Super-Resolution dSTORM Imaging of Human Galectin-1 Interacting with Neuroblastoma Cells

Galectins are a family of carbohydrate-binding proteins with an affinity for β-galactosides. They share a core sequence consisting of 130 amino acids, and the β-sandwich fold. Human Galectin-1 (hGal-1) is a well studied representative of prototype galectins, non-covalently linked homodimers with two specific carbohydrate recognition domains (CRD). It is differentially expressed by various normal and pathological tissues and is involved in intra- and extracellular processes like cell adhesion, formation of galectin-glycoprotein lattices, signal transduction and regulating immune responses, inflammation, allergies, and host-pathogen interactions. Furthermore oxidized galectins are associated with the regeneration of the central nervous system after injury.

We use direct stochastic optical reconstruction microscopy (dSTORM) to study the spatial organization of hGal-1 interacting with glycans like ganglioside GM1 presented on the membrane of human SK-N-MC neuroblastoma cells. Using the photoswitchable fluorophore ALEXA 647 as specific galectin marker, we employ fluorescence on/off switching with standard widefield microscopy and spot analysis of single molecules in order to resolve clustering, localization, and cross-linking of galectins on the cell surface with a spatial resolution of less than 50 nm.

We study spatial organization and its dependence on galectin concentration and oxidation state, as well as inhibition of the specific recognition. 

Gaining More Information with CW-STED Microscopy by Time-gated Detection

COLOR CODED OPTICAL NANO-SECTIONING (COCOS) REVEALS FOCAL ADHESION DYNAMICS

We present a fast imaging technique for performing high contrast microscopy and optical nanosectioning in the sub-100 nm vicinity of a biocompatible metal/dielectric coated substrate. The technique makes use of the distance dependent interactions of excited fluorophors with surface plasmons and polaritons of the coating: The fluorophor's emission spectrum changes with its distance from the substrate and thus allows inferring the molecule-substrate distance with a 50 times higher precision than the diffraction limit.

Qualitatively, a COCOS image has a similar appearance to a typical Total Internal Reflection Fluorescence Microscopy (TIRFM) image however contains spectral information that provides an effective axial resolution in the nanometer over the range of the evanescent (surface plasmon polariton) field.

The technique is demonstrated for tracking the axial dynamics of GFP-Paxillin at the adhesion sites of migrating fibroblasts with approximately 10 nm axial localization precision. Our results for the average separation of the protein from the intercellular matrix are consistent with suggestions from previous studies and show periodic fluctuations in the axial position previously inaccessible. The proposed technique is likely to find a wide range of applications due to its practical simplicity and compatibility with established fluorescence methods. 

Superresolution Optical Fluctuation Imaging: Towards Rapid, Multicolor and Three-dimensional Superresolution Fluorescence Microscopy

Superresolution Optical Fluctuation Imaging (SOFI) is one of most recent additions to the field of superresolution techniques. Besides superresolution SOFI provides inherent background elimination and 3D sectioning on a conventional widefield microscope. In comparison to localization-based superresolution methods, such as Stochastic Optical Reconstruction Microscopy (STORM) or Photo-Activated Localization Microscopy (PALM), SOFI can be performed on lower signal to noise ratios, on higher concentrations of fluorophores and SOFI requires less control over the ‘on’ and ‘off’ states of the fluorophores. We will present recent progress and new applications regarding our technology towards rapid, multicolor and three-dimensional superresolution microscopy.

Next Generation TCSPC Detection

More than 20 years ago single photon counting based single molecule detection started with cooled photomultiplier tubes (PMT). Already 5 years later the single photon avalanche photodiode (SPAD) starts to replace the PMT especially due to its higher detection efficiency and became the workhorse in confocal single molecule microscopy. In the last decade step by step SPAD technology improvements enabled to meet most of the requirements of modern Time-Correlated Single Photon Counting (TCSPC) for ultrasensitive detection.

Recently a new detector module became available which allows to merge the still remaining timing performance advantages of the PMT with the photon processing efficiency of the SPAD. We incorporated this novel hybrid photomultiplier detector module (PMA hybrid) in a cooled, self-contained housing and will present its outstanding performance like narrow and ultrastable IRF, low darkcounts and almost negligible afterpulsing. Our experiments will demonstrate the advantages for several applications like FLIM and FCS.

Beneath this new single point detector we will show recent results for different types of TCSPC detector arrays for highly parallel and / or spectrally resolved detection together with high throughput counting electronics and new approaches towards a robust and efficient multidimensional data analysis. 

Modifying the fluorescence properties and determining the quantum yield of a single molecule with a tunable optical subwavelength microcavity

We present experimental and theoretical results on changing the fluorescence emission spectrum of a single molecule by embedding it within a tunable planar microcavity with subwavelength spacing [1]. The cavity length is changed with nanometer precision by using a piezoelectric actuator. By varying its length, the local mode structure of the electromagnetic field is changed together with the radiative coupling of the emitting molecule to the field. Because mode structure and coupling are both frequency dependent, this leads to a renormalization of the emission spectrum of the molecule. We develop a theoretical model for these spectral changes and find excellent agreement between theoretical prediction and experimental results.

We also demonstrate controlled modulation of the radiative transition rate of a single molecule, which is measured by monitoring its fluorescence lifetime [2]. By comparing the experimental data with a theoretical model, we extract both the pure radiative transition rate as well as the quantum yield of individual molecules. We observe a broad scattering of quantum yield values from molecule to molecule, which reflects the strong variation of the local interaction of the observed molecules with their host environment.

[1] Chizhik, A. I.; Schleifenbaum, F.; Gutbrod, R.; Chizhik, A. M.; Khoptyar, D.; Meixner, A. J.; Enderlein, J. Phys. Rev. Lett. 2009, 102, 073002-1.

[2] Chizhik, A. I.; Chizhik, A. M.; Khoptyar, D.; Bär, S.; Meixner, A. J.; Enderlein, J. Nano Lett. 2011, 11, 1700-1703. 

