Quantum yield fluorescence measurements, technique

Quimtua Yields of Fluorescence Measurements. All of the quantum yields of fluorescence were measured by the relative fluorescence measurement technique of Parker and Rees (24). This method compares the fluorescence of the compound of interest to the fluorescence of some known compound. All of the fluorescence quantum yields were measured using I as a reference. Compound I had previously been measured by this same method using rhodamine B as a standard. 

By using the techniques mentioned before, room-temperature x s for about 50 alkanes were determined. In Fig. 2, we show the fluorescence quantum yields as a function of lifetimes. The 4>[ values were generally taken from the work of Rothman et al. [25] most of the fluorescence quantum yields were measured using 165-nm photons for excitation. This wavelength is close to the absorption onset of most alkanes and (with the exception of the smaller molecules) the measured quantum yield is close to the fluorescence quantum yield of the relaxed Si molecules [26]. The plot in Fig. 2 is similar to the plot we published in Ref. 59 using only our measurements. Here we use practically all the data that are available in the literature. For most of the alkanes, several lifetime measurements were published. When... 

Calibration. Many approaches have been used to calibrate flow cytometric measurements. Including the comparison of flow and nonflow techniques (radiolabels, spectrofluorometry). In recent years, commercial standards have been introduced which are calibrated in fluorescein equivalents/particle (e.g., 3,000 or 500,000). With labeled ligands, calibration requires determining the relative quantum yield of the ligand compared to pure fluorescein and using the standards to analyze the amount bound on cells. Our ligands (fluorescein isothiocyanate derivatives) are typically 50% as fluorescent as fluorescein. 

Before being able to study the nonlinear optical properties of any material, it is necessary to have a complete understanding of its linear optical properties. Therefore, we start this section with a brief discussion of the techniques used to measure some of the most important linear properties, e.g., linear absorption, fluorescence, anisotropy, and fluorescence quantum yield. 

The 2PF technique measures the upconverted fluorescence induced by 2PA. This technique allows for the measurement of 2PA cross sections that are less than tens of GMs when the fluorescence quantum yield is large. The technique was first proposed by Kaiser et al. [76] in 1961 and represents an indirect way to measure the 2PA cross section, which can be calculated by comparing the integrated fluorescence signal... 

The experiment is performed with a spectrofluorometer similar to the ones used for linear fluorescence and quantum yield measurements (Sect. 2.1). The excitation, instead of a regular lamp, is done using femtosecond pulses, and the detector (usually a photomultiplier tube or an avalanche photodiode) must either have a very low dark current (usually true for UV-VIS detectors but not for the NIR), or to be gated at the laser repetition rate. Figure 11 shows a simplified schematic for the 2PF technique. 

Intercalation of BPDE. Several groups have studied the reversible intercalative binding of BPDE to DNA. The fluorescence quantum yield of BPDE is much lower than that of BP derivatives which do not contain an epoxide group and fluorescence techniques have not been widely used to study BPDE physical binding to DNA (4). Association constants for the DNA intercalation of BPDE have been obtained by measuring red shifts in the UV absorption spectra of BPDE which occur upon the formation of intercalated complexes and from fluorescence studies (8) of the kinetics of DNA catalyzed hydrolysis of BPDE. The hydrolysis reaction is conveniently monitored by following the fluorescence of the hydrolysis product, BPT, which has a quantum yield many times greater than BPDE. 

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

In another study, Kondrat eva (103) made a determination of the luminescent quantum yield of the 5D4 state of the terbium ion in aqueous solution. The method used was based upon fluorescent-lifetime measurements and had previously been used by Rinck (96) and Geisler and Hellwege (96) to determine the quantum yield of rare earths in crystals. Kondrat eva made his studies on chloride and sulfate solutions, using the electronic shutter technique of Steinhaus et al. (66). 

To study the excited state one may use transient absorption or time-resolved fluorescence techniques. In both cases, DNA poses many problems. Its steady-state spectra are situated in the near ultraviolet spectral region which is not easily accessible by standard spectroscopic methods. Moreover, DNA and its constituents are characterised by extremely low fluorescence quantum yields (<10 4) which renders fluorescence studies particularly difficult. Based on steady-state measurements, it was estimated that the excited state lifetimes of the monomeric constituents are very short, about a picosecond [1]. Indeed, such an ultrafast deactivation of their excited states may reduce their reactivity something which has been referred to as a "natural protection against photodamage. To what extent the situation is the same for the polymeric DNA molecule is not clear, but longer excited state lifetimes on the nanosecond time scale, possibly of excimer like origin, have been reported [2-4],... 

Measurements of phytochrome with this time-resolved LIOAS technique [82] allowed the quantum yield d>r-,70o of the photoreaction Pr - I 700 to be calculated using Eq. (1) [135] (and neglecting the small contribution of fluorescence [76]). 

Much larger RISC values were found for the cyanine dyes 56-58 [38]. A similar two-color technique was used to measure these yields with the exception that meyo-tetraphenylporphyrin was used in place of Aberchrome 540 as the two-laser actinometer. As Table 4 shows, there is an inverse relationship between the triplet depletion (bleaching) quantum yield, 4>B1, and the 5 —> T ISC yield, 1SC. Thus a large value for 4>B1 was accompanied by a small 4>ISC. This reflects the cyclic flow of energy following excitation of Tx to Tn. Once RISC has occurred, decay of the S, state partitions between ISC and fluorescence. If 4>ISC is small, relatively few Sj states will be cycled back to T and 4>B1 will be large. This effect masks the actual efficiency of RISC. Thus the ratio < B1/(1 — isc) s giyen as 311 indicator of RISC efficiency. 

Cf,F4H2(B"B, ). They measured fluorescence quantum yields and lifetimes using photoelectron-photon coincidence techniques and determined laser-... 

The direct chemiluminescence quantum yield is given by Eq. 35, where is the singlet excitation quantum yield and 0 is the fluorescence quantum yield of the singlet excited carbonyl product. The latter is directly responsible for the observed chemiluminescence. If 0 is known from photoluminescence work, determination of 0° allows us to calculate the desired 0 -parameter. Frequently 0 is not known and it is necessary to measure it, using routine fluorescence techniques. 

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