Transient State Monitoring by Total Internal Reflection Fluorescence Microscopy

Triplet, photo-oxidized and other photoinduced, long-lived states Of fluorophores are sensitive to the local environment and thus attractive for microenvironmental imaging purposes. In this work, we introduce an approach where these states are monitored in a total internal reflection (TIR) fluorescence microscope, via the characteristic variations of the time-averaged fluorescence occuring ill response to different excitation modulation schemes. The surface-confined TIR excitation field generates a signal from the fluorescent molecules Close to the glass surface. Thereby, a high selectivity and low background noise is obtained, and in combination with IOW duty Cycles Of excitation, the overall photodegradation of the fluorescent molecules of the sample call be kept low, To verify the approach. the kinetics of the triplet and radical states of the dye Rhodamine 110 were imaged and analyzed in aqueous solutions at different concentrations of dissolved oxygen and of the reducing agent ascorbic acid. The experimental results Were compared to data from corresponding fluorescence correlation spectroscopy (FCS) measurements and simulations based oil finite element analysis. The approach was found to accurately determine relative populations and dynamics of triplet and photooxidized states, Overcoming passage time limitations seen ill FCS measurements. The method circumvents the need for time resolution ill the fluorescence detection, allowing simultaneous readout over the whole SLII-face area subject to excitation. It call be applied over a broad range of concentrations and does not require I strong fluorescence brightness of the sample molecules. Given the sensitivity of the triplet and photooxidized states to oxygen concentrations and not the least to local redox environments, we expect the approach to become an attractive tool for imaging cell metabolism.QC 20100525