Femtosecond Dynamics of Single Chromophores and Single Light-Harvesting Complexes at Room Temperature

Ultrafast excitation-energy transfer is at the heart of both natural and artificial light-harvesting, and plays a key role in the initial steps of photosynthesis as well as in photovoltaic applications of organic functional materials. However, a detailed understanding of energy-transfer processes on nanoscopic scales is hampered because molecular systems are often strongly heterogeneous with highly disordered environments and current ultrafast techniques intrinsically average over large ensembles.

Here, we present our recent advances in combining femtosecond pulse-shaping techniques with single-molecule detection schemes at room temperature [1-3]. We address electronic coherences in a model system, individual terrylenediimide (TDI) molecules embedded in a polymer matrix, by phase-controlled double-pulse excitation. The coherence decay times are found to be broadly distributed around 60 fs reflecting interactions with highly disordered local surroundings. Despite these rapid dephasing processes we are able to induce Rabi-oscillations as well as to fully control the coherent superposition state by manipulating the inter-pulse phase difference. Finally, we discuss how these techniques can be extended to multichromophoric molecular systems. We present preliminary results on the ultrafast intra-complex dynamics of electronic excitations in individual light-harvesting complexes of purple bacteria. 

[1] R. Hildner, D. Brinks, and N. F. van Hulst, Nature Phys. 7, 172 - 177 (2011).

[2] R. Hildner, D. Brinks, F. D. Stefani, and N. F. van Hulst, Phys. Chem. Chem. Phys. 13, 1888 - 1894 (2011).

[3] D. Brinks, F. D. Stefani, F. Kulzer, R. Hildner, T. H. Taminiau, Y. Avlasevich, K. Müllen, and N. F. van Hulst, Nature 465, 905 - 908 (2010). 

Single-molecule fluorescence in bacterial cells

Most single-molecule fluorescence experiments are performed in vitro, using tightly controlled conditions and well-defined concentrations of a limited number of interacting components. However, in order to understand biological mechanisms as they occur in vivo while taking advantage of the extra information provided by single-molecule detection, there is a growing need for performing single-molecule fluorescence measurements in cellular contexts, and in particular in living cells.

Towards this goal, we have been developing physical methods for delivering fluorescent biomolecules in bacteria and observing single-molecule fluorescence and single-molecule FRET in the bacterial cytoplasm; we use both confocal and wide-field imaging approaches for detection. We have also been using localization-based super-resolution imaging approaches to study the subcellular localization, mobility and abundance of DNA binding proteins in bacterial cells. These approaches are general and should be useful for studying many processes in gene replication, expression, and maintenance. 

Single-molecule fluorescence studies of site-specific DNA recombination

Site-specific DNA recombination is crucial for bacterial cell division and other biological processes. We study Xer site-specific recombination, during which two separate DNA sites on the bacterial chromosome recombine through four Xer proteins activated by the ATP-dependant DNA translocase FtsK; recombination proceeds via a Holliday junction intermediate, and strand exchanges between two DNA duplexes eventually separate chromosome dimers. Despite substantial genetic and biochemical work, the mechanism of the reaction and activation of the Xer-DNA complex by FtsK remain unclear.

Using single-molecule FRET we observe, for the first time, the formation of the Xer-mediated synaptic complex, a key intermediate prior to the recombination reaction. Formation of a synaptic complex between the ends of a long surface-immobilized DNA molecule can be observed via FRET. Moreover, a novel implementation of alternating laser excitation allows observation of the fluorescence intensity and point-spread-function width of the FRET acceptor, which in turn determines the lateral and axial freedom (and thus conformational state) of the DNA strand. Our results report directly on the complex stability and uncover interesting dissimilarities to crystal structures for the closely related Cre recombinase.

The generality of our assay makes it attractive for studying other processes that involve long-range DNA looping. 

Photon statistics of single donor-acceptor pair fluorescence as a new method for studying Fӧrster Resonance Energy Transfer (FRET)

Fluorescence of a single molecule excited by CW-laser light always fluctuates, because time instants of absorption and emission of photons are random. Therefore, number N of fluorescence photons counted on equal time intervals T will be distributed with the probability w(N,T). Photon distribution function w(N,T) (photon statistics) of a single polymer molecule has already been used for studying conformational changes in a polymer molecule [1,2]. Operating fluctuations of fluorescence intensity gives an advantage as compared to the usage of mere fluorescence intensity, because the intensity I=/T=T-1∑(Nw(N,T)) is only the first moment of the photon distribution function.

In this report we show the way how the single donor-acceptor pair fluorescence fluctuations, i.e. the distributions in donor/acceptor fluorescence, can be used for finding the exact value F of FRET rate. We show that our method of studying FRET has an advantage as compared to the equation E=(F/D)/(1+F/D) for FRET efficiency that is in common use. Here D is the rate of donor fluorescence.

[1] I.S.Osad'ko, V.V.Fedyanin, J.Chem.Phys.130, 064904 (2009)

[2] I.S.Osad’ko, V.V.Fedyanin, Phys.Rev.A (accepted for publication)

Protein Folding Dynamics from Single Molecule Spectroscopy

We use single molecule Förster resonance energy transfer (FRET) in combination with other biophysical methods to investigate the structure and dynamics of proteins, especially in non-native conformations. We obtain quantitative distance information from transfer efficiencies and fluorescence lifetimes, and combine it with dynamic information from correlation spectroscopy on time scales from nanoseconds to minutes. This approach allows us to determine structural and dynamic properties of proteins under conditions that have been largely inaccessible to classical structural biology methods. Examples include unfolded and intrinsically disordered proteins, proteins interacting with molecular chaperones, and transiently populated misfolded states. 

Ion-specific effects on α-helices

Salt-specific effects on protein structure remain controversial despite decades of research. Recent efforts using molecular dynamic (MD) simulations suggest a direct interaction between ions and the protein backbone [1]. Here, we combine accurate single-molecule multiparameter FRET experiments, CD and explicit-water MD to investigate salt-specific effects on short charged α-helices.

Using CD and MD, we explored the influence of five salts on the structure of a positively charged α-helix. Surprisingly, NaClO4behaved similar to KF in CD measurements, contradictory to what is observed in the MD simulations and at the same time expected from its ranking in the Hofmeister series. To resolve this contradiction we carried out spFRET experiments under varying salt concentrations.  With the help of photon distribution analysis, not only the average end to end distance of the protein could be tracked but also the width of the FRET histogram was used to investigate the flexibility of the peptide.

