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Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novel super-resolution microscopy techniques achieve nanometer resolution of fluorescently labeled molecules. The cellular restriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1 (HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resolution analysis of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherin that preserved HIV-1 particle formation and Vpu-dependent restriction, respectively. Multicolor super-resolution microscopy revealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin at the plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors. Tetherin clusters containing on average 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combined biochemical and super-resolution analysis revealed that extended tetherin dimers incorporate both N-termini into assembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichment of the raft marker GM1. Together, our super-resolution microscopy analysis of HIV-1 interactions with tetherin provides new insights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-host interactions. [ABSTRACT FROM AUTHOR]

This tutorial reviewsummarizes the most important results and developments in the field of polymer science by means of single molecule fluorescence spectroscopy (SMFS) at ambient temperatures. A broad range of topics will be addressed and it will be discussed which single molecule methods are suitable to get the maximum amount of information about polymer structure, polymer dynamics and the photophysics of incorporated or embedded dye molecules. In particular, we will report on the use of polymer films for immobilization of molecules, the visualization of dynamics near the glass transition temperature Tg, the reptation of polymer chains, the conformation adopted by polymer chains and the in situobservation of the polymerization reaction itself. [ABSTRACT FROM AUTHOR]

A chemically synthesized silver nanowire was used for atomic-resolution STM imaging and tip-enhanced Raman scattering (TERS) spectroscopy, yielding excellent reproducibility. This TERS tip will open a new venue to surface analysis, such as molecular finger printing at nanoscales. [ABSTRACT FROM AUTHOR]

Tip-enhanced Raman scattering (TERS) microscopy is a technique that combines the chemical sensitivity of Raman spectroscopy with the resolving power of scanning probe microscopy. The key component of any TERS setup is a plasmonically-active noble metal tip, which serves to couple far-field incident radiation with the near-field. Thus, the design and implementation of reproducible probes are crucial for the continued development of TERS as a tool for nanoscopic analysis. Here we discuss conventional methods for the fabrication of TERS-ready tips, highlighting the problems therein, as well as detailing more recent developments to improve reducibility. In addition, the idea of remote excitation-TERS is enlightened upon, whereby TERS sensitivity is further improved by using propagating surface plasmons to separate the incident radiation from the tip apex, as well as how this can be incorporated into the fabrication process. [ABSTRACT FROM AUTHOR]

Tip-enhanced Raman scattering (TERS) microscopy is a promising technique for use in surface analysis, allowing both topographic and spectroscopic information to be obtained simultaneously at a scale below 10 nm. One proposed method to further improve spatial resolution is the use of propagating surface plasmons as an excitation light source (i.e., remote excitation). However, this requires a specialized tip that can only be fabricated via expensive procedures, such as electron-beam lithography. Here, we propose a new method for fabricating silver nanowire-based tips that are suitable for remote excitation-TERS, removing the need for such techniques. A silver nanowire was fixed onto a tungsten-tip using a micromanipulator, before gold nanoparticles were attached in a site-specific manner using AC-dielectrophoresis. All the processes were completed using an optical microscope in the ambient. The background intensities in TERS spectra were suppressed with remote excitation relative to the conventional excitation configuration, indicating an increase in TERS sensitivity. [ABSTRACT FROM AUTHOR]