Absolute quantum yield measurements of fluorescent proteins using a plasmonic nanocavity.

One of the key photophysical properties of fluorescent proteins that is most difficult to measure is the quantum yield. It describes how efficiently a fluorophore converts absorbed light into fluorescence. Its measurement using conventional methods become particularly problematic when it is unknown how many of the proposedly fluorescent molecules of a sample are indeed fluorescent (for example due to incomplete maturation, or the presence of photophysical dark states).

Time-resolved MIET measurements of blood platelet spreading and adhesion.

Human blood platelets are non-nucleated fragments of megakaryocytes and of high importance for early hemostasis. To form a blood clot, platelets adhere to the blood vessel wall, spread and attract other platelets. Despite the importance for biomedicine, the exact mechanism of platelet spreading and adhesion to surfaces remains elusive.

Dual-Color Metal-Induced Energy Transfer (MIET) Imaging for Three-Dimensional Reconstruction of Nuclear Envelope Architecture.

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and control transport of macromolecules between the two compartments. Recently, it has been shown that the axial distance between the inner nuclear membrane and the cytoplasmic side of the NPC can be measured using dual-color metal-induced energy transfer (MIET).

Three-dimensional single-molecule localization with nanometer accuracy using Metal-Induced Energy Transfer (MIET) imaging.

Our paper presents the first theoretical and experimental study using single-molecule Metal-Induced Energy Transfer (smMIET) for localizing single fluorescent molecules in three dimensions. Metal-Induced Energy Transfer describes the resonant energy transfer from the excited state of a fluorescent emitter to surface plasmons in a metal nanostructure. This energy transfer is strongly distance-dependent and can be used to localize an emitter along one dimension.

Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm.

Dual-color metal-induced and Förster resonance energy transfer for cell nanoscopy.

We report a novel method, dual-color axial nanometric localization by metal--induced energy transfer, and combine it with Förster resonance energy transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells. Our approach is based on fluorescence-lifetime-imaging microscopy and allows for precise determination of the three-dimensional architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell-matrix mechanics.

Three-Dimensional Reconstruction of Nuclear Envelope Architecture Using Dual-Color Metal-Induced Energy Transfer Imaging.

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs), which are embedded in the nuclear envelope, control transport of macromolecules between the two compartments. Here, using dual-color metal-induced energy transfer (MIET), we determine the axial distance between Lap2β and Nup358 as markers for the inner nuclear membrane and the cytoplasmic side of the NPC, respectively.

Quantum Yield Measurements of Fluorophores in Lipid Bilayers Using a Plasmonic Nanocavity.

Precise knowledge of the quantum yield is important for many fluorescence-spectroscopic techniques, for example, for Förster resonance energy transfer. However, to measure it for emitters in a complex environment and at low concentrations is far from being trivial. Using a plasmonic nanocavity, we measure the absolute quantum yield value of lipid-conjugated dyes incorporated into a supported lipid bilayer.

Photoactivation of Luminescent Centers in Single SiO2 Nanoparticles.

Photobleaching of fluorophores is one of the key problems in fluorescence microscopy. Overcoming the limitation of the maximum number of photons, which can be detected from a single emitter, would allow one to enhance the signal-to-noise ratio and thus the temporal and spatial resolution in fluorescence imaging. It would be a breakthrough for many applications of fluorescence spectroscopy, which are unachievable up to now.

Dead-time correction of fluorescence lifetime measurements and fluorescence lifetime imaging.

We present a comprehensive theory of dead-time effects on Time-Correlated Single Photon Counting (TCSPC) as used for fluorescence lifetime measurements, and develop a correction algorithm to remove these artifacts. We apply this algorithm to fluorescence lifetime measurements as well as to Fluorescence Lifetime Imaging Microscopy (FLIM), where rapid data acquisition is necessarily connected with high count rates. There, dead-time effects cannot be neglected, and lead to distortions in the observed lifetime image.