Addition of KCl allowed the peptide to adopt an α-helical conformation whereas GndCl denatured by swelling the chain. NaClO4 condenses the chain into a rapidly fluctuating collapsed state. With the help of CD, MD and spFRET we could show that two denaturing ions (Gnd, ClO4-) work in two  different mechanisms

Joachim Dzubiella, J. Am. Chem. Soc., 130 (42), pp 14000–1400 (2008)

Quantitative extraction of structural and dynamic properties in the protein hGBP-1 by single molecule FRET, fluorescence correlation spectroscopy and ensemble fluorescence measurements

Results on ensemble lifetime measurements, photon distribution analysis (PDA), dynamic-PDA [2] and FCS on measurements in the of the human immuno-active GTPase hGBP-1 are presented. hGBP1 basically consists of three domains: LG-, middle-, and an helical-domain. First FRET-measurements from the LG-domain to the helix α12,13 revealed dynamical properties by several different fluorescence based techniques (FCS, dynamic PDA, lifetime shift in 2D-histograms). To interpret the measurements on the FRET-samples in a quantitative manner the lifetimes of the donor- and acceptor-only samples were considered in the data analysis to account for non-FRET related donor quenching and to estimate the lower limit of the peak-width in PDA.

To describe the measurements in terms of quantitative structural/dynamical protein properties further double mutants from the LG to the middle domain, along the axis of the helix α12,13 and from the middle domain to the helix α12,13 were measured (see figure). The measurements from LG- to middle-domain and along the helix reveled that dynamics is mainly associated to movement of helix α12,13 with respect to the middle/LG-domain. A structural dynamical model of the protein is derived using experimental FRET-constraints in conjuncture with trilateration (FRET positioning / FPS) and accessible volume calculations to describe the fluorophore position distributions. Dynamical properties are obtained by fluorescence crosscorrelation (green/red) lifetime filtered species crosscorrelation and dynamic PDA. 

[1] Sindbert S, Kalinin S, Kienzler A, Clima L, Bannwarth W, Nguyen H, Appel B, Müller S, Seidel CA, J. Am. Chem. Soc., 133(8), 2463–2480 (2011)

[2] Kalinin S, Valeri A, Antonik M, Felekyan S, Seidel CA, J. Phys. Chem. B , 114(23), 7983-95 (2010)

Nonlinear Correlation Spectroscopy (NLCS)

We present nonlinear correlation spectroscopy (NLCS) of diffusing and flowing nanoparticles. NLCS is a method closely related to fluorescence correlation spectroscopy (FCS), however instead of fluorescence intensity fluctuations, NLCS analyses coherent field fluctuations of the second and third harmonic light generated by nanoparticles passing through the focal field of a pulsed femtosecond laser. In bulk material, the third harmonic contribution vanishes due to the destructive interference of the third harmonic generation in front and behind the focal field (Guoy phase shift). On the other hand, nanoparticles with dimensions below the wavelength can generate strong higher harmonic signals. In particular, particles based on non-centrosymmetric non-linear materials such as KNbO3 or BaTiO3 show a strong second and third harmonic signal.

We show NLCS results of polystyrene spheres (PS), gold nanoparticles as well as KNbO3 particles with a detailed analysis of the forward and backward scattering contributions. 

Size and shape determination of macromolecules by fluorescence correlation spectroscopy

Size and shape of biomolecules is often closely related to their function: Molecular interactions or biochemical reactions can change one or both of them. However, detailed structural information under native conditions is usually challenging to measure. Although NMR is the method that comes to mind first in this relation, it requires copious amounts of sample and requires expensive and labor-intensive technology. Similar to NMR, light scattering techniques require large sample concentrations and become increasingly difficult to apply with decreasing molecular size. An alternative for measuring the size and shape of a molecule is to probe its rotational diffusion by observing the fluorescence anisotropy of an attached fluorescent label. However, this technique is only applicable if the fluorescence lifetime is on the same order of magnitude as that of the rotational diffusion time of the molecule of interest.

Recent developments of single photon detectors, high-speed detection electronics, and advanced microscopy optics have led to a renaissance of fluorescence correlation spectroscopy (FCS). A very promising application of FCS is the measurement of the rotational diffusion on time scales of dozen of nanoseconds up to several microseconds, where fluorescence anisotropy with nanosecond fluorescent dyes will no longer work. We present an extension of conventional FCS that uses orthogonally polarized pulsed interleaved laser excitation and four polarization-sensitive detection channels which enables us to record all possible polarization-resolved correlation curves, event those which cannot be measured with conventional CW-excitation. We show that this full correlation information enables us to extract not only the global size of a macromolecule but also its shape, which is rather impossible to achieve with fluorescence anisotropy or conventional FCS measurements. We exemplify our method by measuring the size and shape of large proteins, and compare the experimental results with theoretical predictions. Moreover, we systematically study the impact of fluorescence depolarization by high-aperture optics, non-collinearity of absorption and emission dipole orientation, or labeling stoichiometry on the measurement results. 

Single Molecule Imaging Reveals Intranuclear Structure and Dynamics

Numerous problems in biology and biophysics have been studied by single-molecule techniques, but the application to the interiour of living cells is still just beginning. For the first time ever, single molecule imaging provides a direct, real-time view to molecular processes within living cells at almost „molecular resolution". New insights into the activation of transcription factors (1) and the trafficking and export of native mRNA molecules (2, 3) within living cells will be presented. These examples prove that single molecule imaging allows fascinating insights into the intracellular pathways of single mRNAs and functional proteins, and the structure and dynamics of supramolecular complexes in vivo. 

[1] Speil et al., submitted.

[2] Veith et al., 2010. Balbiani Ring mRNPs diffuse through and bind to clusters of large intranuclear molecular structures. Biophys. J. 99: 2676-2685.

[3] Siebrasse et al., 2008. Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin, Proc Natl Acad Sci USA 105:20291-6. 

Single-molecule investigation of the B-Z transition under mechanical control.

