Rhodamine emission spectra

Since the same dye molecules can serve as both donors and acceptors and the transfer efficiency depends on the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, this efficiency also depends on the Stokes shift [53]. Involvement of these effects depends strongly on the properties of the dye. Fluoresceins and rhodamines exhibit high homo-FRET efficiency and self-quenching pyrene and perylene derivatives, high homo-FRET but little self-quenching and luminescent metal complexes may not exhibit homo-FRET at all because of their very strong Stokes shifts. 

The laser emission peak from R6G doped ORMOSIL gels occurred at 571 nm with a bandwidth of 4 nm. The laser emisison band is narrower than the FWHM fluorescence band. The doped ORMOSIL sample exhibited a luminescence peak at 565 nm with a bandwidth of 55 nm (FWHM) In contrast to the C153 gel, the solid state rhodamine doped sample did not oscillate over the FWHM range of die fluorescence emission spectrum. The R6G samples exhibited detectable oscillation over a total range of about 38 nm (559 to 587 nm). 

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. 

For the measurement only two materials were selected rhodamine B, which shows a beautiful orange luminescence, and luminol—i.e., 3-aminophthalhydrazide or 2,3-phthalazdione. Luminol was preferred because the yield seemed to be higher and its emission spectrum corresponded better to the spectral sensitivity curve of the photomultiplier. The rhodamine type of compound—i.e., the compound without any substitution—prepared in the authors laboratory, was not found suitable. 

FITC is excited at 490 nm and emits light at 520 nm. Rhodamine has an absorption maximum at 550 nm, and emits at 580 nm. Figure 7.8(b) shows the overlap between the emission spectrum of FITC and the absorption spectrum of rhodamine. 

Figure 1. The emission spectrum of rhodamine 6G excited at 480 nm and the absorption spectrum of rhodamine B in aqueous solutions of PA-I8K2 0.01 g/L). The dashed line is the emission spectrum of rhodamine B excited...

Figure 3.17. Fluorescence emission spectrum of Rhodamine B dissolved in ethanol. The spectrum was taken by R. A-Fiii on 6/20/9S uang an excitation wavelet ofSIO nm See Du, H., Fuh, R.A., Coikan, Li. A, Lindsey. J. S, 1998. Photochem. PhetobioL 68, 141-142. Curtos from Oregon Medical Lastf Center.

Steady-state emission spectra of a donor-acceptor labeled sample and a donor-only labeled sample are taken. The donor emission is removed from the donor-acceptor emission spectrum by subtracting the normalized donor-only emission spectrum. This leaves the fluorescence of the acceptor due to direct excitation and due to energy transfer (see Fig. 4). Clegg and co-workers call this the extracted acceptor emission spectrum, Fen,. Note that this process does not require the concentration of donor-only sample to be the same as the donor-acceptor sample—only the shape of the donor spectrum is used. This spectrum is divided by a fluorescence value (often the maximum) of an emission spectrum taken on the donor-acceptor complex excited at a wavelength where only the acceptor absorbs (565 nm for fluorescein-tetramethylrhodamine). Alternatively, one can divide by the maximum of the excitation spectrum of the donor-acceptor complex (excitation at 400-590 nm, emission in the range 580-600 nm, for fluorescein-rhodamine). In either case, the resultant ratio spectrum, (ratio), is normalized for quantum yield of acceptor, for concentration of total molecules, and for incomplete acceptor labeling. 

Fig. 40. Variation of the fluorescence intensity of rhodamine B and rhodamine B isothiocyanate adsorbed onto microcrystalline cellulose measured as the total area under the corrected emission spectrum,, as a function of (1 — 7 )/dye. Curve (1)—Type I samples of rhodamine B prepared from ethanolic solutions. Curve (2)—Type I samples of the reactive dye prepared from ethanol. Curve (3)—Type I samples of the reactive dye prepared from water. Curve (4)—Type II or dyed samples. Samples from curves 3 and 4 were repeatedly washed after the initial solvent evaporation.

Figure 7. Steady-state and phase-resolved emission spectra for 1 1 binary mixture of rhodamlne 6G and rhodamine B. Laser excitation at 1 57.9 nm. Curve a steady-state emission spectrum for mixture ...

