Photon Antibunching Reveals Static and Dynamic Quenching Interaction of Tryptophan with Atto-655

Fluorescence correlation spectroscopy (FCS) of photoinduced electron transfer (PET) between the dye Atto-655 and the amino acid tryptophan has been extensively used for studying fast conformational dynamics of small disordered peptides and proteins. However, a precise understanding of the quenching mechanism and its exact rates that would explain ensemble as well as single-molecule spectroscopy results is still lacking. In this contribution, a general unified model for intermolecular PET between Atto-655 and tryptophan is developed, which involves ground-state complex formation, quenching sphere of action, and dynamic quenching at the single-molecule level. We present measurements of fluorescence antibunching, fluorescence lifetime, and steady-state fluorescence intensity and absorbance and demonstrate that our model is capable to describe all results in a global and coherent manner

Photon Antibunching in a Cyclic Chemical Reaction Scheme

The direct observation of chemical reactions on the single-molecule level is an ultimate goal in single-molecule chemistry, which also includes kinetic analyses. To analyze the lifetime of reaction intermediates, very sophisticated excitation schemes are often required. Here we focus on the kinetic analysis of the ground-state proton transfer within the photocycle of a photoacid. In detail, we demonstrate the determination of the bimolecular rate constant of this process with nanosecond resolution. The procedure relies on the exploration of a purely quantum-optical effect, namely, photon antibunching, and thus on evaluating interphoton arrival times to extract the reaction rate constant

Quantifying Microsecond Transition Times Using Fluorescence Lifetime Correlation Spectroscopy

Many complex luminescent emitters such as fluorescent proteins exhibit multiple emitting states that result in rapid fluctuations of their excited-state lifetime. Here, we apply fluorescence lifetime correlation spectroscopy (FLCS) to resolve the photophysical state dynamics of the prototypical fluorescence protein enhanced green fluorescent protein (EGFP). We quantify the microsecond transition rates between its two fluorescent states, which have otherwise highly overlapping emission spectra. We relate these transitions to a room-temperature angstrom-scale rotational isomerism of an amino acid next to its fluorescent center. With this study, we demonstrate the power of FLCS for studying the rapid transition dynamics of a broad range of light-emitting systems with complex multistate photophysics, which cannot be easily done by other methods

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. So far, the only approach for diminishing the effect of photobleaching has been to enhance the photostability of an emitter. Here, we present a fundamentally new solution for increasing the number of photons emitted by a fluorophore. We show that, by exposing a single SiO₂ nanoparticle to UV illumination, one can create new luminescent centers within this particle. By analogy with nanodiamonds, SiO₂ nanoparticles can possess luminescent defects in their regular SiO₂ structure. However, due to the much weaker chemical bonds, it is possible to generate new defects in SiO₂ nanostructures using UV light. This allows for the reactivation of the nanoparticle’s fluorescence after its photobleaching

Excitation Intensity-Dependent Quantum Yield of Semiconductor Nanocrystals

One of the key phenomena that determine the fluorescence of nanocrystals is the nonradiative Auger-Meitner recombination of excitons. This nonradiative rate affects the nanocrystals' fluorescence intensity, excited state lifetime, and quantum yield. Whereas most of the above properties can be directly measured, the quantum yield is the most difficult to assess. Here we place semiconductor nanocrystals inside a tunable plasmonic nanocavity with subwavelength spacing and modulate their radiative de-excitation rate by changing the cavity size. This allows us to determine absolute values of their fluorescence quantum yield under specific excitation conditions. Moreover, as expected considering the enhanced Auger-Meitner rate for higher multiple excited states, increasing the excitation rate reduces the quantum yield of the nanocrystals

DNA-Based Optical Sensors for Forces in Cytoskeletal Networks

Mechanical forces are relevant for many biological processes, from wound healing and tumor formation to cell migration and differentiation. Cytoskeletal actin is largely responsible for responding to forces and transmitting them in cells, while also maintaining cell shape and integrity. Here, we describe a FRET-based hybrid DNA-protein tension sensor that is designed to sample transient forces in actin networks by employing two actin-binding motifs with a fast off-rate attached to a central DNA hairpin loop. Such a sensor will be useful to monitor rapidly changing stresses in the cell cytoskeleton. We use fluorescence lifetime imaging to determine the FRET efficiency and thereby the conformational state of the sensor, which makes the measurement robust against intensity variations. We demonstrate the applicability of the sensor by confocal microscopy and by monitoring crosslinking activity in in vitro actin networks by bulk rheology

Charge-Driven Fluorescence Blinking in Carbon Nanodots

This study focuses on the mechanism of fluorescence blinking of single carbon nanodots, which is one of their key but less understood properties. The results of our single-particle fluorescence study show that the mechanism of carbon nanodots blinking has remarkable similarities with that of semiconductor quantum dots. In particular, the temporal behavior of carbon nanodot blinking follows a power law both at room and at cryogenic temperatures. Our experimental data suggest that static quenching via Dexter-type electron transfer between surface groups of a nanoparticle plays a major role in the transition of carbon nanodots to off or gray states, whereas the transition back to on states is governed by an electron tunneling from the particle’s core. These findings advance our understanding of the complex mechanism of carbon nanodots emission, which is one of the key steps for their application in fluorescence imaging

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. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups