Intensity ratio, fluorescence modulation

Figure 1.1. Schemes for fluorescence sensing intensity, intensity ratio, time-domain, and phase-modulation, from left to right.

The fluidity in the neighborhood of probe molecules can be tested by use of probes capable of intramolecular excimer formation. The probe molecules contain the two excimer-forming moieties linked by an alkyl chain. The extent of excimer formation depends on the viscosity of the environment and can be monitored by measuring the excimer/monomer fluorescence intensity ratio. The dependence of this ratio on reciprocal viscosity for the probe molecule dipyrenylpropane is shown in Fig. 18, in which the obtained microfluidities for surfactant systems are indicated. The fluidities decrease in the order SHS microemulsion, SDS, CTAC, Triton X-100 cf. Ref. 167 (for abbreviations see Tables 6 and 7). The same sequence order was found by Kano et al. (68). In systems containing heavy counterions the method leads to data that must be evaluated carefully, since heavy atom interactions may be different with excited monomers and excimers. The intramolecular excimer technique is also useful in biological studies. For instance, Almeida et al. investigated the sarcoplasmic reticulum membrane in which the activity of the Ca -pumping enzyme is modulated by the membrane fluidity (197). 

Chemical Mechanisms for Fluorescence Modulation. While UV/visible signalling almost always results from the ionization of a conjugated substituent, there exists a plethora of mechanisms by which fluorescence signal transduction may be engendered. It is useful to categorize the mechanisms for fluorescence modulation described to date via the type of measurement that is made. These are intensity, intensity-ratio, and lifetime. A pictoral summary of each is found in Figure 1 of the chapter by Szmacinski and Lakowicz. 

Figure 1. Schemes for fluorescence sensing. A - single excitation or emission wavelength intensity, B - dual excitation or emission wavelengths intensity ratio C - measurement of intensity decay D -measurement of fluorescence phase angle and/or modulation.

Fig. 9 Fluorescence emission spectra (n-hexane) of photo-stationary states of c/s-17a and trans-17b in 90 10 and 30 70 ratios (A.U. = arbitrary units, relative intensities). Inset modulated emission signal during alternating irradiation at 365 and 435 nm (excitation 300 nm, irradiation time 60s).

The modulation ratio M is another valuable parameter that describes the modulation of the fluorescence intensity relative to the excitation intensity ... 

Field studies of modulated chlorophyll a fluorescence emissions and chlorophyll content are not always in accordance with studies carried out under controlled conditions A study of Parmelia quercina in northern Castelon (Spain) detected an unaffected Fy/Fm ratio whereas the air pollution was found to be quite intense (Calatayud et al., 1996). On the other hand, significant changes in the maximal quantum yield of PSII were found for the lichen Ramalina lacera exposed in polluted sites in Israel and an application of heavy-metal containing solutions and simulated acid rain yielded an additional proof of change (Garty, Weissman, Tamir and Beer, unpublished). 

Fluorescent intensity across the channel width is used to define quality of separation. For example, as particles travel in the modulated aspect-ratio device [10], the 20 pm-diameter particles focus at the top and bottom of the channel, while the 10 pm-diameter particles focus at the side-walls (Fig. 5d). The intensity plot across the channel width shows a complete separation of streaks of 20 and 10 pm-diameter particles (Fig. 5e). The FWHM of the fluorescent intensity peak is nearly equivalent to the corresponding particle diameter, illustrating that microparticles are fully focused. These examples show the approaches for visualizing microparticle... 

A spectrometer is an instrument that provides information about the intensity of radiation as a function of wavelength or frequency. The dispersing modules in some spectrometers are multichannel so that two or more frequencies can be viewed simultaneously. Such instruments are sometimes called polychromators. A spectrophotometer is a spectrometer equipped with one or more exit slits and photoelectric transducers that permit the determination of the ratio of the radiant power of two beams as a function of wavelength as in absorption spectroscopy. A spectrophotometer for fluorescence analysis is sometimes called a speciroftttorometer. 

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