Room-temperature fluorescence spectroscopy

Sanchez E J, Novotny L, Floltom G R and Xie X S 1997 Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation J. Chem. Phys. A 101 7019-23... 

The room-temperature fluorescence properties of a series of 2-substituted, 10-alkylated phenothiazine derivatives, including emission spectra, Stokes shifts, fluorescence quantum yields (< p) and lifetimes (tp), were investigated by Garcia et al. [4,28] in the same solvents as for the absorption spectroscopy study. Comparative values were also reported by Elisei et al. [29] for two similar promazines, namely fluphenazine hydrochloride and perphenazine hydrochloride. All these data are gathered in Table 2. 

A number of less commonly used analytical techniques are available for determining PAHs. These include synchronous luminescence spectroscopy (SLS), resonant (R)/nonresonant (NR)-synchronous scan luminescence (SSL) spectrometry, room temperature phosphorescence (RTP), ultraviolet-resonance Raman spectroscopy (UV-RRS), x-ray excited optical luminescence spectroscopy (XEOL), laser-induced molecular fluorescence (LIMP), supersonic jet/laser induced fluorescence (SSJ/LIF), low- temperature fluorescence spectroscopy (LTFS), high-resolution low-temperature spectrofluorometry, low-temperature molecular luminescence spectrometry (LT-MLS), and supersonic jet spectroscopy/capillary supercritical fluid chromatography (SJS/SFC) Asher 1984 Garrigues and Ewald 1987 Goates et al. 1989 Jones et al. 1988 Lai et al. 1990 Lamotte et al. 1985 Lin et al. 1991 Popl et al. 1975 Richardson and Ando 1977 Saber et al. 1991 Vo-Dinh et al. 1984 Vo- Dinh and Abbott 1984 Vo-Dinh 1981 Woo et al. 1980). More recent methods for the determination of PAHs in environmental samples include GC-MS with stable isotope dilution calibration (Bushby et al. 1993), capillary electrophoresis with UV-laser excited fluorescence detection (Nie et al. 1993), and laser desorption laser photoionization time-of-flight mass spectrometry of direct determination of PAH in solid waste matrices (Dale et al. 1993). 

Of the first 31 chlorina mutants characterised in the Copenhagen mutant collection, 9 were found to be characterised by unusually high F680/F740 and Fm/Fo ratios, when examined by low temperature fluorescence emission spectroscopy and room temperature fluorescence induction kinetics (1). Of these nine, four showed these properties when grown in the glasshouse at 17°C, but not at 22°C. This paper describes one of these temperature-sensitive mutants, chlorina- y which is the first barley mutant in which mutant leaf tissue returns to normal as the result of being shifted from the restrictive temperature. 

Fluorescence spectrometry is a well-known method for quantitative analysis of various classes of molecules. Compared to absorption spectrometry it provides in general higher sensitivity and selectivity. However, conventional room-temperature fluorescence excitation and emission spectra are usually broad (e.g., 10 nm or more) and show no or hardly any fine structure. Obviously, its potential for qualitative analysis would be strongly extended if an increase in spectral resolution could be obtained, so that the vibrational fine structure of the spectra became visible (see Figure 1). This has been realized in high-resolution fluorescence spectroscopy, which has a sensitivity similar to that of conventional fluorescence spectroscopy and a selectivity comparable to that of infrared (IR) spectroscopy. 

This agrees quite well with the rate constants for intramolecular proton transfer in 2,4-bis(dimethyl-amino )-6-(2-hydroxy-5-methylphenyl)-5-triazine which had been measured by Shizuka et al. ( l6) using laser picosecond spectroscopy. The fluorescence decay constant t of (TIN) was found to be 60 20 ps. Because of the weak intensity all fluorescence lifetimes refer to the pure substance in crystalline form at room temperature. 

Fluorescence spectroscopy forms the majority of luminescence analyses. However, the recent developments in instrumentation and room-temperature phosphorescence techniques have given rise to practical and fundamental advances which should increase the use of phosphorescence spectroscopy. The sensitivity of phosphorescence is comparable to that of fluorescence and complements the latter by offering a wider range of molecules for study. 

It is now clear that in the absence of molecular oxygen most proteins phosphoresce in aqueous solutions at ambient temperature.(10) In this chapter we discuss the use of phosphorescence of tryptophan to study proteins, with emphasis on measurements at room temperature. Comparisons between phosphorescence and the more commonly used fluorescence spectroscopy are made. Comprehensive reviews of protein luminescence have been written by Longworth.(n 12 1 A discussion on the use of phosphorescence at room temperature for the study of biological materials was given by Horie and Vanderkooi.(13)... 

Solid and solution phase fluorescent spectra at room temperature exhibit relatively broad, often mostly featureless excitation (absorption) and emission spectra, particularly when compared to mid- and far-infrared spectroscopies. These spectra are often mirror images of each other but there are several exceptions as a result of either disparate molecular geometries between the ground and excited states or when the fluor is an excimer. ... 

Room temperature ILs have been the object of several Raman spectroscopy studies but often ILs emit intensive broad fluorescence. In our own experiments, the use of visible laser light (green 514.5 nm or red 784 nm) resulted in strong fluorescence [29,46]. Similar observations have been reported for many IL sysfems. Our experimental spectra needed to be obtained by use of a 1064 nm near-IR exciting source (Nd-YAC laser at 100 mW of power). The scattered light was filtered and collected in a Bruker... 

An additional piece of information can be obtained by studying a synthetic compound derived from the GFP chromophore (1-28) fluorescing at room temperature. In Fig. 3a we show the chemical structure of the compound that we studied in dioxan solution by pump-probe spectroscopy. If we look at the differential transmission spectra displayed in Fig. 3b, we observed two important features a stimulated emission centered at 508 nm and a huge and broad induced absorption band (580-700 nm). Both contributions appear within our temporal resolution and display a linear behavior as a function of the pump intensity in the low fluences limit (<1 mJ/cm2). We note that the stimulated emission red shifts with two characteristic time-scales (500 fs and 10 ps) as expected in the case of solvation dynamics. We conclude that in the absence of ESPT this chromophore has the same qualitative dynamical behavior that we attribute to the relaxed anionic form. 

The extremely small cross sections for conventional Raman scattering, typically 10 111 to 10-25 cm2/molecule has in the past precluded the use of this technique for single-molecule detection and identification. Until recently, optical trace detection with single molecule sensitivity has been achieved mainly using laser-induced fluorescence [14], The fluorescence method provides ultrahigh sensitivity, but the amount of molecular information, particularly at room temperature, is very limited. Therefore, about 50 years after the discovery of the Raman effect, the novel phenomenon of dramatic Raman signal enhancement from molecules assembled on metallic nanostructures, known as surface-enhanced Raman spectroscopy or SERS, has led to ultrasensitive single-molecule detection. 

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