Fluorophore intracellular

Chen AK, Cheng ZL, Behlke MA, Tsourkas A (2008) Assessing the sensitivity of commercially available fluorophores to the intracellular environment. Anal Chem 80 7437-7444... 

Intracellular reduced pyridine nucleotides NAD(P)H are the primary suppliers of reducing power to anabolic and catabolic pathways. They can be measured because of their fluorescent properties.<16) The fluorescence is caused by the presence of the reduced forms of the pyridine nucleotides NADH and NADPH (jointly referred to as NAD(P)H). These fluorophores absorb light in a wide band around 340 nm, and reemit, or fluoresce, light in a wide band around 460 nm. The phosphorylated and nonphos-phorylated nucleotides have essentially equivalent fluorescence properties while the oxidized forms of these nucleotides are nonfluorescent. 

In order to evaluate binding, uptake, and intracellular localization of targeted drugs via CLSM, nonfluorescent molecules of interest, as well as the appropriate structures of the specimen, have to be labeled with suitable fluorophores. This technique, however, requires a number of preparative steps, including fixation and permeabilization of the sample, staining and counterstaining, as... 

Proteomic Fluorescence microscopy In situ visualisation of cellular/ECM protein with fluorescence-labelled antibody Multiplexing capability (typically 3 fluorophores). Extra- and intracellular antigen location on opaque biomaterials. Issues with bleaching and autofluorescence. Yes/no... 

A direct comparison of the abilities of Angeli s salt and synthetic ONOO- to affect intracellular targets was probed with the fluorophore diaminofluorescein (DAF) (239). Exposure of cells to synthetic ONOO- at a rate comparable to Angeli s salt decomposition resulted in only 4% of the intracellular signal from Angeli s salt. These data show that the reactivity of HNO-derived species in an aerobic cellular milieu cannot be readily reconciled with an intermediacy involving ONOO-. Importantly, cells acted as a sink for HNO-derived species, effectively sequestering intermediates from reaction with DAF when the probe was present in the extracellular medium. 

Figure 4 Examples of mimics of ligands and receptors used to deliver cargo into cells. (a,b) Ligands such as folate can be linked to macromolecules (e.g., nucleic acids and proteins) and small molecules (e.g., drugs, radiochemicals, and fluorophores) to promote cellular uptake, (c) Peptides and small molecules linked to N-alkyl derivatives of 3p-cholesterylamine or 3p-dihydrocholesterylamine mimic cell-surface receptors by cycling rapidly between the plasma membrane and the intracellular endosomes. The synthetic Fc receptor shown in (c) enables treated mammalian cells to internalize human igG.

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