Z-DNA has fascinated biological scientists for decades by its unusual structure and potential biological roles. Although it is less stable than B-DNA, Z-DNA is stabilized in vivo by negative supercoiling. Here, we have investigated the B-Z transition in short purine-pyrimidine repeats (GC and TG repeats) in the presence of controlled tension and superhelicity via a hybrid technique of single-molecule FRET and magnetic tweezers. The hybrid scheme enabled us to identify the states of the specific region under mechanical control and trace conformational changes synchronously at local and global length scales. Minute negative superhelicity can facilitate the B-Z transition at low tension, indicating that tension, as well as torsion, plays a critical role in the transition[1]. Dynamic transitions between the states at elevated temperatures yielded thermodynamic and kinetic constants of the transition. Interestingly, the TG repeat showed markedly different kinetic and energetic behaviors compared to the GC repeat, implying an intriguing biological role of the former sequence. Our single- molecule studies shed light on the understanding of Z-DNA formation by highlighting the highly cooperative and dynamic nature of the B-Z transition. 

[1] M. Lee, S. H. Kim, S.-C. Hong, PNAS, 107, 4985-4990 (2010)

Dimerization of organic fluorophores reports on conformational dynamics ofbiopolymers

Dimerization of organic fluorophores has to be considered in single-molecule and ensemble experiments that rely on two intramolecular labels. Two identical fluorophores can serve as a reporter system for monitoring conformational dynamics by homodimerization. However, the same quenching interaction can compromise Förster Resonance Energy Transfer measurements by heterodimerization of both acceptor and donor fluorophores. We report here on using dimerization of organic fluorophores for quantitative analysis of conformational dynamics of unstructured biopolymers. With different fluorophores forming dimers of distinct stability, this quenching process allows optimizing the complex stability for a given environment. We show that the oxazine fluorophore MR121 exhibits the right dimer stability for following nanosecond dynamics of unstructured polypeptides under strongly denaturing conditions using fluorescence correlation spectroscopy. Furthermore, heterodimers-formation of organic flourophores that are typically used in single-molecule experiments is characterized. 

Single molecule studies of Bodipy-FTY720 (an immune suppressor analog) in biomembranes

FTY720, a synthetic analog of sphingosine that has immuno-suppressive properties, is the first oral drug to be approved by the U.S. FDA for treatment of multiple sclerosis (under the trade name GilenyaTM). Our hypothesis is that a new fluorescent analog Bodipy-FTY720 (hereafter, Bdp-FTY720) will be phosphorylated by sphingosine kinase 2 (SphK2), just as the parent FTY720, prior to binding to sphingosine-1-phosphate receptor 1 (S1PR1) followed by internalization and degradation. To test this hypothesis, we examined the uptake of Bdp-FTY720 by epithelial cells (Hs578T) as well as its partitioning in the plasma membrane and ternary-phased giant unilamellar vesicles (GUVs). Based on two-channel confocal co-localization imaging with DiI-C12, a liquid-disordered phase marker, Bdp-FTY720 has an affinity for the liquid-disordered lipid phase in GUVs. Bdp-FTY720 resides in both the cytoplasm and the plasma membrane of Hs578T cells; but excluded from the nucleus. Multiscale diffusion of Bdp-FTY720 in different environment was carried out using fluorescence correlation spectroscopy (µs-s) and time-resolved anisotropy (ps-ns). Complementary measurements on Triton X-100 micelles were also carried out to examine the interaction of Bdp-FTY720 with surfactant molecules. These studies represent a step towards elucidating the action mechanism of FTY720 as an immune suppressor at the single-cell level. 

Dynamic disorder in enzyme catalyzed reactions:a general phenomenon?

Single molecule fluorescence experiments allow for recording the sequence of individual enzymatic turnover reactions. Many experiments with different enzymes have shown that the dwell times follow a stretched exponential distribution interpreted as dynamic disorder. Although easily explained with the existence of several enzyme conformations with different activity the question remains if dynamic disorder is a general property of enzymes.

Studying the enzyme a-chymotrypsin we have observed deviations from a stretched exponential distribution. The shape of the dwell time distributions differs depending on the data analysis method used. In order to analyze this effect we have simulated sets of data based on a kinetic scheme with two a-chymotrypsin conformations. We have subsequently analyzed this data with the commonly used threshold approach and the more recently developed change point analysis. Indeed, different dwell time distributions are obtained depending on the signal:noise ratio, the overall number of photons detected and the bin size. Applying the criteria learned from the simulation, we can analyse a-chymotrypsin data in order to resolve the question if a‑chymotrypsin catalysis is characterized by dynamic disorder. More importantly, our finding provides a general guideline for the analysis of single molecule fluorescence data ultimately allowing for the construction of kinetic schemes. 

Kerstin Blank, et al., Biotechnology Journal, 4, 465-479 (2009).

Gert De Cremer, et al., Journal American Chemical Society, 129, 15458-15459 (2007).

Labeling freedom for the single molecule spectroscopist

Observation of single molecule fluorescence has matured into a central tool to study biomolecular structure and dynamics. As hardware and data analysis technology dramatically improved over the last decade, site-specific labeling of proteins with small but highly photostable fluorescent dyes has turned into a major bottleneck for biological applications. Traditional approaches to label natural amino acids are the most widely distributed, but due to the high abundance of even the (rare) cystein in larger biological machineries, only few protein systems remain accessible. We have now developed a novel artificial amino acid that is easily and site-specifically introduced into any protein by the natural machinery of the living cell. Expressed proteins only differ from their natural counterparts by very few atoms that constitute a ring-strained cyclooctyne functional group. We show that this completely inert and non-toxic group can be stably incorporated into any protein and readily reacts with commercially available single molecule fluorophores without the need of special reagents, catalyst or non-physiological buffer conditions. In fact, the ability of this method to proceed fast and specific holds great potential for applications of single molecule and super resolution techniques in living cells. 

Genetically encoded copper-free click chemistry. Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E.A. Angew Chem Int Ed Engl. 2011 Apr 18;50(17):3878-81.doi:10.1002/anie.201008178.

