Thorben Cordes

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.

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 ...

Light-triggered β-hairpin folding and unfolding

A light-switchable peptide is transformed with ultrashort pulses from a β-hairpin to an unfolded hydrophobic cluster and vice versa. The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining density functional theory with molecular mechanics is used to interpret the absorption transients. Illumination of the β-hairpin state triggers an unfolding reaction that visits several intermediates and reaches the unfolded state within a few nanoseconds. In this unfolding reaction to the equilibrium hydrophobic cluster conformation, the system does not meet significant barriers on the free-energy surface. The reverse folding process takes much longer because it occurs on the time scale of 30 μs. The folded state has a defined structure, and its formation requires an extended search for the correct hydrogen-bond pattern of the β-strand.

The Hammett Relationship and Reactions in the Excited Electronic State : Hemithioindigo Z/E-Photoisomerization

The photochemical reaction dynamics of a set of photochromic compounds based on thioindigo and stilbene molecular parts (hemithioindigos, HTI) are presented. Photochemical Z/E isomerization around the central double bond occurs with time constants of 216 ps (Z --> E) and 10 ps (E --> Z) for a 5-methyl-hemithioindigo. Chemical substitution on the stilbene moiety causes unusually strong changes in the reaction rate. Electron-donating substituents in the position para to the central double bond (e.g., para-methoxy) strongly accelerate the reaction, while the reaction is drastically slowed by electron-withdrawing groups in this position (e.g., para-nitrile). We correlate the experimental data of seven HTI-compounds in a quantitative manner using the Hammett equation and present a qualitative explanation for the application of ground-state Hammett constants to describe the photoisomerization reaction.

Single-molecule photophysics of oxazines on DNA and its application in a FRET switch

The role and interplay of triplet states and radical ion states in single-molecule fluorescence spectroscopy has recently been elaborated providing us with new insights into the photophysics and photobleaching pathways of fluorescent dyes. Adjustment of fluorophore redox properties in combination with specific redox properties of the environment, i.e. addition of reducing and oxidizing agents, allows control of the emission properties: it has become possible to suppress blinking and to also induce blinking in single-molecule fluorescence transient by selectively opening and closing specific excited state pathways. Induced blinking is, for example, of interest for super-resolution fluorescence microscopy based on the subsequent localization of single fluorophores. For oxazines this control even allowed the separation of the influence of reducing and oxidizing agents, enabling switching the fluorescence of single fluorophores. Here, we study the factors that contribute to the kinetics of the photophysical pathways more closely with a focus on the photophysics of the oxazine ATTO655 labeled to DNA. Our data show that the oxazine ATTO655 interacts with DNA, shielding it efficiently from reagents in solution. Besides redox reactions, the pH also influences the blinking kinetics and especially the off-times. Moreover, we present the extension of ATTO655 as a single-molecule redox sensor to a ratiometric fluorescence-resonance-energy-transfer based sensor. Therefore, we designed FRET probes that showed the highest possible contrast of FRET changes and demonstrate reversible FRET-switching of Cy3B-ATTO655 DNA constructs.

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs.

Molecular Driving Forces for Z/E Isomerization Mediated by Heteroatoms: The Example Hemithioindigo

A combined experimental and theoretical investigation of photoinduced Z/E isomerizations is presented. Unsubstituted Hemithioindigo is selected as a representative minimal model to unravel the reaction mechanism in the presence of heteroatoms on an atomic level. Time-resolved spectroscopy reveals multiexponential reaction dynamics on the few picoseconds time scale, which are interpreted by quantum chemical calculations at the CASSCF/CASPT2 level of theory. Detailed insight into the processes governing the ultrafast decay from the first excited state, mediated by a number of conical intersections, is provided. Charge separation and charge balance recovery on the reaction pathway play the leading role and are controlled by the electron-donating or -withdrawing character of the heteroatoms. The electronic and geometric structures of the individual minimum energy conical intersections governing the reaction are rationalized, and an extended energetically low lying conical intersection seam is extracted. Comparison to the experimental results permits linking the observed time constants to molecular intermediates and pathways. An explanation is provided for the pronounced differences of Z → E and the E → Z photoreactions upon excitation to the first excited singlet state.

Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ

The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.