Fluorescein, properties

The physical properties of the xanthene type dye stmcture in general have been considered. For example, the aggregation phenomena of xanthene dyes has been reviewed (3), as has then photochemistry (4), electron transfer (5), triplet absorption spectra (6), and photodegradation (7). For the fluoresceins in particular, spectral properties and photochemistry have been reviewed (8), and the photochemistry of rhodamines has been investigated (9). 

Shimomura, O., Musicki, B., Kishi, Y., and Inouye, S. (1993a). Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium. Cell Calcium 14 373-378. 

Fluorescein absorbance is sensitive to pH. This property is utilized to measure cytosolic pH changes. Fluorescein derivatives that contain esters on the carboxyl groups have been constructed. These compounds partition through the cell membrane and, once inside the cell, the esters are cleaved by nonspecific esterases in the cytosol, leaving free carboxyl groups thus the probe cannot diffuse out of the cell (or at least does so slowly). Commonly used derivatives are 6-carboxyfluorescein (21-23) and the more recently developed probe 2, 7 -bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (13, 24,25). 

This probe utilizes dual excitation wavelengths and is observed at a single emission wavelength. The intracellular calcium probes Fluo3 and Rhod2, which have recently been introduced, have fluorescein-like and rhodamine-like spectral properties, respectively (Minta, A. Harootunian, A.T. Kao, J.P.Y Tsien, R.J. J. Cell Biol 1987, 105,... 

Different substituents on the carboxy-functionalized fluorescein can be introduced to produce marked alterations in the absorbance and fluorescence emission wavelengths, as well as in other physical properties. The selective substitution of chlorine for aromatic hydrogen has been found to increase fluorescence efficiency and to narrow emission and absorbance maxima when compared with fluorescein 48, which is useful in multicolor imaging. 

Song A-M, Zhang J-H, Zhang M-H et al (2000) Spectral properties and structure of fluorescein and its alkyl derivatives in micelles. Colloids Surf A 167 253-262... 

One of the interesting features in the structure-photophysical property relationship of fluorescein is that the quantum yield of fluorescein increases under the basic condition. Therefore, many of fluorescein derivatives have been used as pH sensors to measure intracellular pH due to their pH-responding photophysical property [53]. Although fluorescein itself is slightly fluorescent in alcoholic solutions, the addition of alkali (pH > 8) to the fluorescein solution produces the very intense fluorescent alkali salt. The salt form of fluorescein... 

Fig. 8 Structure-photophysical properties relationship of fluorescein derivatives. Measured in 0.1 N NaOH(aq). bOxidation potential of corresponding benzene moiety, obtained in acetonitrile containing 0.1 M TBAP. °HOMO energy level of the corresponding benzene moiety, calculated with B3LYP/6-31G(d)//B3LYP/6-31G(d) by Gaussian 98 W...

Fig. 9 Structure-photophysical properties relationship of benzannulated fluorescein derivatives...

Miura T, Urano Y, Tanaka K, Nagano T, Ohkubo K, Fukuzumi S (2003) Rational design principle for modulating fluorescence properties of fluorescein-based probes by photoinduced electron transfer. J Am Chem Soc 125 8666-8671... 

Fluorescein is excited at 494 nm, which fits to the argon-ion laser line at 488 nm, a very convenient feature for many microscopy experiments. It emits at 520 nm and the emission band is far from being sharp. The broad fluorescence emission spectrum varies with pH [18]. The advantageous photochemical properties of fluorescein are its high absorption (emax = 79,000M-1cm-1) and quantum... 

Dyes which are half fluorescein, half rhodamine are called rhodols. Their spectral properties are intermediate with respect to excitation and emission wavelength. Generally, rhodol fluorophores are more... 

A fluorophore can undergo a change in their spectral properties as a result of pH variations or enzymatic activity. For example, fluorescein is such as fluorophore due to its two possible isoforms, lactone, and quinoid form. While the lactone form only absorbs in the UV and is not fluorescent, the quinoid form is excited at 490 nm and fluoresces. Only in this last form, there is... 

The fluorescent properties of NHS-fluorescein are similar to FITC. The wavelength of maximal absorbance or excitation for the reagent is 491nm and its emission maximum is 518nm, exhibiting a visual color of green (Sheehan and Hrapchak, 1980). Its molar extinction coefficient at 491 nm in a pH 8.0 buffer environment is Other components in... 

Fluorescein-5-thiosemicarbazide is soluble in DMF or in buffered aqueous solutions at pH values above 7.0. The reagent may be dissolved in DMF as a concentrated stock solution before adding a small aliquot to an aqueous reaction medium. The compound itself and all solutions made with it should be protected from light to avoid decomposition of its fluorescent properties. 

Aminomethylcoumarin derivatives possess intense fluorescent properties within the blue region of the visible spectrum. Their emission range is sufficiently removed from other common fluorophores that they are excellent choices for double-labeling techniques. In fact, coumarin fluorescent probes are very good donors for excited-state energy transfer to fluoresceins. 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

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