Curve b steady-state emission spectrum for rhodamine B Curve c steady-state emission spectra for rhodamine 6G Curve d phase-resolved emission spectrum nulling rhodamine 6G signal Curve e phase-resolved emission spectrum picked to slightly enhance the rhodamine 6G component over the rhodamine B, but not null it. 

For temperature measurement by single-dye fluorescence, the temperature sensitivity of a dye, specifically its quantum efficiency, effectively defines the temperature resolution of the measurement itself. Rhodamine B is the most common temperature-dependent fluorescent dye used in both macro- and microscale liquid applications because of its relatively strong temperature sensitivity of 2.3 % in water over a temperature range of 0-120 °C. This dye is also soluble in many other organic solvents, like ethanol, making it a practical choice in a variety of microfluidic applications. Moreover, its absorption spectrum is rather broad (470-600 nm with a peak at 554 nm), meaning it can be readily excited with conventional illumination sources like mercury-arc lamps as well as argon-ion (continuous) and Nd YAG (pulsed) lasers. Further, its emission spectrum is also... 

The terbium complexes allow more flexibility in the choice of suitable acceptors and more particularly of multiple acceptors in the scope of multiplexing, since the terbium emission lines are more evenly disposed in the emission spectrum (compared to europium emission) displaying lines at 490, 545, 585, and 620 nm. Thus, either fluorescein, rhodamin, or indocyanines derived acceptors can be used, their respective emission falling in workable windows between successive emission lines or in the NIR window. Furthermore, green fluorescent protein (GFP) or GFP-like acceptors have been recently used to design assay involving a fusion protein substrate [17]. 

Preintercalation of a-ZrP with propylamine, for example, accelerates the binding of rhodamine, (109) and its binding is accompanied by a large blue shift in the emission spectrum of the dye, from 690 nm in the solid state to 675 nm for the intercalated dye. The blue shift in the emission peak is attributed to the aggregation of the dye in the galleries due to the high local concentrations, but a similar scenario is to be expected in the solid state. Clearly, reduced repulsion due to the binding to the matrix and the dielectric properties of the matrix are also to be considered to explain the dye behavior. 

Examples of such continuous absorption and emission line profiles are the optical dye spectra in organic solvents, such as the spectrum of Rhodamine 6G shown in Fig. 3.25b, together with a schematic level diagram [108]. The optically pumped level Ei is collisionally deactivated by radiationless transitions to the lowest vibrational level Em of the excited electronic state. The fluorescence starts therefore from Em instead of Ei and ends on various vibrational levels of the electronic ground state (Fig. 3.25a). The emission spectrum is therefore shifted to larger wavelengths compared with the absorption spectrum (Fig. 3.25b). 

In principle, any couple of fluorophores can be used for FRET, provided that the emission spectrum of the donor overlaps with the absorption of the acceptor. For a review of FRET-couples (and RO values) of chemical dyes see [62]. Furthermore, donors with a high fluorescence quantum-yield and acceptors with a high molar absorbance will display increased FRET. For FLIM it will be important to tune the instrument-performance to ensure maximal sensitivity to small changes in lifetimes at the control donor lifetime. Usually this is easily achieved. Many FRET-pairs have been used for FRET-FLIM including chemical probes as Fluorescein-Rhodamine [54,93],calcein-sulforhodamine B [94], and Cy3-Cy5, [70]. Since 1996, the availability of genetic-encoded fluorophores such as CFP, GFP, YFP has boosted application of FRET-FLIM enormously [95]. Nowadays fluorescent-tagging of proteins no longer depends on laborious protein pu-... 

Fig.3.20a>b. Absorption and emission spectrum of rhodamine 66, solved in ethanol (a). Schematic level diagram to illustrate radiative and radiationless transitions (b)... 

Most modem fluorimeter instruments have a means to monitor the excitation intensity as a function of wavelength and over time. This task is usually performed using a beam splitter to separate a small proportion of the excitation light and record that signal using some form of photon detector such as a photodiode, photomultiplier, or a quantum counter. Originally, such reference detectors used a quantum counter approach in which a concentrated dye solution, often Rhodamine B, would absorb all photons incident upon it and whose emission spectrum and emission intensity are not excitation wavelength dependent. 

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

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