Fluorescence study methods

We will begin by a brief review of the concept of the X-ray fluorescence analytical method widely used in the petroleum industry for studying the whole range of products and for analyzing catalysts as well. 

Aoki, H., Kitamura, M. and Ito, S. (2008) Nanosecond dynamics of poly(methyl methacrylate) brushes in solvents studied by fluorescence depolarization method. Macromolecules, 41, 285-287. 

Horinaka, J., Ono, K. and Yamamoto, M. (1995) Local chain dynamics of syndiotactic poly(methyl methacrylate) studied by the fluorescence depolarization method. Polym. J., 27, 429-435. 

The LB film depositions were performed using a Joyce-Loebl Langmuir Trough IV equipped with a microbalance for measurement of the surface pressure by the Wilhelmy plate method. Filtered deionized water with a pH of 7 was used for the subphase. For the electron beam lithography study, PMMA was spread on the water surface from a dilute benzene solution ( 10 mg PMMA in 20 ml benzene). The novolac/PAC mixtures were spread from solutions ( 20 mg solids in 10 ml solvent) of isopropyl acetate. For the fluorescence studies, the PMMA/PDA mixture was spread on fee water surface from a dilute benzene solution (1.75 mg PDA and 8.33 mg PMMA in 20 ml benzene). Prior to compression, a 20 min interval was allowed for solvent evaporation. The Langmuir film was compressed to the desired transfer pressure at a rate of 50 cm2/min, followed by a 20 minute equilibration period. The Cr-coated silicon wafers and quartz wafers were immersed into fee subphase before... 

Albert H. Coons (Fig. 1.2) was the first who attached a fluorescent dye (fluorescein isocyanate) to an antibody and used this antibody to localize its respective antigen in a tissue section. The concept of putting a visible label on an antibody molecule appeared both bold and original. His initial results were described in two brief papers in the early 1940s (Coons et al. 1941,1942), but the research was halted while he joined the army and spent the next 4 years in the South Pacific. His later studies (Coons and Kaplan 1950) contributed immensely to the use of the fluorescent antibody method in a wide variety of experimental settings. In our time, the use of antibodies to detect and localize individual or multiple antigens in situ has developed into a powerful research tool in almost every field of biomedical research (http //books.nap.edu/html/biomems/acoons.pdf). 

In conclusion, the method of intramolecular excimer formation is rapid and convenient, but the above discussion has shown that great care is needed for a reliable interpretation of the experimental results. In some cases it has been demonstrated that the results in terms of equivalent microviscosity are consistent with those obtained by the fluorescence polarization method (described in Section 8.5), but this is not a general rule. Nevertheless, the relative changes in fluidity and local dynamics upon an external perturbation are less dependent on the probe, and useful applications to the study of temperature or pressure effects have been reported. 

J. R. Lakowicz, Fluorescence studies of structural fluctuations in macromolecules as observed by the time, lifetime and frequency domains, Methods Enzymol. 131, 518-567 (1986). 

Vasilescu, M., Angelescu, D., Almgren, M., Valstar, A. (1999). Interactions of globular proteins with surfactants studied with fluorescence probe methods. Langmuir, 15, 2635-2643. 

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],... 

On the other hand, pyrenyl-L-alanine 184 has also been used as a conformational probe in the characterization of an artificial 4-a-helix bundle protein.11,121 The 53-residue peptide 186 incorporating one residue of 184 in each of two different helical segments was synthesized by solid-phase synthesis using a segment condensation strategy and the oxime resin. Boc-pyrenyl-L-alanine 191 was coupled just like any other amino acid by the BOP/HOBt method in DMF. CD and fluorescence studies demonstrated that the two pyrene groups were in close proximity forming an excimer complex, which is possible only when the polypeptide chain folds into a 4-a-helix bundle structure. 

In order to qualitatively and quantitatively evaluate the various deactivation channels of the electronically excited bilatriene chromophore in Pr, the photophysical and photochemical parameters of this chromophore were determined by stationary fluorescence [76] and time-resolved spectroscopic methods (for in-house developments and first applications of fluorescence and optoacoustics see [77-80] and [81,82], respectively) [2,9,10,83,84], Furthermore, a time-resolved fluorescence study has been carried out also with the selectively excited tryptophane residues of Pt and Pfr. The results are discussed in Sections II.A-C and III.A. Unless specified otherwise, the experiments were performed in vitro with 124-kDa phytochrome isolated from etiolated oat seedlings (Avena sativa L ), and the measurements were carried out with ethylene glycol-containing phosphate buffer solutions at 3s 275 K. 

