Radionuclides are used for sensitive and specific detection of small molecules in vivo and in vitro. Recently, radioluminescence microscopy extended their use to single-cell studies. Here we propose a new single-cell radioisotopic assay that improves throughput while adding sorting capabilities. The new method uses fluorescence-based sensor for revealing single-cell interactions with radioactive molecular markers. This study focuses on comparing two different experimental approaches. Several probes were tested and Dihydrorhodamine 123 was selected as the best compromise between sensitivity, brightness and stability. The sensor was incorporated either directly within the cell cytoplasm (direct approach), or it was coencapsulated with radiolabeled single-cells in oil-dispersed water droplets (droplet approach). Both approaches successfully activated the fluorescence signal following cellular uptake of 18F-fluorodeoxyglucose (FDG) and external Xrays exposure. The direct approach offered single-cell resolution and longtime stability ( > 20 hours), moreover it could discriminate FDG uptake at labelling concentration as low as 300 μCi/ml. In cells incubated with Dihydrorhodamine 123 after exposure to high radiation doses (8-16 Gy), the fluorescence signal was found to increase with the depletion of ROS quenchers. On the other side, the droplet approach required higher labelling concentrations (1.00 mCi/ml), and, at the current state of art, three cells per droplet are necessary to produce a fluorescent signal. This approach, however, is independent on cellular oxidative stress and, with further improvements, will be more suitable for studying heterogeneous populations. We anticipate this technology to pave the way for the analysis of single-cell interactions with radiomarkers by radiofluorogenic-activated single-cell sorting.
We present a new method for calibrating an optical-tweezer setup that is based on Bayesian inference<sup>1</sup>. This method employs an algorithm previously used to analyze the confined trajectories of receptors within lipid rafts<sup>2,3</sup>. The main advantages of this method are that it does not require input parameters and is insensitive to systematic errors like the drift of the setup. Additionally, it exploits a much larger amount of the information stored in the recorded bead trajectory than standard calibration approaches. The additional information can be used to detect deviations from the perfect harmonic potential or detect environmental influences on the bead. The algorithm infers the diffusion coefficient and the potential felt by a trapped bead, and only requires the bead trajectory as input. We demonstrate that this method outperforms the equipartition method and the power-spectrum method in input information required (bead radius and trajectory length) and in output accuracy. Furthermore, by inferring a higher order potential our method can reveal deviations from the assumed second-order potential. More generally, this method can also be used for magnetic-tweezer calibration.
Yttrium vanadate particles doped with europium are studied for their applications as biomolecule labels. Two parts of
our work will be presented. The first concerns the thermal treatment of particles incorporated in a silica matrix. After
annealing at 1000°C and redispersion in water by dissolution of the silica matrix, the structural and optical properties are
greatly improved: without any modification of size, the obtained nanoparticles appear as perfect single crystals of 33 nm
and have the same emission properties as the bulk material, with a quantum yield and emission lifetime increasing up to
40% and 0.8 ms. The second aspect concerns the detection of single nanoparticles and their emission properties as
compared to an ensemble of nanoparticles.