Fluorescence with glycerol

The prism and shde may be optically coupled with glycerol, cyclohexanol, or microscope immersion oil, among other liquids. Immersion oil has a higher refractive index (thereby avoiding possible TIR at the prism/coupling liquid interface at low incidence angles), but it tends to be more autofluorescent (even the extremely low fluorescence types). 

Several studies were performed on the optimization of expression levels of ELP proteins in E. coli. In a recent example, the expression protocol was optimized for an ELP fusion with green fluorescent protein (GFP). This fusion protein was expressed and purified in a yield of 1.6 g/L of bacterial culture, which finally yielded 400 mg GFP/L bacterial culture. This extremely high yield was found after uninduced expression in nutrient-rich medium supplemented with phosphate, glycerol and certain amino acids, such as proline and alanine [234]. The influence of fusion order was also examined and it was found that positioning the ELP at the C-terminus of target protein resulted in significantly higher expression levels [35]. 

Photosensitization of diaryliodonium salts by anthracene occurs by a photoredox reaction in which an electron is transferred from an excited singlet or triplet state of the anthracene to the diaryliodonium initiator.13"15,17 The lifetimes of the anthracene singlet and triplet states are on the order of nanoseconds and microseconds respectively, and the bimolecular electron transfer reactions between the anthracene and the initiator are limited by the rate of diffusion of reactants, which in turn depends upon the system viscosity. In this contribution, we have studied the effects of viscosity on the rate of the photosensitization reaction of diaryliodonium salts by anthracene. Using steady-state fluorescence spectroscopy, we have characterized the photosensitization rate in propanol/glycerol solutions of varying viscosities. The results were analyzed using numerical solutions of the photophysical kinetic equations in conjunction with the mathematical relationships provided by the Smoluchowski16 theory for the rate constants of the diffusion-controlled bimolecular reactions. 

All steady state fluorescence experiments were conducted with the sample placed in a thermostated cell with temperature maintained at 30°C. The concentrations of anthracene and initiator used were 0.000505 and 0.00608 moles per liter, respectively. The relative quantities of solvents (n-propanol and glycerol) were adjusted from 0 to 100% to achieve solutions of different viscosities, while maintaining the same molar concentration of the reactive solutes. 

Fast librational motions of the fluorophore within the solvation shell should also be consideredd). The estimated characteristic time for perylene in paraffin is about 1 ps, which is not detectable by time-resolved anisotropy decay measurement. An apparent value of the emission anisotropy is thus measured, which is smaller than in the absence of libration. Such an explanation is consistent with the fact that fluorescein bound to a large molecule (e.g. polyacrylamide or monoglucoronide) exhibits a larger limiting anisotropy than free fluorescein in aqueous glycerolic solutions. However, the absorption and fluorescence spectra are different for free and bound fluorescein the question then arises as to whether r0 could be an intrinsic property of the fluorophore. 

In most investigations in solvents of medium or high viscosity, or in polymers above the glass transition temperature, the fluorescence quantum yields were in fact found to be a power function of the bulk viscosity, with values of the exponent x less than 1 (e.g. for p-N,N-dimethylaminobenzylidenemalononitrile, x = 0.69 in glycerol and 0.43 in dimethylphthalate). This means that the effective viscosity probed by a molecular rotor appears to be less than the bulk viscosity >/ because of free volume effects. 

A variety of results obtained in studies of dipolar relaxation in the environment of the fluorescence probe 2,6-TNS are illustrated in Figure 2.10. In the model viscous medium (glycerol at 1 °C), the fluorescence spectra exhibit a marked dependence on the excitation wavelength. When 2 varies from 360 to 400 nm, the shift of the fluorescence spectrum maximum is 10 nm with a certain decrease of the half-width. In media with low viscosity, for instance, in ethanol (Figure 2.10a), this effect is never observed. 

Figure 2.10. The dependence of the position of the fluorescence spectrum maximum on excitation wavelength for 2,6-TNS in model media (a) and in complexes with proteins (b). (a) 2,6-TNS (3 x 10-s) M in glucose glass at 20°C (1), glycerol at +1°C (2), and 80% aqueous ethanol at 20°C (3). Excitation spectra are for glycerol (4) and 80% ethanol (5). (b) 2,6-TNS in complexes with / -lactoglobulin (1), tetrameric melittin (2), human serum albumin (3), and lysozyme (4) at 20°C. Excitation spectrum (5) is for human serum albumin.

Figure 8.1S. Spectra of a glycerol coated with a fraction of a monolayer of dil(S). The upper curve is the fluorescence excitation (/,) spectrum, and the lower curve is the 90° elastic scattering (/,) spectrum.

Add 33 pi of fluorescein isothiocyanate (FITC tetramefliylrho-damine isothiocyanate, TRITC, or another fluorescent dye isothiocyanate derivative is used the same way), 50 mg/ml in DMF, to 1 ml of 5 mg/ml IgG in Soln. A. Shake at RT protected from light for 1 h. Remove surplus FITC and its hydrolysis products on a Sephadex G-25 column, equilibrated with PBS. The conjugate appears in the void volume and should be concentrated by ultrafiltration. Add sodium azide to a final concentration of 0.02% (w/v) and glycerol up to 10% (w/v) and store at 4 °C. 

In the delayed emission spectrum of eosin in glycerol or ethanol two bands are present, the relative intensities of which are strongly temperature-dependent (see Fig. 12). The visible band at 1.8 has a contour identical with that of the fluorescence band. It no doubt corresponds to the visible phosphorescence observed by Boudin.26 To interpret the results it was assumed that this band of delayed fluorescence was produced by thermal activation of the eosin triplet to the upper singlet level followed by radiative transition from there to the ground state. The far red band was assumed to correspond to the direct transition from the triplet level to the ground state and was therefore called phosphorescence. To determine the relationship between the intensities of the two bands we write the equations for the formation and consumption of triplet molecules as follows ... 

Fig. 12. Eosin in glycerol (7 X 10 Af) and eosin in ethanol (1.5 X 10 W). (a) Fluorescence emission spectrum at +30°C. (6) delayed emission spectrum (DES) at +69°C. (c) DES at +48°C. (d) DES at + 18°C. (e) DES at -40°C. Delayed emission spectra at a sensitivity 600 times greater than that for the fluorescence emission spectrum. (/) Fluorescence emission spectrum at -J-22°C. (g) delayed emission spectrum (DES) at +71 °C. (h) DES at +43°C. (j) DES at +22°C. (Z) DES at — 7°C. (m) DES at —58°C. (s) Sensitivity of 1)558 photomultiplier with quartz monochromator (unite of quanta and frequency). Delayed emission spectra at a sensitivity 3000 times greater than that for the fluorescence emission spectrum.

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