Fluorescence Of Monolayers Containing Pyrene-Labeled Probes. A fluorescence probe method was also used as a complementary technique to study the thermodynamics of SA film formation. Mixed monolayers containing the fluorescence probe pyrene hexadecanoic acid, Py-C16, in host fatty acids of different lengths were prepared by adsoiption from solutions containing mostly the host fatty acid and a small fraction of Py-C16 (approximately 1 to 5 mol %). All monolayers were prepared under equilibrium adsoiption conditions. For fluorescence measurements only A1 substrate was used because when glass is used an impurity fluorescence from glass interferes with the pyrene fluorescence. 

The strategy we have been following is to pump the v" = 0 - V = 0 vibrational band in the UV and observe the resulting fluorescence. This method reduces complexity caused by eliminating the need to consider vibrational relaxation and results in most of the fluorescence signal appearing in the 0-0 vibrational band. Moreover, the energy levels, transition frequencies and transition probabilities for this band have been studied extensively and can be found in the literature. 

The photochemistry of small molecule LC materials has been an active area of research for many years and has been reviewed recently [9]. The photochemistry of LC polymers, per se, has received much less attention although two brief reviews have appeared [5,10], and there has been a considerable effort to apply some simple photochemical transformations such as trans-cis photoisomerization, to the development of practical devices [1-6]. This section is divided into three parts. In Part A, chromophore aggregation, which seems to be important in almost all the cases in which careful UV-Vis and/or fluorescence studies of films of pure LC polymers have been made, is explicitly discussed. Part B is devoted to a thorough review, organized by chromophore type, of the photochemistry and related photophysics of LC polymers. No attempt has been made to extensively cross-reference the work on LC polymers to the hundreds of papers and reviews on analogous non-LC compounds. However, when it seemed particularly appropriate or interesting, experiments related to optical applications of the photochemistry of LC polymers are briefly described. In Part C, a few experiments are described in which a classical photophysical method, fluorescence spectroscopy, is used to probe the microstructures of some LC polymers. 

Owing to the longer lifetime of the triplet state it is expected that the protolytic reaction will usually reach equilibrium within the lifetime of the state. Unlike the fluorescence titration method for pAr(S1) described above, the triplet-triplet absorption technique leads directly to pA"(T ]) without the necessity for a knowledge of lifetimes. Phosphorescence titration studies, on the other hand, will involve the lifetime term log t0/To just as for fluorescence. 

Because of the superior morphology provided by formalin-fixed paraffin-embedded tissues, this has become the medium of choice for most clinical and research studies. The peroxidase-labeled antibody method, introduced in 1968, was the first practical application of antibodies to paraffin-embedded tissues and overcame some of the limitations of earlier fluorescence antibody methods (1). These pioneering studies using enzyme labels instead of fluorescent dyes opened the door to the development of modern methods of immuohistochemistry. 

On the other hand, when CPO was modified with citraconic, maleic, and phthalic anhydrides, catalytic efficiencies for phenol oxidation were higher compared to native CPO. Those modifications also improved their thermostability by 1-2-fold and tolerance to dimethylformamide (DMF). Circular dichroism studies showed no changes in the secondary structure of CPO, but changes in the environment of the aromatic residues were demonstrated by fluorescence studies [67]. Those findings are in agreement with those obtained for HRP modification using the same compounds. Modification with citraconic, maleic, and phthalic anhydrides represents a simple and powerful method to enhance catalytic properties, thermostability, and organic solvents tolerance of hemeperoxidases. 

Dufour, E., Riaublanc, A. 1997a. Potentiality of spectroscopic methods for the characterisation of dairy products. I. Front-face fluorescence study of raw, heated and homogenised milks. Lait 77, 657-670. 

Sora, D.I. et al. Vahdated ion-pair liquid chromatography and fluorescence detection method for assessing the variability of loratadine metabolism occurring in bioequivalence studies. Biomed Chmmatogr. 2007, 21, 1023-1029. 

Fluorescent tracer techniques hold the promise of improved accuracy in assessing dermal exposures, as they require no assumptions regarding the distribution of exposure across skin surfaces. However, this approach also has several limitations. First, it requires introduction of the tracer compound into the agricultural spray mix. Secondly, there must be demonstration of a correspondence between pesticide deposition and deposition of the fluorescent compound for the production, such that the fluorescence can indeed be considered a tracer of chemical deposition. Thirdly, range-finding and quality assurance studies may be needed to ensure the accuracy of tracer measurements. Fourthly, when protective clothing is worn by workers, the relative penetration of the pesticide and tracer needs to be characterized. All of these limitations make fluorescent tracer methods technically challenging. 

Rice KG. Preparation of fluorescent glycoconjugates for energy-transfer studies. Methods Enzymol. 1994 247 30-43. 

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