Philip Tinnefeld

Fluorescence Enhancement at Docking Sites of DNA-Directed Self-Assembled Nanoantennas

Building a Fluorescent Hotspot When two gold nanoparticles come close together, their overlapping plasmonic fields can create a region that acts as a nanoantenna that can enhance the fluorescent emission of a molecule. Acuna et al. (p. 506) used a surface-anchored DNA origami structure to assemble one or two gold nanoparticles next to a dye trapped within the structure. A > 100-fold enhancement in fluorescent emission was observed when the dye molecules were located in a 23-nm gap between two 100-nm gold nanoparticles. A DNA origami structure enhances the emission of a dye molecule by directing the proximate binding of gold nanoparticles. We introduce self-assembled nanoantennas to enhance the fluorescence intensity in a plasmonic hotspot of zeptoliter volume. The nanoantennas are prepared by attaching one or two gold nanoparticles (NPs) to DNA origami structures, which also incorporated docking sites for a single fluorescent dye next to one NP or in the gap between two NPs. We measured the dependence of the fluorescence enhancement on NP size and number and compare it to numerical simulations. A maximum of 117-fold fluorescence enhancement was obtained for a dye molecule positioned in the 23-nanometer gap between 100-nanometer gold NPs. Direct visualization of the binding and unbinding of short DNA strands, as well as the conformational dynamics of a DNA Holliday junction in the hotspot of the nanoantenna, show the compatibility with single-molecule assays.

Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami.

DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with <30 nm resolution. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and nonfluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by “blinking” with independently controllable on- and off-state lifetimes in both deoxygenated and oxygenated environments. This “continuous switching mode” is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.

Distance Dependence of Single-Fluorophore Quenching by Gold Nanoparticles Studied on DNA Origami

We study the distance-dependent quenching of fluorescence due to a metallic nanoparticle in proximity of a fluorophore. In our single-molecule measurements, we achieve excellent control over structure and stoichiometry by using self-assembled DNA structures (DNA origami) as a breadboard where both the fluorophore and the 10 nm metallic nanoparticle are positioned with nanometer precision. The single-molecule spectroscopy method employed here reports on the co-localization of particle and dye, while fluorescence lifetime imaging is used to directly obtain the correlation of intensity and fluorescence lifetime for varying particle to dye distances. Our data can be well explained by exact calculations that include dipole-dipole orientation and distances. Fitting with a more practical model for nanosurface energy transfer yields 10.4 nm as the characteristic distance of 50% energy transfer. The use of DNA nanotechnology together with minimal sample usage by attaching the particles to the DNA origami directly on the microscope coverslip paves the way for more complex experiments exploiting dye-nanoparticle interactions.

Superresolution microscopy on the basis of engineered dark states.

New concepts for superresolution fluorescence microscopy by subsequent localization of single molecules using photoswitchable or photoactivatable fluorophores are rapidly emerging and provide new ways to resolve structures beyond the diffraction limit. Here, we demonstrate that superresolution imaging can be carried out with practically every single-molecule compatible, synthetic fluorophore by controlling their emission properties. We prepare dark states by removing oxygen that extends the triplet state lifetime to several milliseconds. We further increase the duration of the off-states using electron transfer reactions to create radical ion states of severalfold longer lifetimes. Imaging single molecules, actin filaments, and microtubules in fixed cells as well as simulations demonstrate that the thus created dark states are sufficiently long for resolution of approximately 50 nm.

On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent

Recent advances in photobleaching and blinking prevention have aided the advancement of single-molecule and super-resolution fluorescence microscopy. However, a common mechanism of the action of antifading agents such as Trolox is still missing. In this communication we present evidence that Trolox acts in accordance with a mechanism that involves triplet quenching through electron transfer and subsequent recovery of the resulting radical ion by the complementary redox reaction. The required oxidant for this unifying mechanism based on a reducing and oxidizing system (ROXS) is formed via (photo-) reaction with molecular oxygen. We present evidence that this oxidized form is a quinone derivative of Trolox with strong oxidizing properties. These findings shed light on many contradicting results regarding the action of antifading agents and might lead to a common mechanistic understanding of photobleaching and its prevention. Finally, a recipe on the proper use of Trolox as an antifading agent is provided.

Make them blink: probes for super-resolution microscopy.

In recent years, a number of approaches have emerged that enable far-field fluorescence imaging beyond the diffraction limit of light, namely super-resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to spread into conventional biological laboratories. Nowadays these approaches can be divided into two categories, one based on targeted switching and readout, and the other based on stochastic switching and readout of the fluorescence information. The main prerequisite for a successful implementation of both categories is the ability to prepare the fluorescent emitters in two distinct states, a bright and a dark state. Herein, we provide an overview of recent developments in super-resolution microscopy techniques and outline the special requirements for the fluorescent probes used. In combination with the advances in understanding the photophysics and photochemistry of single fluorophores, we demonstrate how essentially any single-molecule compatible fluorophore can be used for super-resolution microscopy. We present examples for super-resolution microscopy with standard organic fluorophores, discuss factors that influence resolution and present approaches for calibration samples for super-resolution microscopes including AFM-based single-molecule assembly and DNA origami.

The 2015 super-resolution microscopy roadmap.

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio) physical and biomedical research, particularly with respect to ...

Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor-acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Förster radius R(0) could directly be determined from the distance dependence of energy transfer yielding R(0)=5.3±0.3 nm for the Cy3-Cy5 pair.

Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules

We demonstrate measurements of the efficiency of competing Förster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.