Fluorophore selection

The use of an evanescent wave to excite fluorophores selectively near a solid-fluid interface is the basis of the technique total internal reflection fluorescence (TIRF). It can be used to study theadsorption kinetics of fluorophores onto a solid surface, and for the determination of orientational order and dynamics in adsorption layers and Langmuir-Blodgett films. TIRF microscopy (TIRFM) may be combined with FRAP ind FCS measurements to yield information about surface diffusion rates and the formation of surface aggregates. 

Fluorescence polarisation spectroscopy is still very much used to probe the rotational dynamics of single molecules, either on surfaces or in solution [152]. In bioa-nalytical assays the fluorescence emission intensity is measured as a function of rotational speed. When a solution of fluorophores is excited with polarised light, the fluorophores selectively absorb those photons that are parallel to the transition moment of the fluorophore, resulting in photoselective excitation. The fluorophore molecules rotate to varying extents during the fluorophore lifetime. If the fluores-... 

Phenolic copolymers containing fluorophores (fluoroscein and calcein) were synthesized by SBP catalysis and used as array-based metal-ion sensor. Selectivity and sensitivity for metal ions could be controlled by changing the polymer components. Combinatorial approach was made for efficient screening of specific sensing of the metals. 

Fluorescence probes possessing the PyU base 46 selectively emit fluorescence only when the complementary base is adenine. In this case, the chromophore of is extruded to the outside of the duplex because of Watson-Crick base pair formation, and exposed to a highly polar aqueous phase. On the contrary, the duplex containing a PyU/N (N = G, C and T) mismatched base pair shows a structure in which the glycosyl bond of uridine is rotated to the syn conformation. In this conformation, the fluorophore is located at a hydrophobic site of the duplex. The control of base-specific fluorescence emission is based on the polarity change in the microenvironment where the fluorophore locates are dependent on the l>yU/A base-pair formation. 

Three types of reactive spectrally distinct fluorophores, namely lissamine rhoda-mine (LR) 80, 7-dimethylaminocoumarin (DMAC) 81, and bodipy-630 (BDPY) 82 dyes, prepared by coupling 3-azidopropylamine or propargylamine to commercially available amine-reactive dyes were evaluated for the use in selective dye-labeling of newly synthesised proteins in Rat-1 fibroblasts. 

Fig. 24 Discovery of novel fluorophore, l,2-dihydropyrrolo[3,4-(3]indolizin-3-one, using a combinatorial approach (a) Synthetic schemes of fluorescent core skeleton (b) Collected emission spectra of selected compounds covering full-color emission wavelength (c) Table of photophysical properties of all fluorescent compounds. All the photophysical properties were measured in DCM (dichloromethane). Reproduced with permission from [92]...

This procedure involves selecting a fluorophore of known lifetime and placing it in the microscope and measuring the phase and modulation depth [11]. Rearranging Eqs. (2.5 and 2.6) allows the expected phase and modulation to be predicted. These may then be used to compute the position of zero phase and the modulation depth of the light source. An advantage of the method is that it may be done under conditions exactly matching those of a sample. 

When compared to fluorescent proteins, fluorophores and quenchers of fluorescence (short quenchers) are small molecules with sizes varying from 1 to 10 A. They are the main building blocks for constructing small molecule FRET probes. As molecular entities, they might influence the performance of the probe to a great extent. Their fluorescent properties will determine the sensitivity and dynamic range of the sensor. The success of the probe for a specific application will depend on the selection of the right fluorophores... 

An important feature that influences the performance of the probe to a large extent is the selection of the FRET pair. Generally, fluorophores with high excitation wavelengths are preferred over blue fluorophores in single photon microscopy for applications in tissue since there is no autofluorescence in this region and the ratio... 

In a similar fashion, steroids are molecules that have been investigated by disruption of FRET. The sensor is a double labeled peptide with cyclodextrin bound to one side chain. The latter keeps the fluorophores closely together by accommodating the coumarin into its cavity thereby ensuring efficient FRET. Steroids compete for the cavity of cyclodextrin and displace the coumarin reducing FRET efficiency. This model, although useful for in vitro applications, seems to be poorly selective for its application in biological samples [95],... 

Alternatively, proteins can be labeled selectively using amine-reactive dyes. Particularly, cysteine and lysines can be modified covalently with a variety of commercially available fluorophores including Texas Red, Oregon Green, and Cy3 [19] (see also Chapter 6 for small molecule FRET probes, and Chapter 12 depicting a variety... 

The exploitation of the above expression systems in FRET requires the coherent selection of donor and acceptor moieties, from both the spectroscopic and biological perspective relative expression levels, compartmentalization, and temporal evolution of the system under study. Very advantageous are combinations of small fluorophores with VFPs, as well as with fluorescent nanoparticles, particularly QDs. 

Dedecker, P., Hotta, J.-I., Flors, C., Sliwa, M., Uji, I. H., Roefifaers, M. B. J., Ando, R., Mizuno, H., Miyawaki, A. and Hofkens, J. (2007). Subdififraction imaging through the selective donutmode depletion of thermally stable photoswitchable fluorophores Numerical analysis and application to the fluorescent protein Dronpa. J. Am. Chem. Soc. 129, 16132-41. 

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