Fluorescence measurements matter

Kumke, M. U., Abbt-Braun, G., and Frimmel, F. H. (1998). Time-resolved fluorescence measurements of aquatic natural organic matter (NOM). Acta Hydrochim. Hydrobiol. 26, 73-81. 

F.E. Hoge, A. Vodacek, N.V. Blough (1993). Inherent optical properties of the ocean Retrieval of the absorption coefficient of chromophoric dissolved organic matter from fluorescence measurements. Limnol. Oceanogr., 38,1394-1402. 

The previous sections may give the impression that it is an easy matter to establish the mechanism of a multi-substrate enzyme. In fact, more often than not uncertainty and controversy surround such mechanisms for many years despite an abundance of experimental work. We have assumed an ideal situation whereas there are a number of possible obstacles in practice. For example the reaction may be effectively irreversible so that it is only possible to measure the kinetic parameters for one direction of reaction the substrate specificity may be so stringent that it is impossible to apply tests which rely on using a range of alternative substrates the available methods of rate measurement may not be sufficiently sensitive to allow all the kinetic parameters to be determined rehably. The last problem at least is one that allows some hope the kinetic study of NAD-dependent dehydrogenases became much more incisive once fluorescence measurement took over from absorbance measurement as the method of choice [52,57]. Nevertheless there is clearly a need for as many criteria of mechanism as may be mustered and the study of inhibition patterns is a valuable adjunct to the methods already discussed. 

Time-Resolved Fluorescence Measurements on Dissolved Marine Organic Matter... 

Li, J., Busscber, H.J., Swartjes, J.J.T.M., Cben, Y., HarapanabaUi, A.K., Norde, W., et al., 2014. Residence-time dependent cell wall deformation of different Staphylococcus aureus strains on gold measured using surface-enbanced-fluorescence. Soft Matter 10,7638-7646. bttp // dx.doi.org/10.1039/C4SM00584H. 

Belzile, C., Roesler, C.S., Christensen, J.R, Shakhova, N., and Semiletov, 1. (2006). Fluorescence measured using the WETStar DOM fluorometer as a proxy for dissolved matter absorption. Estuar, Coast. Shelf Sci., 67,441 449. 

CDOM fluorescence measurements are a powerful tool to study dynamics of organic matter in the oceans. The apphcation of fluorescence to remote sensing of CDOM is limited to the use of airborne LIDAR systems that can measure g(A) accurately. Contribution of FCDOM to R and Ailing of Fraunhofer lines can be important only during special conditions of high CDOM and low scatterers. FCDOM can be extremely useful in the validation of remote sensing estimates of flg(A). 

Figure 9.11. A 1000-year record of organic matter humification preserved in a cave stalagmite, (a) The annual hand width (annual accumulation rate) of the stalagmite as determined hy counting annual hands formed from fluorescent organic matter, (b) The fluorescence ratio as determined by measuring the intensity of emitted fluorescence at 420 and 470 nm. Low ratios (note the inverse scale) correlate with periods of faster stalagmite growth due to a decrease in water table in the overlying peat and increased COj production and limestone dissolution. Low ratios therefore correspond to periods of high humification. (Adapted from Proctor et al., 2000.)...

Luminescence measurements on proteins occupy a large part of the biochemical literature. In what surely was one of the earliest scientific reports of protein photoluminescence uncomplicated by concurrent insect or microorganism luminescence, Beccari (64), in 1746, detected a visible blue phosphorescence from chilled hands when they were brought into a dark room after exposure to sunlight. Stokes (10) remarked that the dark (ultraviolet) portion of the solar spectrum was most efficient in generating fluorescent emission and identified fluorescence from animal matter in 1852. In general, intrinsic protein fluorescence predominantly occurs between 300 nm and 400 nm and is very difficult to detect visually. The first... 

The physical basis of spectroscopy is the interaction of light with matter. The main types of interaction of electromagnetic radiation with matter are absorption, reflection, excitation-emission (fluorescence, phosphorescence, luminescence), scattering, diffraction, and photochemical reaction (absorbance and bond breaking). Radiation damage may occur. Traditionally, spectroscopy is the measurement of light intensity... 

Now, we are not particular experts in X-ray and gamma-ray spectroscopy (nor mass spectroscopy, for that matter), but our understanding of those technologies is that they are used mainly in emission mode. Even when the exciting source is a continuum source, such as is found when an X-ray tube is used to produce the exciting X-rays for an X-ray Fluorescence (XRF) measurement, the measurement itself consists of counting the X-rays emitted from the sample after the sample absorbs an X-ray from the source. These measurements are themselves the equivalent of single-beam measurements and will thus also be Poisson-distributed in accordance with the basic physics of the phenomenon. 

Other near-IR techniques that have been used to measure lifetimes, though not to the same extent as the aforementioned methods, include fluorescence up-conversion,(19 21) parametric amplification, 22 streak camera detection,(23) and two-photon excitation,1(24) The latter technique is particularly useful as it enables the greater penetration depth of near-IR radiation in organic matter to be used to obtain a well-defined region of excitation, e.g., in single cells or mammalian tissue. 

The fluorescence decay is multiexponential.(199 200) This is unequivocal evidence that the wyebutine base can be bound in at least two different conformations with different solvent shielding. Wells and Lakowicz(200) resolved two exponential components. They measured the normalized amplitudes and lifetimes for the wyebutine fluorescence at two different concentrations of added Mg2+ S° = 0.50, t, = 1.7 ns, = 0.50, and t2 = 5.9 ns with no added Mg2+ present, and S°i = 0.16, t, =0.6 ns, S2 = 0.84, and r2 = 6.0ns with 10 nM Mg2+. Since the 6ns component is the longest lifetime present, it must represent the conformation that shields the wyebutine to the greatest extent and is generally believed to involve a 3 stack of bases 34-38.w 199-2011 The fraction of the tRNA in this conformation increases when Mg2 + is added to the solution. This structure is also observed in crystal structures which include Mg2+.(202 204) In the other conformation(s), the wyebutine is more exposed to the solvent. A 5 stack, which does not include bases 37 and 38, is one possibility. The wyebutine base would be more exposed to the solvent and have a shorter fluorescence lifetime as a result. However, both NMR data(205 206) and chemical modification studies(207) are inconsistent with a 5 stack. For the moment, this matter is unresolved. 

The mby fluorescence method allows us to perform pressure measurements in a short time scale (1-10 s), providing a real-time access for pressure control comparing to the time scale of many solid-state chemical processes. As a matter of fact, real-time pressure measurements are necessary when studying kinetic processes [117], but it is also important to minimize the laser power used for measuring the mby fluorescence in order to avoid undesired photochemical effects on the sample, whenever these are possible. In the case of IR absorption studies, which are commonly used for kinetic purposes, the advantage of using the mby fluorescence method, once photochemical effects are prevented, with respect to the employment of vibrational gauges is that no additional absorption bands are introduced in the IR spectmm. 

The surface sensitivity is ensured by detecting the decay products of the photoabsorption process instead of the direct optical response of the medium (transmission, reflection). In particular one can measure the photoelectrons, Au r electrons, secondary electrons, fluorescence photons, photodesorbed ions and neutrals which are ejected as a consequence of the relaxation of the system after the photoionization event. No matter which detection mode is chosen, the observable of the experiment is the interference processes of the primary photoelectron with the backscattered amplitude. 

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