Fluorophore photobleaching

TABLE 11.2 Calculated Sensor Lifetimes Based on the Fluorophore Photobleaching Rate Constants from the Literature... 

With such small amounts of fluorophore, photobleaching of individual molecules could have significant effects. To avoid these difficulties. Tan etal [23,... 

The approach to standardization used by Haaijman (53) and others (66,67), in which the fluorophor is incorporated within or bound to the surface of a plastic sphere, is more versatile than the use of inorganic ion>doped spheres, since the standard can be tailored exactly to the specifications required by the analyte species. However, this approach increases the uncertainty of the measurement because the photobleaching characteristics of both the standard and the sample must be considered. The ideal approach is to employ both types of standards. The glass microspheres can be used to calibrate instruments and set instrument operating parameters on a day-to-day basis, and the fluorophor-doped polymer materials can be used to determine the concentration-instrument response function. 

One of the most popular applications of molecular rotors is the quantitative determination of solvent viscosity (for some examples, see references [18, 23-27] and Sect. 5). Viscosity refers to a bulk property, but molecular rotors change their behavior under the influence of the solvent on the molecular scale. Most commonly, the diffusivity of a fluorophore is related to bulk viscosity through the Debye-Stokes-Einstein relationship where the diffusion constant D is inversely proportional to bulk viscosity rj. Established techniques such as fluorescent recovery after photobleaching (FRAP) and fluorescence anisotropy build on the diffusivity of a fluorophore. However, the relationship between diffusivity on a molecular scale and bulk viscosity is always an approximation, because it does not consider molecular-scale effects such as size differences between fluorophore and solvent, electrostatic interactions, hydrogen bond formation, or a possible anisotropy of the environment. Nonetheless, approaches exist to resolve this conflict between bulk viscosity and apparent microviscosity at the molecular scale. Forster and Hoffmann examined some triphenylamine dyes with TICT characteristics. These dyes are characterized by radiationless relaxation from the TICT state. Forster and Hoffmann found a power-law relationship between quantum yield and solvent viscosity both analytically and experimentally [28]. For a quantitative derivation of the power-law relationship, Forster and Hoffmann define the solvent s microfriction k by applying the Debye-Stokes-Einstein diffusion model (2)... 

In choosing a fluorescent tag, the most important factors to consider are good adsorption (high extinction coefficient), stable excitation without photobleaching, and efficient, high quantum yield of fluorescence. Some fluorophores, such as fluorescein, exhibit rapid fluorescent quenching which lowers the quantum yield over time. Up to 50 percent of the fluorescent intensity observed on a fluorescein-stained slide can be lost within 1 month in storage. AMCA and... 

Antifade agents (free-radical scavengers) in aqueous mounting media prevent photobleaching of fluorophore labels under exposure to excitation light. 

Photobleaching (fading) Photochemical reaction of fluorophore, light and oxygen that causes the intensity of the fluorescence emission to decrease with time. 

Since highly fluorescent water soluble gold clusters have been synthesized by various routes, they can be used in biology-related experiments such as imaging. These clusters may possess additional benefits over organic fluorophores and fluorescent semiconductor quantum dots. Organic fluorophores are prone to photobleaching,... 

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