E.M.H.P. van Dijk

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2–1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.

On the role of electromagnetic boundary conditions in single molecule fluorescence lifetime studies of dyes embedded in thin films

Single molecule fluorescence lifetime studies are generally performed in thin polymer films, where the influence of the interface on the behaviour of fluorescing molecules is not negligible. In order to describe this influence, we investigate annealed films of different thickness. We show that the distribution of fluorescence lifetimes of the embedded dyes is shifted to lower values as the thickness of the film increases. We explain this shift by simple electromagnetic arguments related to the boundary conditions at the interfaces of the polymer film with air and glass, respectively. The conclusion is that extreme care must be taken in order to interpret single molecule data with respect to the true chemical nature of the phenomena.

Single-molecule pump-probe experiments reveal variations in ultrafast energy redistribution

Single-molecule pump probe (SM2P) is a novel, fluorescence-based technique that allows the study of ultrafast processes on the single-molecule level. Exploiting SM2P we have observed large variations (from 1 ps to below 100 fs) in the energy redistribution times of chemically identical molecules in the same sample. Embedding the molecules in a different matrix or changing the excitation wavelength does not lead to significant changes in the average redistribution time. However, chemically different molecules exhibit different characteristic redistribution times. We therefore conclude that the process measured with the SM2P technique is dominated by intramolecular energy redistribution and not intermolecular transfer to the surrounding matrix. The matrix though is responsible for inducing conformational changes in the molecule, which affect the coupling between electronic and vibrational modes. These conformational changes are the main origin of the observed broad distribution of redistribution times.

Coherent imaging of local fields in photonic crystals

Summary form only given. In the past years, much research has been focused on so called photonic crystals. Different ways of fabricating and simulating these structures have been introduced. Characterization is performed mostly with black box experiments, where reflected or transmitted light is detected. We demonstrate a different approach, which allows us to take a direct look inside such structures. With a heterodyne interferometric photon scanning tunneling microscope (PSTM), we are able to visualize not only the amplitude of the local optical field, but also the phase information of light propagating inside the crystal. Heterodyne interferometric photon scanning tunneling microscopy gives detailed insight in reflected and transmitted waves as they develop through periodic structures. Ultimately, this method will allow us to visualize the opening of a stop gap in one or two-dimensional photonic crystals.