High-order photobleaching of pyrylium salts under two-photon excitation
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Pyrylium salts were found to be attractive candidates for applications based on two-photon absorption effect. Especially, they have been used for three-dimensional data storage through photobleaching process. In the present work the photobleaching, under two-photon excitation, is investigated for a representative pyrylium salt hosted in a polymethylmethacrylate film. The photobleaching dependence on time, power and wavelength irradiation as well as on the film concentration is presented. Our results reveal the existence of two photobleaching mechanisms and indicate the presence of high-order interactions in these processes.Keywords:
Photobleaching
Two-photon excitation microscopy
Excitation wavelength
Photobleaching
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Green fluorescent proteins (GFPs) are widely used tools to visualize proteins and study their intracellular distribution. One feature of working with GFP variants, photobleaching, has recently been combined with an older technique known as fluorescence recovery after photobleaching (FRAP) to study protein kinetics in vivo. During photobleaching, fluorochromes get destroyed irreversibly by repeated excitation with an intensive light source. When the photobleaching is applied to a restricted area or structure, recovery of fluorescence will be the result of active or passive diffusion from fluorescent molecules from unbleached surrounding areas. Fluorescence loss in photobleaching (FLIP) is a variant of FRAP where an area is bleached, and loss of fluorescence in surrounding areas is observed. FLIP can be used to study the dynamics of different pools of a protein or can show how a protein diffuses, or is transported through a cell or cellular structure. Here, we discuss these photobleaching fluorescent imaging techniques, illustrated with examples of these techniques applied to proteins of the Saccharomyces cerevisiae pheromone response MAPK pathway.
Photobleaching
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Abstract The mobility of nuclear proteins can be studied by photobleaching techniques. The three main advantages of photobleaching are fast experimental turn around, good spatial and temporal resolution, and the ability to measure kinetics inside of living cells. The main disadvantage of these techniques is the requirement for fluorescently tagged proteins that have rigorously tested to ensure it has the same properties and function as its native counterpart. Three major methods of photobleaching microscopy are described: fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and inverse fluorescence recovery after photobleaching (iFRAP). Each of these techniques has characteristics permitting the determination of distinct parameters of protein behavior in vivo.
Photobleaching
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Photobleaching
Trypan blue
Rhodamine
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Confocal and two-photon fluorescence microscopy techniques using genetically encoded fluorescent probes are widely used in cell biology. Beyond the common problems of photobleaching and phototoxicity, we present evidence that photounbinding also has the potential to compromise such methods, especially in quantitative studies. We show that laser intensities within excitation regimes typical for imaging approaches such as as fluorescence recovery after photobleaching (FRAP), photolysis, or fluorescence correlation spectroscopy (FCS) experiments can cause the dissociation of antibodies from their ligands. Indeed, both one- and two-photon excitation of a fluorescent anti-GFP antibody caused its dissociation from immobilized GFP in vitro. Importantly, with two-photon excitation, the laser intensity threshold for photobleaching was the same as for photounbinding. By contrast, with single-photon excitation, we found a range of laser intensities where photobleaching can be separated from photounbinding. This photounbinding effect was visualized and measured by rebinding a second fluorescent anti-GFP (Green Fluorescent Protein) antibody, indicating that the GFP remained functional for reassociation following the photoinduced dissociation. Finally, we show that this unbinding effect occurs only when at least one binding partner carries a fluorescent label. Our results show that this photounbinding effect can readily remain masked or be misinterpreted as photobleaching, which can compromise the quantitative interpretation of binding studies made using fluorescence microscopy.
Photobleaching
Two-photon excitation microscopy
Fluorescence Correlation Spectroscopy
Fluorescence cross-correlation spectroscopy
Autofluorescence
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This unit describes fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) using commercially available confocal scanning laser microscopy (CSLM). Photobleaching is the photo-induced change in a fluorphore that abolishes that molecule's fluorescence. The different characteristics of green fluorescent protein (GFP) chimeras in a cell can be studied by FRAP, in which a selected region of the cell is photobleached with intense light. The movement of unbleached molecules into a photobleached region is quantified by imaging with an attenuated light source. The movement of molecules between cellular compartments can be determined by FLIP, in which the same region of a cell expressing a GFP chimera is repeatedly photobleached. The loss of fluorescence from regions outside the photobleached region is monitored to characterize the movement of a protein. Together these two techniques are providing fundamentally new insights into the kinetic properties of proteins in cells.
Photobleaching
Chimera (genetics)
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Photobleaching
Live cell imaging
Endomembrane system
Transport protein
Lipid microdomain
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Photobleaching
Dynamics
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The analytic expression of fluorescence correlation spectroscopy(FCS) based on twophoton excitation(TPE) was modified by introducing a photobleaching factor(θ).Using the modified expression,the effect of photobleaching for the measurement of dynamic characteristics can be eliminated,and the photobleaching lifetime can also be obtained.The simulated results for the rhodamine B(RB) in sucrose solution showed that the experiments data can be fitted accurately by the modified expression rather better than the conventional expression,and that the photobleaching lifetime of different excitation conditions can also be obtained simultaneously.
Photobleaching
Fluorescence Correlation Spectroscopy
Two-photon excitation microscopy
Rhodamine
Fluorescence cross-correlation spectroscopy
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