Fluorescence imaging pathways

Fluorescence imaging with genetically encoded fluorophores is widely being used to study the dynamics and turnover of diverse proteins involved in different plant signal transduction pathways... 

Watson, P., Jones, A.T., Stephens, D.J., Intracellular trafficking pathways and drug delivery Fluorescence imaging of living and fixed cells. Adv Drug Deliv Rev 57, 43-61 (2005). 

The carpanone-based hbrary was screened to discover small molecules that perturb a secretory pathway that involves the transport of properly folded proteins from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane. Whole-cell fluorescence imaging with temperature-sensitive vesicular stomatitis virus glycoprotein fused to green fluorescent protein (VSVGts-GFP) ... 

Since endocytosis ofLDH was confirmed by TEM images (Figure 13.9), forthe next step, its specific endocytic pathway for membrane entry was determined by immunofluorescence and confocal microscopy. Cells were incubated with LDH-FITC, fixed with 3.7% freshly made formaldehyde, and then stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to the red fluorescent dye Texas Red (TR). The confocal microscopic images showed that green fluorescent... 

A combinatorial library of fluorescent NBD molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution <2004MI414>. 

Alternatively, arene displacement can also be photo- rather than thermally-induced. In this respect, we studied the photoactivation of the dinuclear ruthenium-arene complex [ RuCl (rj6-indane) 2(p-2,3-dpp)]2+ (2,3-dpp, 2,3-bis(2-pyridyl)pyrazine) (21). The thermal reactivity of this compound is limited to the stepwise double aquation (which shows biexponential kinetics), but irradiation of the sample results in photoinduced loss of the arene. This photoactivation pathway produces ruthenium species that are more active than their ruthenium-arene precursors (Fig. 18). At the same time, free indane fluoresces 40 times more strongly than bound indane, opening up possibilities to use the arene as a fluorescent marker for imaging purposes. The photoactivation pathway is different from those previously discussed for photoactivated Pt(IV) diazido complexes, as it involves photosubstitution rather than photoreduction. Importantly, the photoactivation mechanism is independent of oxygen (see Section II on photoactivatable platinum drugs) (83). 

The labeling can also be done by fluorescence lifetime differences, e.g., introduced by quenching pathways connected with different surroundings. This can be used for lifetime imaging methods (Chapter 1, this volume) or for distinguishing complexes of one and the same fluorescence label with, e.g., different DNA-bases. In this case, the fluorescence label is not only a label but incorporates a function which senses the environment and can therefore be regarded as a sensing fluorescence probe. 

Combining fluorescence spectroscopy with fluorescence microscopy, confocal microscopy could be used to elucidate the pathway of siderophore-mediat iron uptake in the fungus Ustilago maydis, and visualize this pathway by providing unique fluorescent microscopic images. Using these techniques, clear images of two independent iron-uptake mechanisms have become visualized as well as their cellular compartment locahzed. 

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