Alexander Kiel

Far-Field Nanoscopy with Reversible Chemical Reactions**

These methods separate overlapping point-spread functions of nearby objects in time by light-inducedswitching of the fluorescent probe between a bright (“on”)and a dark (“off”) state.This principle is used in a spatially defined manner inSTED microscopy to decrease the size of the point-spreadfunction by a donut-shaped depletion pulse.

Fluorescent probes and delivery methods for single-molecule experiments.

The recent explosion in papers utilising single-molecule experiments pushes the envelope further for increased spatial and temporal resolution. In order to achieve this, a combination of novel fluorescent probes and spectroscopy techniques are required. Herein, we provide an overview on our contribution to developments in the field of fluorescent probes along with a palette of alternative delivery methods for introducing the probes into living cells. We discuss probe requirements arising from the use of single-molecule spectroscopy methods and the customisation of probes that depends on the target molecule, the chemical state of the molecule as well as the distance and the type of interaction between sensor and ligand. We explain how Förster resonance energy transfer (FRET) and photon-induced electron transfer (PET) can increase the probe customisation. We also discuss additional requirements that arise when performing experiments in living cells like toxicity and cell permeability. Regarding the latter, we devote a special paragraph on the different ways to introduce the desired probe into the cell and how the different properties of each probe and cell type may require different delivery methods. We offer insights based on our experience working with a variety of single-molecule methods, fluorescent probes and delivery systems. Overall, we encompass the latest developments on probe design and delivery and illustrate that the wealth of information provided by single-molecule studies goes along with increased complexity.

Ensemble and single-molecule studies on fluorescence quenching in transition metal bipyridine-complexes.

Beyond their use in analytical chemistry fluorescent probes continuously gain importance because of recent applications of single-molecule fluorescence spectroscopy to monitor elementary reaction steps. In this context, we characterized quenching of a fluorescent probe by different metal ions with fluorescence spectroscopy in the bulk and at the single-molecule level. We apply a quantitative model to explain deviations from existing standard models for fluorescence quenching. The model is based on a reversible transition from a bright to a dim state upon binding of the metal ion. We use the model to estimate the stability constants of complexes with different metal ions and the change of the relative quantum yield of different reporter dye labels. We found ensemble data to agree widely with results from single-molecule experiments. Our data indicates a mechanism involving close molecular contact of dye and quenching moiety which we also found in molecular dynamics simulations. We close the manuscript with a discussion of possible mechanisms based on Förster distances and electrochemical potentials which renders photo-induced electron transfer to be more likely than Förster resonance energy transfer.

Single-Molecule Fluorescence Studies Reveal Long-Range Electron-Transfer Dynamics Through Double-Stranded DNA

Complex dynamics of combined long-range fluorescence resonance energy transfer (FRET) and short-range electron transfer (ET) processes in a single double-stranded DNA (dsDNA) molecule (see figure) reveals that FRET remains almost unaltered in the presence of ET. Present systems also demonstrate the potential for long-range ET studies through the base stack in single dsDNA molecules.

Observation of Unusual Molecular Diffusion Behaviour below the Lower Critical Solution Temperature of Water/2-Butoxyethanol Mixtures by using Fluorescence Correlation Spectroscopy

The effect of solute affinity on solute diffusion in binary liquids well below the lower critical solution temperature (LCST) was studied by using fluorescence correlation spectroscopy. We measured the hydrodynamic radii of a hydrophobic and an amphiphilic fluorescent dye under systematic variation of the relative molar fractions of water/2-butoxyethanol and, for comparison, of water/methanol mixtures, which do not show phase separation. We found that the apparent hydrodynamic radius of the hydrophobic dye almost doubled in water/2-butoxyethanol, whereas it remained largely unchanged for the amphiphilic dye and in water/methanol mixtures. Our results indicate that the translational diffusion of solutes is influenced by transient local solution structures, even at temperatures well below the LCST. We conclude that, even far below LCST, different solutes can experience different environments in binary liquid mixtures depending on both the solute and solvent properties, all of which impact their reactivity.