Design of single-molecule optical devices : unidirectional photonic wires and digital photoswitches

Heilemann M. Design of single-molecule optical devices : unidirectional photonic wires and digital photoswitches. Bielefeld (Germany): Bielefeld University; 2005.Molecular photonics is a new emerging field of research around the premise that it is possible to develop optical devices using single molecules as building blocks. Currently used waveguides, applied for example in telecommunication, rely on the classical physics of bulk materials: Maxwell's equations allow propagating modes in the far field, and the wavelength of light imposes a fundamental lower limit on device size. However, nature has evolved several examples of photonic nanostructures to guide light over much smaller length scales for "light harvesting" in plants and photosynthetic bacteria. This fundamentally quantum mechanical solution is most often based on near-field dipole-dipole interactions, i.e. fluorescence resonance energy transfer (FRET). As a consequence, light-harvesting complexes have inspired researchers to engineer molecular optical devices, such as molecular photoswitches or molecular photonic wires. A molecular photonic wire is distinguished from an electronic wire by supporting excited-state energy transfer rather than electron- (or hole-) transfer processes and could find application in, for example, optical computing as short-range interconnects in dense optical circuits. In this work an alternative access to molecular photonic wires was elaborated. This approach was based on (I) the use of conventional, single molecule compatible chromophores, (II) an energy cascade as the driving force for the excited-state energy, and (III) an arrangement of chromophores such that strong electronic interactions promoting fluorescence quenching are prevented. The main requirement the chromophores have to fulfil is their compatibility with single molecule spectroscopy, i.e. photostability and photophysics. To achieve a very regular arrangement of chromophores, DNA was used as rigid scaffold. Well-developed labelling and post-labelling strategies of DNA were exploited to introduce a variety of different chromophores in a modular conception. Best results could be obtained when dye-labelled oligonucleotides were hybridised against a long DNA-strand already carrying a primary donor chromophore and a biotin for specific immobilization. The distance between subsequent chromophores was adjusted to 3.4 nm, i.e. 10 bases, which ensured efficient FRET and prevented direct orbital interaction. Photonic wires were synthesised carrying up to 5 chromophores and covering a spectral range from 488 nm to 750 nm. In ensemble experiments the maximum overall transfer efficiency was determined to be 21 percent, but as indicated by steady state and time-resolved measurements, a broad heterogeneity within the samples was suspected. To disentangle the complexity of the photophysics of so-built photonic wires, a confocal fluorescence microscope, single molecule sensitive on four spectrally separated detectors, was developed. For the first time, a quadruple jump of energy transfer along a single photonic wire was demonstrated with an overall transfer efficiency of about 90 percent. Confirmation that the energy is transferred stepwise comes from prolonged excitation of single molecules, which results in sequential photobleaching and a shift in the emission from the red back towards the blue. Fluorescence lifetime information revealed further aspects of energy transfer, and complemented spectral data in order to identify fluorophores involved in particular energy transfer steps. Leakages in energy transfer, created by photodestruction of a fluorophore inside the chain, were revealed. Polarization modulation of the excitation light in combination with fluorescence lifetime gave insight into the rotational mobility of the fluorophore serving as input unit, i.e. Rhodamine Green. After the accomplishment of the photonic wire, a further goal was the development of a molecular photoswitch. Hitherto, only one demonstration of chemically synthesized photoswitching of single molecules at room temperature had been reported. In the context of this work, it was shown that commercially available unmodified carbocyanine dyes such as Cy5 and Alexa647 could be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation in the range of 488 - 532 nm. In oxygen-free environment and in the presence of 100 mM [beta]-mercaptoethanol (MEA) as triplet quencher, more than 20 switching cycles could be achieved for single Cy5 molecules with a reliability of more than 90 percent. Single pair FRET experiments with high time resolution revealed the existence of three intermediates prior to fluorescence restoration. In addition to the importance of such single-molecule photoswitches e.g. for optical data storage, the results presented in this work imply limitations for the use of carbocyanine dyes in sp-FRET experiments

