is a major problem with many organic fluorophores. Pho-tobleaching generally refers to chemical degradation of a fluorophore, leading to the disappearance of fluorescence. This photodegradation may result from photochemistry in the excited state, photooxidation in the presence of oxygen, or thermal decomposition due to local heating by nonradiative processes following light absorption.
A practical consideration in the selection of a fluorophore is that it should be efficiently excited at the wavelengths of the common laser sources available for microscopy. Furthermore, the emission wavelength of the fluorophore should be compatible with the emission filters on the microscope.
Some situations may require multiple labeling with more than one fluo-rophore to simultaneously probe different target molecules/organelles. In such cases, an additional consideration is that these fluorescence spectra (peaks) be sufficiently separated so that appropriate cutoff (or narrow bandpass) filters
FLUOROPHORES AS BIOIMAGING PROBES
can be used to discriminate their emission from each other. In some cases, such as in FRET imaging (see Section 8.5.5), it may be desirable that their excitation peaks are also well-separated, so that excitation wavelengths can be judiciously selected to excite only one fluorophore.
A wide array of fluorescence probes for most imaging applications as well as light sources most suitable to excite them are commercially available. Some of the commonly used fluorophores for bioimaging and their applications are shown in Table 8.1. The wavelengths of their one-photon excitation and resulting emission maxima are listed, along with convenient light sources used to excite them.
Using fluorescence labeling, bioimaging of a specific organelle or a site in a cell can be accomplished to study its structure and function. Fluorescent probes used for labeling specific sites and organelles in a cell can be divided into two categories:
1. Fluorophores targeting biological molecules, sites, or organelles without any prior coupling to a biomolecule. For example, some fluorophores in their commercially available form show selective staining of specific organelles. These features are also listed in Table 8.1, wherever applicable. Hence they can be conveniently used to probe the structure of an organelle and various biophysical and biochemical processes occurring in them.
2. Fluorophores that need to be conjugated to a biomolecule in order to acquire specificity for certain biological sites.
In the first case, the labeling characteristic is often derived from electrostatic and hydrophilic/hydrophobic interactions of a probe with the biomole-cule or organelle of interest.
In the second case, fluorescent probes, which require conjugation with another biomolecule for selective staining, are often used for histological applications. They are chemically conjugated with oligonucleotides or proteins to allow targeting and imaging specific sites in cells. Examples are Alexa Fluor dyes, Cy dyes and Texas red, which are also listed in Table 8.1. In this case, high fluorescence quantum yield, low pH sensitivity, and high photostability are among the necessary demands on the fluorophore. Also, fluorescence of the probe should not be significantly reduced or quenched on conjugation to biological molecules. This approach has been used to obtain a multicolor image of bovine pulmonary artery epithelial cells that were stained with three different Alexa Fluor conjugates (see http://www.iwai-chem.co.jp/products/ m-probes/alexa.pdf, a Molecular Probes website).
It is also worth noting that the same probe sometimes can be used for a number of purposes. For example, ethidium bromide, used as an intercalating dye for DNA studies, is also useful for monitoring cell viability, since dye permeation to the cells increases with a decrease in the cell viability.
TABLE 8.1. Some Commonly Used Fluorophores for Bioimaging
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