Fluorophores

Abstract

FluorophoresFluorescence probes represent the most important area of fluorescence spectroscopy. One can spend a great deal of time describing the instrumentation for fluorescence spectroscopy, including light sources, monochromators, lasers, and detectors. However, in the final analysis, the wavelength and time resolution required of the instruments are determined by the spectral properties of the fluorophores. Furthermore, the information available from the experiments is determined by the properties of the probes. Only probes with nonzero anisotropies can be used to measure rotational diffusion, and the lifetime of the fluorophore must be comparable to the correlation time of interest. Only probes which are sensitive to pH can be used to measure pH. And only probes with reasonably long excitation and emission wavelengths can be used with tissues which display autofluorescence at short excitation wavelengths.Thousands of fluorescent probes are known, and it is not practical to describe them all. This chapter contains an overview of the various types of fluorophores, their spectral properties, and their applications. Fluorophores can be broadly divided into two main classes, intrinsic and extrinsic. Intrinsic fluorophores are those which occur naturally. These include the aromatic amino acids, NADH, flavins, and derivatives of pyridoxal and chlorophyll. Extrinsic fluorophores are added to the sample to provide fluorescence when none exists or to change the spectral properties of the sample. Extrinsic fluorophores include dansyl chloride, fluorescein, rhodamine, and numerous other substances. 3.1.A. Fluorescent Enzyme CofactorsEnzyme cofactors frequently are fluorescent (Figure 3.1). NADH is highly fluorescent, with absorption and emission maxima at 340 and 460 nm, respectively (Figure 3.3). The oxidized form of NADH, NAD+, is nonfluorescent. The lifetime of NADH in aqueous buffer is near 0.4 ns. The 63 J. R. Lakowicz, Principles of Fluorescence Spectroscopy

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

“…5(b)) is much lower compared to bound NAD(P)H (Fig. 5(c)), partly because free NADH has lower fluorescence quantum yield (0.02) as compared to bound NADH (0.1) [39,40]. This is consistent with earlier reports that more than 80% of total NADH fluorescence is due to bound NADH [41,42].…”

Near-infrared autofluorescence for the detection of parathyroid glands

“…9 Tissue autofluorescence is typically observed in the ultraviolet-visible (UV/VIS) wavelength range. 12 However, studies performed across multiple tissues in vitro (unpublished data) as well as in vivo (presented here) indicate that the nearinfrared spectra measured from thyroid and parathyroid tissues are repeatable (within the same tissue) and highly reproducible (across all patients) reinforcing the validity of the detected signal. The observed signal exhibits the typical Stokes' shift associated with fluorescence and the peak emission wavelength does not vary, indicating that this is a form of luminescence and is most likely due to tissue autofluorescence.…”

Enzymatic activity and conformational and morphological studies of four commercial lipases treated with supercritical carbon dioxide

“…Fluorescence spectroscopy is an available technique for studying the conformational structure of free enzymes, in which the intrinsic fluorescence at 280 nm of Trp and Tyr residues is influenced by the environment [20]. Therefore, differences in the intrinsic fluorescence intensity can indicate changes in the tertiary structure of the lipases.…”