Self-Assembly of Highly Ordered Conjugated Polymer Aggregates with Long-Range Energy Transfer

Conjugated polymers (CP) are a promising new class of materials for low-cost electronic devices, such as organic photovoltaics (OPV) and organic light-emitting diodes (OLED).[1-3] For over a decade single molecule spectroscopy (SMS) is used to elucidate morphologic, spectroscopic and electronic properties of single CP chains.[4-6] The question arises, how these findings are connected to the properties of a neat solid.[7] Therefore we elaborated a general concept based on solvent vapor annealing (SVA) for the controlled and directly observable aggregation of single CP chains into aggregates of different sizes. This concept is used together with SMS techniques to study the evolution of morphology and electronic transport dynamics during the transition from single molecules to bulk materials. It is shown that the highly ordered morphology of single CP chains persist to aggregate sizes at least 25 times bigger then the single CP chain. Blinking depth studies on the aggregates together with a simple model suggest that the previously reported large energy transfer mechanism in single CP chains is also present in these highly ordered aggregates, due to efficient inter-chain energy transfer.

[1]          H. L. Wang, A. G. MacDiarmid, Y. Z. Wang, D. D. Gebler, A. J. Epstein, Synth. Met., 78, 33 (1996).

[2]          F. Hide, M. A. DiazGarcia, B. J. Schwartz, A. J. Heeger, Accounts Chem. Res., 30, 430 (1997).

[3]          R. H. Friend, R. W. Gymer, A. B. Holmes, J. H. Burroughes, R. N. Marks, C. Taliani, D. D. C. Bradley, D. A. Dos Santos, J. L. Bredas, M. Logdlund, W. R. Salaneck, Nature, 397, 121 (1999).

[4]          J. M. Lupton, Advanced Materials, 22, 1689 (2010).

[5]          J. C. Bolinger, M. C. Traub, T. Adachi, P. F. Barbara, Science, 331, 565 (2011).

[6]          T. Adachi, J. Brazard, P. Chokshi, J. C. Bolinger, V. Ganesan, P. F. Barbara, Journal of Physical Chemistry C, 114, 20896 (2010).

[7]          C. Bardeen, Science, 331, 544 (2011).

Surface-immobilization strategies for observing real-time dynamics of DNA polymerase

Most DNA polymerases achieve high fidelity for DNA synthesis by selecting the correct nucleotide substrate during early mechanistic steps that precede the chemical reaction of incorporation. These steps involve protein conformational changes directly observed and analysed using single-molecule fluorescence assays on diffusing molecules.

To obtain real-time trajectories of the conformational changes or the reaction pathways, we are studying immobilized polymerase molecules and complexes. A prerequisite for such studies is that the surface supports the full activity of the immobilized molecules and is protected from excessive non-specific adsorption; moreover, the conditions should support the formation of complexes for a significant fraction of the observation time. Towards this end, we are developing surface-coating protocols that involve improved surface PEGylation and coating with lipid bilayers. In parallel, we are evaluating the activity of DNA polymerase and its partners inside diffusing or immobilized lipid vesicles. The methods are general and should be useful for studying many protein-nucleic acid and protein-protein interactions. 

Metalnanoparticles as labels: application to optical coherence microscopy and cell imaging

A molecular-size thermometer

Accurate measurement of temperature is gaining increasing importance due to the wide application range (electronic devices, biology, medical diagnostics). Nowadays, the technological frontier to cross is the measurement of local temperature in an extremely small volume. The thermometric response must be highly accurate, sensitive, stable, and presenting high spatial/temporal resolution [1-2]; in this context, one of the most intriguing challenge is to found suitable probes for ultrasensitive spectroscopic techniques such as confocal fluorescence microscopy.

We present an uncommon zwitterionic metallate cluster fulfilling these requirements. Its photophysics, both in solid and in solution, is characterized by a temperature dependent emission and excited state lifetime that changes remarkably in a wide working range. This allows for an unprecedented accuracy in temperature determination, and the lifetime dependence on temperature makes this compound a suitable probe for FLIM (fluorescence lifetime imaging microscopy) analysis; the emission lifetime, in fact, in contrast with its intensity, is insensitive to the concentration of the fluorophore, allowing temperature detection also in environments where an homogeneous distribution of dyes is hard to achieve, such as in biological matrices or complex devices. 

[1] J. Lee, N. A. Kotov, Nanotoday 2, 48 (2007).

[2] S. Uchiyama, A. P. De Silva, et al. J. Chem. Ed. 83, 720 (2006) 

A new Silicon SPAD with improved red efficiency and 100 ps timing resolution

Many applications require high performance Single Photon Avalanche Diodes (SPAD) either as single pixels or as small arrays of detectors. Although currently available silicon devices reached remarkable performance, further improvements are needed in order to meet the requirements of most demanding time-resolved techniques. In particular, one of the most significant challenges today is the development of a planar silicon technology, compatible with the fabrication of arrays, capable of reaching a high Photon Detection Efficiency (PDE) in the near infrared region while maintaining a good temporal resolution.

We will present a new device structure aimed at attaining the aforementioned performances. In particular, experimental characterization showed a significant increase in the PDE with a remarkable value of 40% at 800nm; a photon timing jitter as low as 93ps FWHM has been also attained, while other device performances, such as Dark Count Rate (DCR) and Afterpulsing Probability (AP) are essentially unchanged, compared to classical thin SPAD. Being planar, the new technology is also intrinsically compatible with the fabrication of arrays of detectors.

Plasmamembrane organization

The composition of the plasma membrane has long been modeled as a mosaic fluid. (Singer and Nicolson, 1972) Studies in the last few years have identified microdomains like lipid rafts and caveolae that constrain membrane proteins within a small region of the cellular plasma membrane. These domains facilitate anchoring of different signaling proteins for example H-Ras that has been shown to co-localize with lipid rafts upon activation (Lommerse et al. 2005, Rotblat et al. 2004). The signaling cascade on the plasma membrane of cells is dependent on the location of different membrane proteins. Therefore it is of high scientific interest to further investigate these domains.

Photo Activated Localization Microscopy (PALM) is used with Dendra2 as the photo convertable fluorescent protein. For this research 3T3-cells have been transfected to express H-Ras-Dendra2. Using Photo Activated Localization Microscopy (PALM) the position of H-Ras can be determined with a precision of 30 nm. By analyzing the trajectories of single molecules of H-Ras it is shown that a percentage of H-Ras molecules is confined within small (+- 200 nm) domains.

