Fluorochrome fluorescein

Immunocytochemistry The presence of the labeled probe bound to the in situ nucleic acid target is detected by means that relate specifically to the nature of the label. The most frequently used labels are enzymes (alkaline phosphatase, hydrogen peroxidase) and fluorochromes (fluorescein, rhodamine, hydroxycoumarin). 

The choice of a fluorochrome to conjugate to the second antibody preparation depends upon the particular application to be undertaken. Chapter 15 (3) reviews the relevant properties of commonly employed fluorochromes. Fluorescein, rhodamine, Texas Red (4), and phycoerythrin (5, 6) have been common choices, but the more recently introduced fluorochrome families, such as the Alexa series, offer advantages in photostability and other optical properties (7). 

Fluorochromes are visualized by excitation with light of one (excitation) wavelength and imaging emitted fluorescence at another (emission) wavelength using appropriate filters. The properties and methods of visualization of the fluorochromes fluorescein and rho-damine, which are used to detect digoxigenin, are the same as for direct fluorochrome-labeled probes and are outlined in Chapter 26. 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

Fluorochromes have been introduced that offer excitation and emission spectra similar to those of fluorescein, but that overcome some of fluorescein s limitations. BODIPY FL has a short Stokes shift, but offers higher fluorescence intensity, and is claimed to be more photostabile and less pH-sensitive than fluorescein. Oregon Green 488 and the newly introduced Alexa 488 fluorochromes have spectra nearly identical to those of fluorescein, but are considerably more photostabile, and produce less quenching of fluorescence with higher numbers of fluorochromes per antibody than does fluorescein. 

Recently, cyanine fluorochromes covering a wide spectral range have become available for immunofluorescence (13,14). The red-emitting fluoro-chrome Cyanine 3.18, which was shown to give a significantly brighter image than TRITC, lissamine rhodamine, Texas Red, or fluorescein under specific conditions of microscopy (7), provides a useful alternative to the rhodamines. Other useful substitutes for the rhodamines include the BODIPY TR and TMR, and Alexa 568 and 594 fluorochromes. The latter are newly introduced and appear to offer superior photostability. 

Rhodamine is a preferred fluorochrome over fluorescein because of its slower bleach rate and its emission in a spectrum that shows less cellular autofluorescence. Also, this spectrum produces less autofluorescence in plastic substrata (see Chapter 14). Rhodamine requires a mercury vapor light source, since other sources, such as xenon, do not have sufficient emission in the green spectrum. 

The second strategy uses combinations of different antibodies coupled to fluorochromes with distinct emission maxima (5,9). The most relevant fluoro-chromes for combined antigen detection are fluorescein isothiocyanate (FITC abs. max. 494 nm, emiss. max. 517 nm), rhodamine isothiocyanate (TRITC ... 

A limitation of the flow cytometric binding assay has been the precise determination of the receptor affinity and calculation of the receptors per cell. This limitation appears to have been overcome by the development of fluorescein and phycoerythrin compensation-calibration standards (Flow Cytometry Standards Corp., Research Triangle Park, NC). These standards have made it possible to quantify the fluorescence intensity of samples labeled with fluorescein or phycoerythrin, and relate the intensity to molecules of equivalent soluble fluorochrome. These standards have been utilized in quantitative studies of neutrophil chemoattractant-ligand interaction (4). 

The quantum efficiency of a given fluorochrome ultimately determines the sensitivity attainable. Thus protein fluorochromes derived from marine algae, such as phycoerythrin, have very high quantum efficiency in comparison to small chemical fluorochromes, such as fluorescein. For analysis of low antigen densities, phycoerythrin is to be preferred. 

Small chemical fluorochromes, such as fluorescein, have an advantage in this case because of the stability and predictability of their conjugates. Although methods for calibration of phycoerythrin-labeled antibodies are now available, a wider range of options is available for fluorescein (see Section 3.3.5.). 

The level of fluorescein modification in a macromolecule can be determined by measuring its absorbance at or near its characteristic excitation maximum (—498 nm). The number of fluorochrome molecules per molecule of protein is known as the F/P ratio. This value should be measured for all derivatives prepared with fluorescent tags. The ratio is important in predicting the behavior of antibodies labeled with FITC (Hebert et al., 1967 Beutner, 1971). Using the known extinction coefficient of FITC in solution at pH 13 (e498nm = 8.1-8.5 x 104 Jobbagy andjobbagy, 1972 McKinney et al., 1964), a determination of derivatization level can be made after excess FITC is removed. At pH 7.8, the absorbance of protein-coupled FITC decreases by 8%. 

Because multiple photodetectors are available, a flow cytometer has the ability to measure two or more fluorescence signals simultaneously from the same cell. To use several fluorochromes at the same time, cytometrists with only one laser required a group of stains, all of which absorb 488 nm light but which have different Stokes shifts so that they emit fluorescent light at different wavelengths and thereby can be distinguished from each other by the color of their fluorescence. Propidium iodide and fluorescein are a pair of fluorochromes that fulfill these criteria (having different Stokes shifts) and can be... 

To be of use in microscopy or flow cytometry, this bond needs to be visualized (to the eye or to the photodetector) by the addition of a fluorescent tag. Visualization can be accomplished by one of two different methods. With direct staining, cells are incubated with a monoclonal antibody that has been previously conjugated to a fluorochrome (for example, fluorescein or phycoerythrin or any fluorochrome with appropriate absorption and emission spectra). This procedure is quick and direct it merely involves a half-hour incubation of cells with antibody (at 4°C), followed by several washes to remove weakly or nonspecifically bound antibodies. Cells thus treated are ready for flow analysis (although final fixation with 1% electron microscopic-grade formaldehyde will provide a measure of biological safety and long-term stability). 

One further step toward calibration has been taken with the use of a calibration curve made with sets of beads with known numbers of fluorochromes on their surface. Such calibrated beads are available with known numbers of PE molecules. Similar, but less direct, beads are available with fluorochrome molecules that have been calibrated in units equivalent to the intensity of fluorochrome molecules in solution ( MESF units = molecular equivalents of soluble fluorochrome ). With these beads, a curve can be obtained (Fig. 6.6), giving each channel on the ADC a calibration in number of fluorochrome molecules (for PE) or MESF values (for fluorescein). In this way, the background fluorescence of a control sample can be expressed as an equivalent number of fluorochrome (or MESF) molecules and can be subtracted from the number of fluorochrome molecules of a stained sample. The fluorescence of the stained sample can then be expressed as, for example, PE molecules over and above the background level. 

Fig. 6.6. The fluorescence histogram of a mixture of fluorochrome-conjugated calibration beads and the calibration line for channel numbers and their equivalence in soluble fluorescein molecules derived from that histogram. From Givan (2001).

Fluorochrome A fluorochrome is a dye that absorbs light and then emits light of a different color (always of a longer wavelength). Fluorescein, propidium iodide, and phycoerythrin, for example, are three fluorochromes in common use in flow cytometry. Flu-orophore is an equivalent term. 

Phycoerythrin Phycoerythrin is a fluorochrome derived from red sea algae. It is particularly useful in flow cytometric applications requiring dual-color analysis because, like fluorescein, it absorbs 488 nm light from an argon laser. However, it has a longer Stokes shift than fluorescein, and therefore the fluorescences of the two fluorochromes can be distinguished. 

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