Normalized steady-state emission spectra

Figure 2. Normalized steady-state emission spectra for 10 /tM PRODAN in normal liquids. Xejt = 351 nm.

In order to calculate the TRES, one confutes a new set of intensity decays, which are normalized so that the time-integrated intensity at each wavelength is equal to the steady-state intensity at that wavelength. Suppose F(X) is the steady-state emission spectrum. One calculates a set of H X) values using... 

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. 23 Normalized absorption spectra of the free BSA protein (1), BSA-dye 50 conjugate (2) and steady state fluorescence emission spectrum of the BSA-dye conjugate (2 )...

Steady-State Spectral Convolution. The steady state absorption and emission spectra of dilute dye samples can be measured using standard spectroscopic techniques. Once the extinction coefficient, e( ), and the normalized luminescence spectrum, f(v), are known for a particular dye, the self—absorption probability r over a pathlength L in the sample containing the dye at a concentration C is given by... 

Time-resolved emission spectra Although there have been several attempts to simplify the characterisation of the SR process, the determination of time-resolved emission spectra (TRES) is certainly the most general and most precise way to quantitatively describe the solvent response. The time-resolved emission spectra are usually determined by spectral reconstruction [96, 97, 106]. The time-resolved emission spectrum at a given time t is calculated from the wavelength dependent time-resolved decays by relative normalization to the steady-state spectrum [107]. By fitting the TRES at different times t by the empirical log-normal function, the emission maximum frequencies i (t) (or 2(t) see Fig. 6.26) and the total Stokes-shift Ac (or A2) are usually derived [106]. Since c(t) contains both information about the polarity (Ac) and the viscosity of the reported environment, the spectral shift c(t) may be normalized to the total shift Ac. The resulting correlation functions C(t) (Eq. (7)) describe the time course of the solvent response and allow for comparison of the SR-kinetic and, thus, of relative micro-viscosities, reported from environments of different polarities [96, 97, 106, 108, 109, 116, 117, 122]... 

So far presented results are connected with so-called steady-state spectroscopy. Conventionally studied steady state or CW (continuous-wave) luminescence is a process where the excitation sources pump the sample at constant intensity over time necessary to perform the measurement. The end result is emission spectrum, namely the distribution of energy emitted by an excited system in terms of the intensity of emitted optical photons as a function of wavelength or photon energy. Such spectroscopy in many cases is inadequate because the discriminatory power of the normal emission spectra is somewhat limited. Therefore, most of the previously provided emission spectra of minerals present overlapping features of several types of luminescence centers. 

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