The measurement of tissue and cell oxygenation is important for understanding cell metabolism. We have addressed this problem with a novel optical technique, called triplet imaging, that exploits oxygen-induced triplet lifetime changes and is compatible with a variety of fluorophores. A modulated excitation of varying pulse widths allows the extraction of the lifetime of the essentially dark triplet state using a high-fluorescence signal intensity. This enables the monitoring of fast kinetics of oxygen concentration in living cells combined with high temporal and spatial resolution. First, the oxygen-dependent triplet-state quenching of tetramethylrhodamine is validated and then calibrated in an L-ascorbic acid titration experiment demonstrating the linear relation between triplet lifetime and oxygen concentration according to the Stern-Volmer equation. Second, the method is applied to a biological cell system, employing as reporter a cytosolic fusion protein of beta-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg(8)]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays monoexponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and the subcellular level.

PostprintThis is the accepted version of a paper published in Analytical Chemistry. This paper has been peerreviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the original published paper (version of record):Chmyrov, V., Spielmann, T., Hevekerl, H., Widengren, J. (2015) Trans-Cis isomerization of lipophilic dyes probing membrane microviscosity in biological membranes and in live cells. Corresponding AuthorJerker Widengren: jerker@biomolphysics.kth.se.ABSTRACT Membrane environment and fluidity can modulate the dynamics and interactions of membrane proteins, and can thereby strongly influence the function of cells and organisms in general. In this work, we demonstrate that trans-cis isomerization of lipophilic dyes is a useful parameter to monitor packaging and fluidity of biomembranes. Fluorescence fluctuations, generated by trans-cis isomerization of the thiocarbocyanine dye Merocyanine 540 (MC540) was first analyzed by Fluorescence Correlation Spectroscopy (FCS) in different alcohol solutions. Similar isomerization kinetics could then also be monitored of MC540 in lipid vesicles, and the influence of lipid polarity, membrane curvature and cholesterol content was investigated. While no influence of membrane curvature and lipid polarity could be observed, a clear decrease in the isomerization rates could be observed with increasing cholesterol contents in the vesicle membranes. Finally, procedures to spatially map photo-induced and thermal isomerization rates on live cells by transient state (TRAST) imaging were established. Based on these procedures, MC540 isomerization was studied on live MCF7 cells, and TRAST images of the cells at different temperatures were found to reliably detect differences in the isomerization parameters. Our studies indicate that trans-cis isomerization is a useful parameter for probing membrane dynamics, and that the TRAST imaging technique can provide spatial maps of photo-induced isomerization as well as both photo-induced and thermal back-isomerization, resolving differences in local membrane micro-viscosity in live cells.

Triplet, photo-oxidized and other photo-induced, 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 occurring in 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 low duty cycles of excitation, the overall photo-degradation of the fluorescent molecules of the sample can be kept low.To verify the approach, the kinetics of the triplet and radical states of the dye Rhodamine110 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 on finite element analysis. The approach was found to accurately determine relative populations and dynamics of triplet and photo-oxidized states, overcoming passage time limitations seen in FCS measurements.The method circumvents the need of time resolution in the fluorescence detection, allowing simultaneous readout over the whole surface area subject to excitation. It can be applied over a broad range of concentrations and does not require a strong fluorescence brightness of the sample molecules. Given the sensitivity of the triplet and photo-oxidized 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.3