Choleratoxin B (CtxB) is the ligand of ganglioside GM1 which is used as a marker for lipid rafts at the outside of the lipid membrane. Since it binds to five GM1 molecules it is suggested that CtxB enlarges the size of lipid rafts.

It is hypothesized that treatment of the cells with CtxB enlarges both the outside and inside lipid raft and therefore the confinement of H-Ras should be enlarged upon CtxB treatment. However, our data suggests only increase of the density of H-Ras upon introduction of CtxB.

One Pot Functionalization of Graphene with Porphyrin using Cycloaddition Reactions

Two types of graphene based hybrid materials, graphene-TPP (TPP:

tetraphenylporphyrin) and graphene-PdTPP (PdTPP: palladium tetraphenylporphyrin) were prepared directly from pristine graphene using one pot cycloaddition reactions. The hybrid materials were characterized by TGA, UV/Vis, FTIR, TEM, Raman, luminescence spectroscopy and fluorescence/phosphorescence lifetime measurements. The covalent linkage between graphene and porphyrin was confirmed by FTIR, Raman spectroscopy and further supported by control experiments. The presence of TPP (or PdTPP) in the hybrid material was demonstrated by UV/Vis spectroscopy, and TGA results indicate that graphene-TPP and graphene-PdTPP hybrid materials contain approximately 18% TPP and 20% PdTPP, respectively. The quenching of fluorescence (or phosphorescence) and reduced lifetime suggests excited state energy/electron transfer between graphene and the covalently attached TPP (or PdTPP) molecules. 

[1] A. K. Geim, K. S. Novoselov, Nat. Mater. 2007, 6, 183-191; (b) A. K. Geim, Science, 324, 1530-1534 (2009);

[2] M. D. Stoller, S. J. Park, Y. W. Zhu, J. H. An, R. S. Ruoff,

Nano Lett. 8, 3498-3502 (2008);

[3] C. Jozsa, M. Popinciuc, N. Tombros, H. T. Jonkman, B. J. van Wees, Phys. Rev. Lett. 100,  236603 (2008);

[4] F. Schedin, A. K. Geim, S. V. Morozov, E. W. Hill, P. Blake1, M. I.

Katsnelson, K. S. Novoselov, Nat. Mater. 6, 652-655 (2007)

Use of arrays of single liposomes to spatially confine PSI complexes and monitor their activity with single molecule resolution and in a massive parallel manner.

Photosystem I (PSI) is a unique photoelectronic nanomachine that produces the largest negative potential in nature, and principally sets the global enthalpy amount in all lifeforms [1].

Single molecule measurements are ideally suited to elucidate molecular level details underlying PSI activity, that remain masked in conventional ensemble PSI activity assays. These assays are usually carried out on native thylakoid membranes, containing different sizes and compositions of proteins and lipids, or on PSI solubilised in detergent, thus yielding an uninformative average activity.

Here, we have employed our recently developed arrays of surface tethered single liposomes [2] on reconstituted single PSI, allowing us to monitor its activity at the single molecule level and in a massive parallel manner [3]. Liposomes constitute an ideal 3D scaffold to spatially confine single PSI in a native like environment, and can efficiently encapsulate the prefluorescent electron acceptor, resazurin [4], that upon reduction by PSI becomes highly fluorescent, thus directly yielding single PSI activity. Our studies allow us for the first time to correlate membrane characteristics (lipid composition, curvature, phase state, etc.), to regulation of PSI activity studied on the single molecule level. 

1. Nelson, N. & Yocum, C., Annu. Rev. Plant Biol., 57, 521-65 (2006).

2.

Hatzakis, N.S., Bhatia, V.K., Larsen, J., Madsen, K.L., Bolinger, PY.,

Kunding, A.H., Castillo, J., Gether, U., Hedegård, P., & Stamou, D.G., Nat. Chem. Biol., 5, 835-41 (2009).

3.

Hatzakis, N.S., Wei, L., Jorgensen, S.K., Kunding, A.H., Bolinger, PY., Makarov, I., Skjot, M., Svendsen, A., Hedegård, P., Stamou, D.G., Submitted

4. Lohse, B., Bolinger, P. & Stamou, D. J. Am. Chem. Soc, 130, 14372-73 (2008). 

Scaling Form of Viscosity at all Length-scales in Poly(Ethylene Glycol)s Mixtures Studied by Fluorescence Correlation Spectroscopy

Relative viscosity of solutions of complex liquids is described by the scaling formula exp(Reff/bξ)a [1]. We measured viscosities of aqueous mixtures of poly(ethylene glycol)s with different molecular weights at different length-scales. We investigated the macroscopic viscosity using standard rheometry. We also performed measurements at the nanoscale using Fluorescence Correlation Spectroscopy with fluorescent dyes as a probes. We found that the relative viscosity at the semi-dilute regime is described by the product of viscosities of every component of the mixture: exp(Reff,1/bξ1)a * exp(Reff,2/bξ2)a.

Acknowledgement: The work was supported by the Project operated within the Foundation for Polish Science Team Programe co-financed by the EU "European Regional Development Fund" TEAM/2008- 2/2 and from the budget of the Ministry of Science and Higher Education 2007-2010. The research was co-financed by the European Regional Development Fund under the Operational Programme Innovative Economy NanoFun POIG.02.02.00-00-025/09. 

[1] T. Kalwarczyk, N. Ziębacz, A. Bielejewska, E. Zaboklicka, K. Koynov, J. Szymański, A. Wilk, A. Patkowski, J. Gapiński, H.-J. Butt, and R. Hołyst, Nano Lett., 11, 2157 (2011)

Superresolution Imaging Using Fluctuations

The interest in superresolution microscopy techniques has dramatically increased in the last years due to the unprecedented insight into cellular structure which has become possible [1]. All superresolution techniques rely on probes, which can be switched between a bright and a dark state. For all traditional camera-based techniques, such as STORM or PALM, it is crucial to have perfect control over these 'on' and off' states, which renders sample preparation sometimes difficult.

A recently developed superresolution technique, namely Superresolution Optical fluctuation Imaging (SOFI) [2] offers the possibility to record superresolved images within short acquisition times, while allowing switching rates of the probes to be almost arbitrary.

