Fluorochrome phycoerythrin

Prior to 1982, the measurement of more than one andgen amultaneously by flow cytometry required two lasers—an argon-ion laser to excite fluorescein (at 488 nm) and a krypton or a dye laser to excite rhodamine or one of its deriva-dves. The discovery of a naturally occurring fluorochrome, phycoerythrin (PE), changed this (i). PE is a phycobiloprotein found in red adgae. It can be excited efficiently at 488 nm (simultaneously with fluorescein) and has a peak fluorescence at 578 nm, sufficiently removed from the peak of 520 nm from fluorescein. There is some overlap in the emission spectra from the two dyes (in pardcular, there is sdll some emission from fluorescein above 580 nm) and this must be corrected, either electronically or by the computer software. 

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.). 

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). 

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. 

Four fluorochromes are commonly used fluorescein, rhodamine, Texas red, and phycoerythrin see Chapter 14). They differ in optical properties, such as the intensity and spectral range of their absorption and fluorescence. Choice of fluorochrome depends on the particular application. For maximal sensitivity in the binding assays, fluorescein is the fluorochrome of choice because of its high quantum yield. If the ligand is to be used in conjunction with fluorescence microscopy, rhodamine coupling is advised, since it has superior sensitivity in most microscopes and less photobleaching than fluorescein. Texas red (5) is a... 

Fluorescence resonance energy transfer (FRET) is a technique that has been used to measure distances between pairs of proximal fluorochromes. A suitable pair consists of a donor fluorochrome, which has an emission spectrum that significantly overlaps with the absorption spectrum of an acceptor fluorochrome (2). With the availability of monoclonal antibodies to many cell-surface determinants, intramolecular distances between nearby epitopes and intermolecular distances between adjacent cell-surface macromolecules can be investigated to analyze molecular interactions influencing important cellular events. Such monoclonal antibodies can be conjugated to fluorescein-isothiocyanate (FITC) as the donor, and either tetramethyl-rhodamine-isothiocyanate (TRITC) or phycoerythrin (PE) as the acceptor. 

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). 

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