Browsing by Author "Dedecker, Peter"

Today’s miniature devices and biological systems share the fact that their dynamics and properties cannot be understood in terms of macroscopic concepts, but require a thorough understanding of the nanoscale structuring. This structuring is often very heterogeneous, both in time and in space, and is difficult or impossible to resolve with traditional approaches. In this Progress Report, we willdiscuss how single-molecule microscopy—and defocused wide- field imaging in particular—can be used to shed light on these phenomena.

"Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coil colonies grown at 37 degrees C and more than 4-fold higher fluorescence when expressed in photoswitching HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of similar to 70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off'-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.

The interactions between single molecules and three-dimensional donut modes in fluorescence microscopy are discussed based on the vector diffraction theory of light. We find that the use of donut modes generated from a linearly polarized laser beam can yield information about the orientation of immobilized single molecules, allowing for their use in orientational imaging. While fairly insensitive over a range of orientations, this technique is seen to be very sensitive for the subset of orientations where the transition dipole of the molecule is oriented close to the optical axis of the microscope and perpendicular to the input polarization. In a second part of the paper we discuss the impact of the molecular orientation on the resolution improvement in STED microscopy. We find that, even for circularly polarized excitation light, the expected resolution improvement depends on the orientation of the molecule relative to the optical axis of the microscope.

We report a rationale for identifying superior dyes for stimulated-emission depletion (STED) microscopy. We compared the dyes pPDI and pTDI, which displayed excellent photostability in single-molecule spectroscopy. Surprisingly, their photostability and performance in STED microscopy differed significantly. While single pTDI molecules could be visualized with excellent resolution (35 nm), pPDI molecules bleached rapidly under similar conditions. Femtosecond transient absorption measurements proved that the overlap between the stimulated-emission band and the excited-state absorption band is the main reason for the observed difference. Thus, assessment of the excited-state absorption band provides a rational means of dye selection and determination of the optimal wavelength for STED.