Dirk-Peter Herten

Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states τoff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of ∼0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

Structure, dynamics, and energetics of siRNA-cationic vector complexation:a molecular dynamics study

The design and synthesis of safe and efficient nonviral vectors for gene delivery has attracted significant attention in recent years. Previous experiments have revealed that the charge density of a polycation (the carrier) plays a crucial role in complexation and the release of the gene from the complex in the cytosol. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with six cationic carrier systems of varying charge and surface topology. The simulations reveal detailed molecular-level pictures of the structures and dynamics of the RNA-polycation complexes. Estimates for the binding free energy indicate that electrostatic contributions are dominant followed by van der Waals interactions. The binding free energy between the 8(+)polymers and the RNA is found to be larger than that of the 4(+)polymers, in general agreement with previously published data. Because reliable binding free energies provide an effective index of the ability of the polycationic carrier to bind the nucleic acid and also carry implications for the process of gene release within the cytosol, these novel simulations have the potential to provide us with a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance the rational design of nonviral gene delivery systems.

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.

Quenched Substrates for Live-Cell Labeling of SNAP-Tagged Fusion Proteins with Improved Fluorescent Background

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

The Shape of Protein Crowders is a Major Determinant of Protein Diffusion

As a model for understanding how molecular crowding influences diffusion and transport of proteins in cellular environments, we combined experimental and theoretical approaches to study the diffusion of proteins in highly concentrated protein solutions. Bovine serum albumin and γ-Globulin were chosen as molecular crowders and as tracers. These two proteins are representatives of the main types of plasma protein and have different shapes and sizes. Solutions consisting of one or both proteins were studied. The self-diffusion coefficients of the fluorescently labeled tracer proteins were measured by means of fluorescence correlation spectroscopy at a total protein concentration of up to 400 g/L. γ-Globulin is found to have a stronger influence as a crowder on the tracer self-diffusion coefficient than Bovine serum albumin. Brownian dynamics simulations show that the excluded volume and the shape of the crowding protein have a significantly stronger influence on translational and rotational diffusion coefficients, as well as transient oligomerization, than hydrodynamic or direct interactions. Anomalous subdiffusion, which is not observed at the experimental fluorescence correlation spectroscopy timescales (>100 μs), appears only at very short timescales (<1 μs) in the simulations due to steric effects of the proteins. We envision that the combined experimental and computational approach employed here can be developed to unravel the different biophysical contributions to protein motion and interaction in cellular environments by systematically varying protein properties such as molecular weight, size, shape, and electrostatic interactions.

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.

A Conformational Change in the α-subunit of Coatomer Induced by Ligand Binding to γ-COP Revealed by Single-pair FRET

Formation of transport vesicles involves polymerization of cytoplasmic coat proteins (COP). In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of γ‐COP by the Golgi membrane protein p23 induces a conformational change that causes polymerization of the complex. Using single‐pair fluorescence resonance energy transfer, we find that this conformational change takes place in individual coatomer complexes, independent of each other, and that the conformational rearrangement induced in γ‐COP is transmitted within the complex to its α‐subunit. We suggest that capture of membrane protein machinery triggers cage formation in the COPI system.

Counting fluorescent dye molecules on DNA origami by means of photon statistics.

Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single-molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1-20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well-defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non-invasively within 150 ms. The method might become a quantifying add-on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT-type microscopy.

Fluorescence Quenching of Quantum Dots by DNA Nucleotides and Amino Acids

Quantum dots found widespread application in the biosciences as bright and highly photo-stable fluorescent probes, i.e. for single-particle tracking. In this work we used ensemble spectroscopy and single-molecule techniques to study the quenching of quantum dots by various biochemical compounds that are usually present in living cells and might thus influence the experiments. We found not only nucleotides such as cytosine, guanine, and thymine can significantly influence the fluorescence emission of CdSe and CdTe quantum dots, but also amino acids, like asparagine and tryptophan. Bulk studies on fluorescence quenching indicated a static quenching mechanism. Interestingly, we could also show by single-molecule fluorescence spectroscopy that quenching of the quantum dots can be irreversible, suggesting either a redox-reaction between quantum dot and quencher or strong binding of the quencher to the surface of the bio-conjugated quantum dots.

An extended scheme for counting fluorescent molecules by photon-antibunching

Acquisition of quantitative information from microscopic biological samples is highly desirable in the context of the emerging field of systems biology. We derive a statistical approach to estimate the number of fluorescent molecules in the observation volume based on a confocal microscope for single-molecule detection. The method employs ps-pulsed laser sources for excitation and time-correlated single-photon counting with 4 avalanche photon diodes (APDs) for detection of individual photons. The feasibility for estimating the number of molecules is shown based on simultaneous emission and detection of multiple photons (photon-antibunching) under realistic experimental conditions. In theory, it should be possible to estimate the number of molecules with errors of less than 1% by using novel photo-stabilizing agents. The proposed method puts into perspective its application for high-resolution microscopy without the need for photo-switching or photo-activation of fluorescence dyes.