Fluorescence ratio

Raman scattering is essentially undelayed with respect to the arrival of the incident light, in this technique the detector is activated only during each laser pulse and deactivated at all other times. This allows only Raman signals to be recorded but fluorescence signals and detector noise are gated out (Fig. 19). Improvement in Raman signal to fluorescence ratio has been achieved as illustrated in Fig. 20. The technique, however, at present seems to be restricted by several instrumental limitations [37). 

The quantum yield of fluorescence (ratio between the number of photons emitted by Si and the number of absorbed photons) and phosphorescence (ratio between the number of photons emitted by Tj and the number of absorbed photons) can range between 0 and 1 and are given by the following expressions ... 

How pectic signals were transduced was unknown, but information suggested that cytosolic free Ca might be involved [22]. Fluorescence ratio imaging has then been used to follow the evolution of free calcium concentrations ([Ca " ] ) after stimulation of carrot protoplasts by oligogalacturonides [23]. 

Various optical detection methods have been used to measure pH in vivo. Fluorescence ratio imaging microscopy using an inverted microscope was used to determine intracellular pH in tumor cells [5], NMR spectroscopy was used to continuously monitor temperature-induced pH changes in fish to study the role of intracellular pH in the maintenance of protein function [27], Additionally, NMR spectroscopy was used to map in-vivo extracellular pH in rat brain gliomas [3], Electron spin resonance (ESR), which is operated at a lower resonance, has been adapted for in-vivo pH measurements because it provides a sufficient RF penetration for deep body organs [28], The non-destructive determination of tissue pH using near-infrared diffuse reflectance spectroscopy (NIRS) has been employed for pH measurements in the muscle during... 

FIG. 5. Maintenance of the superficial buffer barrier depends on NCX-assisted Ca2+ transport from the SR lumen to the extracellular space. (A) Rate of loss of SR Ca2+ content, measured as a caffeine transient, into Ca2+ free perfusate at room temperature. (B) Rate of decline in [Ca2+ I from an elevated level, measured as fura 2 fluorescence ratio, into Ca2+ free superfusate, which is either Na+ free or contains 10 /rM CPA or is Na+ free and contains CPA. (C) This cartoon represents a model for maintained buffering by the superficial SR of Ca2+ entry. Ca2+ taken up by SERCA is subsequently released into the SR-PM junctional space from where it is extruded by the NCX. 

The third group ofpolychromophoric compounds to be discussed are homopolymers in which the pendant rings are separated from the backbone by one or more atoms. The polymers of allyl arenes, which lack only the n = 3 ring spacing of aryl vinyl polymers, have been studied very little. The fluorescence spectrum of poly(l-allyl-naphthalene) in dilute dichloromethane solution has been reported 28). Like 1-ethyl-naphthalene, the maximum intensity was seen at 337 nm, but a weak, broad shoulder was also recorded for the polymer at 410 nm. The fluorescence ratio Iu/IM for poly(l-allylnaphthalene) was only 1/100 th the value for P1VN 28). The excimeric nature of the 410 nm emission in the allyl-based polymer has not been confirmed, since neither the lifetime nor the excitation spectrum of this fluorescence band are known. 

In order to determine whether energy migration makes a significant contribution to the photophysical behavior of P2VN and PS in dilute miscible blends, it is instructive to calculate the expected exdmer-to-monomer fluorescence quantum yield ratio in the absence of energy migration. To do so, it is first necessary to assume that intermolecular and non-adjacent intramolecular EFS are absent. In addition, the adjacent intramolecular EFS are assumed to be frozen into the aryl vinyl polymer and must be excited by direct absorption of a photon. Since the absorption spectrum of an EFS is no different from that of non-EFS chromophores, then the calculated fraction of rings within EFS is sufficient to determine the fluorescence ratio. 

The excimer formation and dissociation rates in the above are primed, to show that they are due to backbone rotations and to distinguish them from the rigid-matrix processes described in Table 4. Each chromophore has only one neighbor, and the values of M for the two chromophores are identical. Energy migration cannot increase the fluorescence ratio of bichromophoric compounds, since there is only one dyad. 

Thus, Eqs. (l)-(4) and the stated substitutions provide the means to approximate the fluorescence ratio of aryl vinyl polymers in solution, in the absence of energy migration. 

P2VN, and the d/-pentane compound, respectively, assuming no energy migration. The experimental values for these respective compounds 12,166) follow the proportion 4.8 8.0 1, so that the fluorescence ratio of P2VN is nearly twice as large as expected. 

The calculated value of experimental values for these respective compounds 17 37 135) fall into two conflicting proportions, because of a wide range in the fluorescence ratio of atactic PS. The proportion 28 6 1 reflects (Pd/ = 17 for PS 17) yields the proportion 28 24 1. The data for PS are inconclusive, but it will be shown later that energy migration in PS is probably slower and less effective than in P2VN. 

We conclude that the difference between the experimental value and the no-transfer value of the fluorescence ratio of P2VN and PS is less in solution than in dilute miscible blends, because energy migration must compete with rotational processes in the generation of excimers in solution. This difference is also present when the effect of molecular weight on aryl vinyl polymers in solution and in dilute miscible blends is considered in the next section. 

The fluorescence ratio of samples of P2VN and PS that are 56 % isotactic and have N = IQ will be computed and compared with the infinite molecular-weight value, assuming no energy migration. First, Mcnd for P2VN and PS chain ends is calculated from the data in Table 10 to be 0.162 and 0.157, respectively. Second, recall that... 

M, for atactic P2VN and PS samples was found to be 0.074 and 0.0545, respectively. Third, the value of M for P2VN and PS samples having N = 10 is given by Eq. (5) as 0.092 and 0.075. Finally, we compute from Eq. (1) that the value of (Pd/polymer relative to the infinite molecular-weight value is 0.79 1 and 0.71 1 for P2VN and PS, respectively. Similar calculations for the N = 100 polymers show that the fluorescence ratios are within 96 % of their infinite molecular-weight values. 

A wide variety of copolymers of vinyl arenes with spectroscopically inert monomers have been synthesized since the first study of P(S-co-MMA) in 1963 20). The fluorescence of copolymers derived from styrene 178-180,184), 1-vinyl naphthalene 31,179-i8i-is3), an(j 2-vinylnaphthalene41 158,164,179) has been studied, primarily in fluid solution. Numerous functions relating cpD, q>M, and the fluorescence ratio to the copolymer composition have been proposed, none of which can be applied over the entire composition range of the copolymer. 

Fluorescence quenching of PS 17,161,190,191), P2VN 157,192 194-> poly(l-naphthyl methacrylate) 149,199, poly(2-naphthylmethyl methacrylate 1SS>, P(S-co-MMA)161), and P(2VN-co-S)194) in solution has been examined. In all cases except one for PS 161, plots of IDi0/Id versus [Q] showed upward curvature similar to Eq. (7). Excimer fluorescence was quenched more rapidly than monomer fluorescence with increasing [Q]. In addition, the observed quenching of the fluorescence ratio and of the monomer fluorescence was roughly linear in [Q]. 

Fig. 11.4. The time course of a calcium response (measured by the indo-1 405/485 fluorescence ratio) induced in lymphocytes by the addition of a stimulus. The upper plot is a two-dimensional dot plot of the calcium ratio versus time. The lower plot shows data binned in 5 s intervals and processed to give both the median ratio and the percentage of cells above the base line as they change with time.

This possibility has been explored by Pappayee and Mishra [130], It was found that in DMPC and DPPC liposomes, simultaneous presence of both P and DP form fluorescence is observed in a pH interval of 11-13. Within this pH interval, the maximal fluorescence sensitivity (largest changes in neutral to anionic form fluorescence ratio) was observed at pH 12. At this pH, CBZ partitions well into the liposome membrane with a large Kp value of 2 X 106 M 1 for DMPC and 3 X 106 M 1 for DPPC liposomes. Fluorescence quenching studies with the hy-... 

Tsien, R.Y. Poenie, M. (1986). Fluorescence ratio imaging A new window into intracellular ionic signaling. Trends Biochem. Sci. 11,450-455. 

D Amax fluorescence ratio at maximal calcium concentration... 

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