Fluorescein excitation and emission

Fluorescein (excitation and emission maxima of 492 nm and 520 nm, respectively) has also been utilized in fluorescence assays. Although its excitation maximum is higher than that of umbelliferone, it suffers from a problem similar to that of umbelliferone in that albumin-bound bilirubin has excitation and emission maxima of 460 nm and 515 nm, respectively. In addition, commercial preparations have been reported to contain two isomers, which may cause heterogeneity during conjugate preparation. 

Mount the cells on a slide with a coverslip, and examine on the fluorescent microscope with the appropriate filters for fluorescein excitation and emission (see Note 3). 

Jablonski (48-49) developed a theory in 1935 in which he presented the now standard Jablonski diagram" of singlet and triplet state energy levels that is used to explain excitation and emission processes in luminescence. He also related the fluorescence lifetimes of the perpendicular and parallel polarization components of emission to the fluorophore emission lifetime and rate of rotation. In the same year, Szymanowski (50) measured apparent lifetimes for the perpendicular and parallel polarization components of fluorescein in viscous solutions with a phase fluorometer. It was shown later by Spencer and Weber (51) that phase shift methods do not give correct values for polarized lifetimes because the theory does not include the dependence on modulation frequency. 

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... 

Figure 1.6 Solvent accessibility and photobleaching behaviour of nanoparticle synthesis intermediates. (A-C) Excitation and emission spectra of nanoparticle intermediates ((A) TRITC (B) core (C) core-shell) in ethanol (blue) and water (red). (D) Photobleaching behaviour of nanoparticle intermediates (blue, TRITC green, core red, core-shell) and fluorescein (black). All curves in (A)-(D) are normalized by the peak values. (Reproduced from ref. 13, with permission.)...

Fig. 3 (a) Normalized excitation and emission spectra of 5-(and-6)-carboxy-fluorescein, succini-midyl ester, rhodamine 6G (R6G), and 6-carboxy-X-rhodamine dyes in pH 7.4 phosphate buffer, (b) Confocal fluorescence image of a mixture of five types of microsphere-DDSN complexes under 488-nm Argon-ion laser excitation. Reproduced with permission from Ref. [12]... 

Fig, (, Excitation and emission spectra of the fluorescein derivative DTAF. Modified from ref. I. EX, excitation spectrum EM, emission spectrum. 

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. 

FITC is probably the most popular fluorescent probe ever created. An isothiocyanate derivative of fluorescein is synthesized by modification of its lower ring at the 5- or 6-carbon positions. The two resulting isomers are nearly identical in their reactivity and spectral properties, including excitation and emission wavelengths and intensities. Their chemical differences, however, may affect the separation of modified proteins from excess reagent or the analysis of tagged molecules by electrophoresis. For this reason, most manufacturers purify the carbon-5 derivative as the FITC reagent of choice. 

Mixing was characterized by an optical microscope with an epifluorescence attachment [160], A 10 4 M fluorescein solution in sodium phosphate buffer (10 mM, pH 7, with trace of methanol) was applied. A filter cube was used for fitting the excitation and emission characteristics, allowing selective passages of the radiation. A CCD digital camera collected real-time fluorescence images. 

Fluorescein (FL) (3, 6 -dihydroxyspiro[isobenzofuran-l[3H], 9 [9H]-xanthen]-3-one) was used for the ORAC assay. Excitation and emission filters of 490 and 520 nm, respectively, were used. A stock solution of 1.2 mM FL was diluted to a working solution of 94 nM made up in 0.75 mM phosphate buffer (pH 7.4). Two pipettors, which were part of the microplate... 

A liquid prism was created on a PDMS chip for detection based on absorption and refractive index shift. The liquid prism was formed by filling a hollow triangular-shaped chamber with a liquid sample. Excitation and emission were arranged at the minimum deviation configuration. At a low concentration of fluorescein (< 100 pM), excitation light from an optical fiber was absorbed by the molecule, but there was no shift in the excitation maximum. In this absorption-only mode, the LOD of fluorescein was 6 pM. At higher concentration (i.e., 100-1000 pM), there is an additional shift in the excitation maximum This leads to a much sharper decrease in the measured intensity, which is more than can be accounted for simply by the absorption effect [714]. 

Fig. 3. Excitation and emission spectra of the I ll chelate with /V./V./V, .V -[(4-plicnvl-2.2 6. 2 -tcrpvndmc-6,6,-diyl)bis(methylenenitrilo)]tetrakis(acetate) (left) and of fluorescein (right).

Following this approach, Kopelman and coworkers have prepared a pH sensor72 using as magnetic material barium ferrite nanocrystals (BaM) and as dye the dextran-linked carboxy SNARF-1, a commercially available fluorescein derivative with a convenient pKa of 7.5 and dual excitation and emission, which allows for ratiometric pH sensing. The two components were incorporated in silica nanospheres of 300 nm... 

Figure 4. Excitation and emission spectrum of fluorescein. When fluorescein is excited at a wavelength other than its peak excitation (470 nm in this example), the shape of the emission curve (darker green) remains the same, but the relative intensity is reduced. The efficiency of the excitation at 470 nm is 45% of peak excitation.

Fluorescein sodium, 3, 6 -dihydroxyspiro[isobenzofuran-l(3H),9 -[9H]xanthen], C2oHioNa205, CAS number 518-47-8, is a yellow acid dye of the xanthene series. Its molecular weight is 376 Da, and its solubility in water at 15°C is 50% (i.e., it is freely soluble). It is generally formulated as its sodium salt (Figure 16-1). When exposed to light, fluorescein maximally absorbs light at approximately 493 nm and emits (fluoresces) at approximately 520 nm. Figure 16-2 illustrates the excitation and emission spectra of dilute fluorescein in phosphate buffer. 

Figure l6-2 Excitation and emission spectra of a 0.00005% solution of sodium fluorescein in KH2PO4-K2HPO4 buffer at pH 8. (Reprinted with permission from Romanchuk KG. Fluorescein. Physiochemical factors affecting its fluorescence. Surv Ophthalmol 1982 26 269-283.)... 

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