Photoswitching can be induced and controlled in most fluorophores using the dyes' oxidation and reduction behaviour by adding oxidizers and reductors (ROXS, Never2FADE [3]).

SOFI is based on the evaluation of stochastic fluctuations of the signal stemming from a single emitter by means of higher-order statistics. Even though in the experiment the fluctuations of many emitters might overlap in a pixel, the algorithm is designed in such way that it is able to extract superresolution information based on these stochastic fluctuations. An experiment consists of taking a movie of these fluctuations and afterwards subject this movie to the SOFI algorithm. Note that this algorithm works completely 'blindly', i.e. no assumptions / fitting of any kind have to be made on sample structure, emitter properties etc. 

[1] N. Blow, Nature, 456, 825-828 (2008)

[2] T. Dertinger, R. Colyer, G. Iyer, S. Weiss, J. Enderlein, Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI), Proceedings of the National Academy of Sciences of USA, Vol.106, p.22287-22292 (2009)

[3] M. Heilemann, S. van de Linde, Mukherjee, M. Sauer, Ang. Chem. Int. Ed. 48, 37, 6903-6908 (2009) 

A SPAD Array Detector for Spectrally and Lifetime Resolved Microscopy

Time-resolved single molecule detection techniques are state-of-the-art. In order to obtain as much as possible information, parallel detection of several polarization and spectrally separated detection channels is necessary. In addition, parallelization of the measurement is a tool to overcome limitations due to the inherent long measurement times.

Fluorescence lifetime measurements are for many single molecule measurements indispensable. The optimal detector type combining an excellent timing resolution together with very high quantum efficiency is the Single Photon Avalanche Diode (SPAD). Therefore SPAD arrays would be an ideal detection tool for either spectral or parallelized time-resolved single molecule measurements.

We investigated a silicon based SPAD array prototype featuring eight detection channels. The array, designed by the Politecnico in Milano (Italy) achieves a detection efficiency of 30 % at 470 nm and 31 % at 670 nm. The timing resolution is 170 ps and 80 ps respectively.

Due to a novel electronic architecture, the crosstalk is reduced and reaches a value below 1 ‰. The sensitive areas have a diameter of 50 µm and a distance of 250 µm between each other.

A special optical layout was developed in order to illuminate the different SPAD areas without light loss after spectrally splitting the fluorescence. To this aim a set of relay lenses and an array of micro-lenses were designed, which focus the light after the spectral separation on the different sensitive areas of the SPAD array.

First results display the feasibility of the planned setup. 

Stress Induced Single Molecule Rotational Motion in Elastomeric Polypropylene

The fluorescence of a single molecule (SM) is a very sensitive probe for its environment and changes of the fluorescence lifetime, emission wavelength, and polarization can report spatial and temporal variations in the surrounding structure of the fluorescent dye [1]. Here we report on SM microscopy and spectroscopy studies of stress relaxation in thin films of elastomeric polypropylene (ePP), a semicrystalline polymer with a complex microstructure of crystalline and amorphous regions on the nanometer scale [2]. The films are stretched using a microtensile testing setup and the temporal evolution of mechanical stress averaged over the whole sample is measured by a micro force sensor. Simultaneously, perylenediimide dyes bound to polymer chains and embedded in the ePP film as isolated dye molecules report molecular dynamics and changes within their local environment via SM polarization dependent microscopy. This experiment allows for insights into dynamical processes within the amorphous regions of ePP which are not accessible with other microscopy techniques. 

[1] S. Krause, P. F. Aramendia, D. Täuber and C. von Borczyskowski, PCCP, 13, 1754-1761 (2011).

[2] M. Franke, R. Magerle, ACS Nano, doi: 10.1021/nn200957g (2011).

SMS - FRET labelling techniques

SMS - FRET spectroscopy has emerged as a versatile tool in life sciences.

The applications range from protein-protein interactions and imaging microscopy to fast dynamic processes such as protein folding.

For SMS-FRET measurements in protein folding studies, site specific labelling of the protein with a donor and acceptor dye is limited to the reaction of cystein and lysine residues.

Depending on the site of the protein two approaches overcome the uncertainty of random labelling.

1) For small proteins or peptides up to 50 aa, solid phase peptide synthesis (SPPS) is the method of choice. By using aa with different protective groups site specific coupling to virtual all residues is possible.

2) For larger proteins two new evolving methods enable the site specific coupling of dyes: a) the so called orthogonal-system allows the insertion of unnatural aa via bacterial expression in the polypeptide chain, the dye is then specifically coupled to the functional side chain of the unnatural aa. (Schulz et al J Am. Chem. Soc., 2008). b) Intein mediated protein ligation can be used to efficiently fuse short peptides with attached fluorophores to expressed proteins (Grant et al, Biol. Chem., 2006). 

J Am Chem Soc. December 31; 130(52): 17664–17665. doi:10.1021/ja807430h. 2008

Grant, J.E., J. Biol. Chem., 281, 6194–6202. 2006

Single molecule study of the intrinsically disordered FG-repeat Nucleoporin 153

Single-molecule fluorescence spectroscopy of the structure and dynamics of macromolecular assemblies

Single-molecule fluorescence spectroscopy is a useful tool to study structural conformations and dynamics of macromolecular assemblies. We are using Dual-Focus Fluorescence Correlation Spectroscopy (2fFCS)[1] and Dual-Color-Fluorescence Cross-Correlation Spectroscopy (2-color-FCCS)[2] to study structural and dynamical properties of proteins and small nuclear ribonucleoproteins in the spliceosomal[3] complex and the ribosomal complex[4].

The spliceosome undergoes compositional and conformational changes during its catalytic action. We are investigating the conditions for the recruitment and release of particular proteins during the splicing steps: How do the changes occur, stepwise or in correlated manner and what are the roles of certain spliceosomal RNA helicases in the restructuring of the complex. 2-color-FCCS in combination with 2fFCS enables the observation of protein-protein interactions and the determination of dissociation constants for protein-protein and protein-mRNA bindings which could not be resolved with standard biochemical methods.

In the ribosomal complex we focus on the translocation of mRNA and tRNA which is catalyzed by the elongation factor G (EF-G). With 2fFCS we observe the binding of EF-G to different tRNA-ribosomal complexes and determine the dissociation constants between these complexes and EF-G in dependence of different nucleotides. 

