How to display data by color schemes compatible with red-green color perception deficiencies.

Visualization of data concerns most scientists. The use of color is required in order to display multidimensional information. In addition, color encoding a univariate image can improve the interpretation significantly. However up to 10% of the adult male population are affected by a red-green color perception deficiency which hampers the correct interpretation and appreciation of color encoded information. This work attempts to give guidelines on how to display a given dataset in a balanced manner. Three novel color maps are proposed providing readers with normal color perception a maximum of color contrast while being a good compromise for readers with color perception deficiencies.

Assessment of transferrin recycling by Triplet Lifetime Imaging in living cells.

An optical method is presented that allows the measurement of the triplet lifetime of a fluorescent molecule. This is a characteristic specific to each fluorophore. Based on differences in triplet lifetimes of two fluorescent species (autofluorescence versus label), this novel approach measures relative quantities of a transmembrane receptor and associated fluorescently labeled ligand during its recycling in living cells. Similarly to fluorescence-lifetime based methods, our approach is almost insensitive to photobleaching. A simple theory for unmixing two known triplet lifetimes is presented along with validation of the method by measurements of transferrin recycling in a model system based on chinese hamster ovarian cells (CHO). Transferrin is the delivery carrier for Fe(3+) to the cell.

Nonlinear correlation spectroscopy (NLCS).

We present a novel concept for optical spectroscopy called nonlinear correlation spectroscopy (NLCS). NLCS analyses coherent field fluctuations of the second and third harmonic light generated by diffusing nanoparticles. Particles based on noncentrosymmetric nonlinear materials such as KNbO(3) show a strong second as well as third harmonic response. The method and the theory are introduced and experimental NLCS results in fetal calf serum are presented showing the promising selectivity of this technique for measurement in complex biological environments.

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 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 photodegradation 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 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 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 for 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.

Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5).

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.