ABSTRACT
Nitric oxide (NO) is an important effector molecule in host defence against bacterial pathogens. The development of fluorescence imaging to monitor NO production in vitro and in vivo will increase our understanding of its biological role. Recently, a novel 'trappable' fluorescent blue 'turn-on' Cu(II)-complexed coumarin-based probe (CB) has been developed to detect NO. In this study, CB was investigated to evaluate its ability to detect NO in macrophages. Using confocal microscopy, NO was successfully detected in macrophages in the presence of stimuli that induce nitric oxide synthase (iNOS), the enzyme responsible for production of NO. The time dependence and subcellular compartmentalisation of CB in macrophages were evaluated. The probe can be trapped within cells and reacts directly and specifically with NO, rendering it a promising tool for imaging NO in response to pharmacological agents that modulate its level, for example during bacterial infections.
Subject(s)
Coumarins/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Macrophages/metabolism , Nitric Oxide/analysis , Animals , Mice , Microscopy, Confocal , Molecular Structure , Nitric Oxide/metabolism , RAW 264.7 Cells , Time FactorsABSTRACT
Two novel cell-permeable and bacteria-compatible 'turn-on' fluorescent probes, designed to be compatible in a multi-dye system and to fluoresce in the blue region exhibiting emission maxima of 440-490 nm, were synthesized. The profluorescent nitroxide probe (11) was developed to visualize and quantify free radical and redox processes, and the Cu(II)-complexed coumarin-based probe (16a) was developed for NO detection. Confocal laser-scanning microscopy and subsequent digital analysis of Pseudomonas aeruginosa biofilms stained with 11 or 16a determined that free radical and redox processes and NO generation occur predominantly in live cells during normal biofilm growth.
Subject(s)
Biofilms , Copper/chemistry , Fluorescent Dyes/pharmacokinetics , Free Radicals/analysis , Nitric Oxide/chemistry , Pseudomonas aeruginosa , Biofilms/growth & development , Cell Membrane Permeability , Coumarins/chemistry , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/classification , Microscopy, Confocal , Oxidation-Reduction , Staining and LabelingABSTRACT
Free radicals and oxidative stress play important roles in the deterioration of materials, and free radicals are important intermediates in many biological processes. The ability to detect these reactive species is a key step on the road to their understanding and ultimate control. This short review highlights recent progress in the development of reagents for the detection of free radicals and reactive oxygen species with broad application to materials science as well as biology.
