Fluorochrome rhodamine

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 ... 

Antibodies labeled with fluorescent molecules have several applications, particularly in cytochemistry and cell sorting. There are many fluorochromes used in labeling (1), such as coumarin derivatives, phycobiliproteins, and rare earth chelates however, fluorescein and rhodamine (Table 1) are the most commonly used. 

Conjugation of antisera with fluoroscein or rhodamine These are the two common fluorochromes and they are covalently linked to proteins by using activated forms (e.g. the isothiocyanates). 

Preparations are incubated with appropriate reagents to allow visualization based upon the detection system associated with the secondary antibody. The secondary antibody may be conjugated to a enzyme (e.g., alkaline phosphatase, horseradish peroxidase). Incubation with the appropriate substrate to the enzyme will result in the production of an insoluble colored product that can be detected upon microscopic analyses of the cells. Secondary antibodies can also be conjugated to fluorochromes (e.g., fluorescein, rhodamine) that can be detected using a microscope equipped to detect fluorescence. Immunohisto-chemistry has proven to be a powerful tool in biochemical toxicology allowing for in situ assessments of protein responses to toxicant exposure. 

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. 

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. 

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). 

Direct method in which the probe-target hybrid can be visualized after the hybridization. When fluorochrome-labeled probes are used, the visualization can be achieved by fluorescence microscopy. Various fluorochromes with different emission colors are now available such as AMA, FITC, fluorescein, rhodamine CY3 and Texas red. The use of different probes labeled by different fluorochromes... 

Tetramethyl rhodamine Isothiocyanate (TRITC) [95197-95-8] 529 (492) 596 (518) Protein fluorochrome, used for immunofluorescence... 

In order to visualize the chemical product in a reacting flow, a Rhod-2 solution and a calcium chloride (CaCl2) solution were used as the reactive species A and B, respectively. Rhod-2 is a fluorochrome that has a Rhodamine moiety, and it fluoresces by chelating with a Ca ion. Hence, the complex ion produced can be visualized as a chemical product by LIF. The reaction rate constant between both reactants Rhod-2 and CaClj is in the order of 10 m mol s , hence the reaction can be regarded as a rapid reaction. 

In addition to minimal perturbation of the original polymer structure, the polymer-fluorochrome bond must be stable and the labelled polymer should have favorable spectral properties. The highly fluorescent derivatives of fluorescein (la), rhodamine (Ila) and coumarin (Ilia) have been studied in detail for analytical purposes in biological material (Fig. 1). Attachment of these substances to a polymer molecule by a co-... 

Digoxigenin is just one of the nucleotide modifying substances. One can simply use biotin. In this case then, rather than an antibody, avidin linked alkaline phosphatase will be used subsequently. Biotin is normally coupled to the C-5 position of a pyrimidine ring through an allylamine linker arm. Other substances that may be used are dinitrophenyl (dinitrophenyl analogue of 8-aminohexyladenosine 5 -triphosphate), and fluorochromes such as fluorescine, rhodamine, and coumarin linked to dUTP. 

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