Multipararneter microscopy and spectroscopy for single-molecule analytics

The ability to monitor several parameters simultaneously from distinct individual fluorescent reporter molecules facilitates the disentanglement of complex and interacting systems and opens new perspectives in areas from basic science to biopharmaceutical technology. By combining annular illumination microscopy, time-correlated singlephoton counting, and multichannel detection, we were able to determine 14 independent parameters from one individual fluorophore. The whole set of parameters was deduced from the few properties of the fluorescence photons, i.e., arrival time, wavelength, and polarization. With this approach, the intensity, the polarization, and the spectral dynamics can be analyzed on a nanosecond time scale and the mean values can be monitored with submillisecond time resolution. Nanosecond spectral dynamics of single molecules has been observed, to the best of our knowledge, for the first time. From our experience, we can determine all parameters for more than 30% of the illuminated fluorophores in biological samples and for more than 80% in doped polymeric films.