[1] Dertinger, T., Pacheco, V., von der Hocht, I., Hartmann, R., Gregor, I., and Enderlein, J., ChemPhysChem 8, 433-443 (2007)

[2] Schwille, P., Meyer-Almes, F. J., Rigler, R., Biophy J. 72, 1878-1886 (1997)

[3] Wahl, M. C., Will, C. L., Lührmann, R., Cell. 136, 4, 701-718 (2009)

[4] Rodnina, M. V., Savelsbergh, A., Katunin, V. I., Wintermeyer W., Nature, 385, 37-41  (1997)

Single-Molecule FRET Reveals DNA Dynamics in the RNA Polymerase-DNA Open Complex

Transcription starts with binding of RNA polymerase (RNAP) to a specific sequence on the promoter DNA. Subsequently, melting of 12-14 base-pairs surrounding the transcription start site forms the catalytically active RNAP-DNA open complex (RPo). During transcription initiation there is flexibility in the position at which Escherichia coli RNAP initiates transcription. It has been suggested that sampling of transcription start sites may proceed via movement of the single-stranded DNA that forms part of the transcription bubble; this DNA movement is suggested to occur prior to the start of any synthesis and may result in "DNA scrunching" within the transcription bubble.

We tested this hypothesis using single-molecule Förster Resonance Energy Transfer (smFRET) of RPo in solution. We provide experimental evidence for conformational dynamics in RPo using different FRET rulers and labelling positions. Further experiments using different subsets of nucleotides allowed us to "re-programme" the transcription start-site according to recently proposed scrunching models. Our investigation marks the first direct experimental observation of DNA dynamics in the transcription bubble of RPo, and supports the hypothesis that these dynamics represent a means of rapid search process for the transcription start-site. 

Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami

DNA origami [1] 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 [2] (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. 

[1] Paul Rothemund, Nature, Vol. 440, Pages 297-302 (2006).

[2] Alexey Sharonov, Robin M. Hochstrasser, Proceedings of the National Academy of Sciences, Vol. 103, Page 18911 (2006).

Fluorescent labeled DNA-Origami as a versatile standard for fluorescence microscopes

The DNA-Origami technique allows to adjust the relative position of single fluorescent dyes with high precision at the nanoscale. The DNA-Origami acts as a molecular breadboard with independent adressable positions for any common dye. Especially for emerging superresolution techniques such as STED, (d)STORM, GSDIM, Blink-Microscopy, and SIM the fluorescent labeled DNA-Origami's are able to serve as a new standard to check the achievable resolution under realistic conditions. 

Influence of temperature on the viscosity at the nanoscale

One of the main factors influencing biochemical processes occurring in living cells is mobility of proteins and other biomolecules. Small proteins (tens of kD, hydrodynamic radii of ca. 1–5 nm) present diffusivities orders of magnitude greater than those predicted by the Stokes-Sutherland-Einstein (SSE) equation D=kT/6πηr. An explanation of this phenomenon is a hereby presented scaling formula [1] for size-dependent viscosity coefficients for proteins, polymers and fluorescent dyes diffusing in complex liquids, which are frequently used as models of the cytoplasm of living cells.

In addition, we analyzed the influence of temperature on viscosity of polymer solutions for all length scales. Although some dependence is clearly noticeable, no rapid changes of viscosity with temperature were found in the investigated systems.

The research was supported by the EU Operational Programme Innovative Economy "Quantum Semiconductor Nanostructures For Applications In Biology and Medicine". 

[1] T. Kalwarczyk, N. Ziębacz, A. Bielejewska, E. Zaboklicka, K. Koynov, J. Szymański, A. Wilk, A. Patkowski, J. Gapiński, H.-J. Butt, R. Hołyst, Nano Letters, 11, 2157 (2011)

Accurate distance and structure determination of RNA molecules via super resolution FRET technique.

Using a confocal fluorescence microscope, multiparameter fluorescence detection (MFD) enables us to collect simultaneously all fluorescence information such as intensity, lifetime and anisotropy in several spectral ranges from picoseconds to seconds. MFD and fluorescence correlation spectroscopy is applied to perform super resolution FRET studies with a high level of precision in determining separations with FRET of 1% of the Förster radius [1][2]. In addition, we can unambiguously distinguish between stochastic processes and broadening due to static or dynamic heterogeneity.

The structures of RNA-junctions (with three or four stems and with an additional loop at the junction) were characterized using 46 (16+30) Donor-Acceptor pairs. This allowed us to prove the existence of several possible stacking conformers simultaneously present in equilibrium. Our studies showed that super resolution FRET measurements are a valuable tool to complement the structural information obtained by X-ray crystallography or NMR spectroscopy as these techniques are limited in detecting minority conformers. 

[1] Antonik, M., et al., J.Phys.Chem., 110, 6970-6978, (2006)

[2] Kalinin, S., J.Phys.Chem., 112, 8361-8374, (2008)

Quantum dot and Cy5.5 labeled nanoparticles to investigate lipoproteinbiointeractions via Förster resonance energy transfer

The study of lipoprotein biology in general and high density lipoprotein's (HDL) biointeractions in particular is of primary importance to better understand, treat and prevent cardiovascular disease. Hybrid nanostructures based on nanocrystals that are stabilized and functionalized by a biocompatible coating have been exploited for various imaging techniques and are specifically suitable for molecular imaging purposes. In this study, we developed a quantum dot core HDL nanoparticle. By detecting Förster resonance energy transfer (FRET) between the quantum dot (QD) core and dye-labeled lipids in the coating, we are able to study lipid exchange dynamics, lipoprotein-lipoprotein interactions, and to visualize the process of HDL uptake by live macrophage cells. 

Skajaa T. ; Zhao Y. ; J. van den Heuvel D. ; Gerritsen H. C. ; Cormode D.P.; Koole R. ; van Schooneveld M.M. ; Post J.A.; Fisher E.A. ; Fayad Z.A. ; de Mello Donega C. ; Meijerink A. ; Mulder W.J.M. Nano Lett., 2010, 10 (12), pp 5131–5138