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Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the bio-molecule(s) of interest to a biocompatible surface. Here we describe a robust surface passivation method by dense polymer-brush grafting, based on optimized polyethylene-glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system. The increased biocompatibility achieved, compared to PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macro-molecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level.

For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution.

Dendritic cells express DC-SIGN, a C-type lectin (CTL) that binds a variety of pathogens and facilitates their uptake for subsequent antigen presentation. DC-SIGN forms remarkably stable microdomains on the plasma membrane. However, inner leaflet lipid markers are able to diffuse through these microdomains suggesting that, rather than being densely packed with DC-SIGN proteins, an elemental substructure exists. Therefore, a super-resolution imaging technique, Blink Microscopy (Blink), was applied to further investigate the lateral distribution of DC-SIGN. Blink indicates that DC-SIGN, another CTL (CD206), and influenza hemagglutinin (HA) are all localized in small (∼80 nm in diameter) nanodomains. DC-SIGN and CD206 nanodomains are randomly distributed on the plasma membrane, whereas HA nanodomains cluster on length scales up to several microns. We estimate, as a lower limit, that DC-SIGN and HA nanodomains contain on average two tetramers or two trimers, respectively, whereas CD206 is often nonoligomerized. Two-color Blink determined that different CTLs rarely occupy the same nanodomain, although they appear colocalized using wide-field microscopy. What to our knowledge is a novel domain structure emerges in which elemental nanodomains, potentially capable of binding viruses, are organized in a random fashion; evidently, these nanodomains can be clustered into larger microdomains that act as receptor platforms for larger pathogens like yeasts.

We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.

The radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time.

The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation – a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP), remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat) pathway, but actively fold in the periplasm following general secretory pathway (Sec) and signal recognition particle (SRP) mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

Recent developments in DNA nanotechnology facilitate a new degree of control to place arbitrary objects. Therefore, DNA is folded into arbitrary 2- and 3-dimensional structures on a nanometer to micrometer scale. In this so-called origami technique, introduced by Paul Rothemund in 2006, one ∼7.3 kbases long single-stranded DNA is hybridized with ∼200 short synthetic DNA “staple” strands to build a desired structure by self-assembly. Objects of interest, e.g. single fluorophores, are attached to individual incorporated DNA strands at specific positions within the structure. Several applications of this approach are shown using single-molecule fluorescence techniques.Revisiting the distance dependence of fluorescence resonance energy transfer (FRET), we used the DNA origami technique to build a spectroscopic ruler. In contrast to double stranded DNA, a commonly used spacer molecule, this technique offers distinct advantages. We designed a rigid DNA origami block, which has a higher persistence length and additionally allows placing the dye molecules all oriented in the same direction on the top surface, limiting static effects of the linker lengths. In contrast to dsDNA, for the origami block the Forster Radius R0 could directly be obtained from the distance dependence of energy transfer based on single-molecule FRET measurements.Guided by the idea to build complex spectroscopic networks by self-assembly, we used rectangular DNA origami as a molecular breadboard to precisely position individual fluorophores. In this artificial system the path of energy transfer can be manipulated on the nanoscale. Fluorophores were incorporated such that the light from the “blue” input dye could either be guided to the “red” or “IR” output dye, by a “green” dye that was placed at two alternative positions. We used a single-molecule four-color FRET approach with alternating laser excitation for analysis of the different energy transfer paths.