When Weak Is Strong:A Plea for Low-Affinity Binders for Optical Microscopy

The exploitation of low-affinity molecular interactions in protein labeling is an emerging topic in optical microscopy. Such non-covalent and low-affinity interactions can be realized with various concepts from chemistry and for different molecule classes, and lead to a constant renewal of fluorescence signals at target sites. Further benefits are a versatile use across microscopy methods, in 3D, live and many-target applications. In recent years, several classes of low-affinity labels were developed and a variety of powerful applications demonstrated. Still, this research field is underdeveloped, while the potential is huge.</p

Real-Time analysis and visualization for single-molecule based super-resolution microscopy

Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy

Super-resolution microscopy reveals specific recruitment of HIV-1 envelope proteins to viral assembly sites dependent on the envelope C-terminal tail

The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread . Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse

Quantitative single-molecule imaging of TLR4 reveals ligand-specific receptor dimerization

In humans, invading pathogens are recognized by Toll-like receptors (TLRs). Upon recognition of lipopolysaccharide (LPS) derived from the cell wall of gram-negative bacteria, TLR4 dimerizes and can stimulate two different signaling pathways, the proinflammatory, MyD88-dependent pathway and the antiviral, MyD88-independent pathway. The balance between these two pathways is ligand-dependent, and ligand composition determines whether the invading pathogen activates or evades the host immune response. We investigated the dimerization behavior of TLR4 in intact cells in response to different LPS chemotypes through quantitative single-molecule localization microscopy (SMLM). Quantitative super-resolved data showed that TLR4 was monomeric in the absence of its coreceptors MD2 and CD14 in transfected HEK 293 cells. When TLR4 was present together with MD2 and CD14, but in the absence of LPS, 52% of the receptors were monomeric and 48% were dimeric. LPS from Escherichia coli or Salmonella minnesota caused the formation of dimeric TLR4 complexes, whereas the antagonistic LPS chemotype from Rhodobacter sphaeroides maintained TLR4 in monomeric form at the cell surface. Furthermore, we showed that LPS-dependent dimerization was required for the activation of NF-κB signaling. Together, these data demonstrate ligand-dependent dimerization of TLR4 in the cellular environment, which could pave the way for a molecular understanding of biased signaling downstream of the receptor

Nanoscopy of bacterial cells immobilized by holographic optical tweezers

Diekmann R, Wolfson D, Spahn C, Heilemann M, Schüttpelz M, Huser T. Nanoscopy of bacterial cells immobilized by holographic optical tweezers. Nature Communications. 2016;7(1): 13711

Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A(Cnp1) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A(Cnp1) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle

Biased signalling is an essential feature of TLR4 in glioma cells

A distinct feature of the Toll-like receptor 4 (TLR4) is its ability to trigger both MyD88-dependent and MyD88-independent signalling, culminating in activation of pro-inflammatory NF-κB and/or the antiviral IRF3. Although TLR4 agonists (lipopolysaccharides; LPSs) derived from different bacterial species have different endotoxic activity, the impact of LPS chemotype on the downstream signalling is not fully understood. Notably, different TLR4 agonists exhibit anti-tumoural activity in animal models of glioma, but the underlying molecular mechanisms are largely unknown. Thus, we investigated the impact of LPS chemotype on the signalling events in the human glioma cell line U251. We found that LPS of Escherichia coli origin (LPSEC) leads to NF-κB-biased downstream signalling compared to Salmonella minnesota-derived LPS (LPSSM). Exposure of U251 cells to LPSEC resulted in faster nuclear translocation of the NF-κB subunit p65, higher NF-κB-activity and expression of its targets genes, and higher amount of secreted IL-6 compared to LPSSM. Using super-resolution microscopy we showed that the biased agonism of TLR4 in glioma cells is neither a result of differential regulation of receptor density nor of formation of higher order oligomers. Consistent with previous reports, LPSEC-mediated NF-κB activation led to significantly increased U251 proliferation, whereas LPSSM-induced IRF3 activity negatively influenced their invasiveness. Finally, treatment with methyl-β-cyclodextrin (MCD) selectively increased LPSSM-induced nuclear translocation of p65 and NF-κB activity without affecting IRF3. Our data may explain how TLR4 agonists differently affect glioma cell proliferation and migration