Publikationen Abteilung Nanooptik

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

We report on the generation and annihilation of color centers in 4H silicon carbide (SiC) by proton irradiation and subsequent annealing. Using low-temperature photoluminescence (PL), we study the transformation of PL spectra for different proton doses and annealing temperatures. Among well reported defect signatures, we observe omnipresent but not yet identified PL signatures consisting of three sharp and temperature stable lines (denoted TS1,2,3) at 768.8 nm, 812.0 nm, and 813.3 nm. These lines show a strong correlation throughout all measurement parameters, suggesting that they belong to the same microscopic defect. Further, a clear dependence of the TS1,2,3 line intensities on the initial implantation dose is observed after annealing, indicating that the underlying defect is related to implantation induced intrinsic defects. The overall data suggest a sequential defect transformation: proton irradiation initially generates isolated silicon vacancies which are transformed into antisite vacancy complexes which are, in turn, transformed into presumably intrinsic-related defects, showing up as TS1,2,3 PL lines. We present recipes for the controlled generation of these color centers. Published by AIP Publishing.

Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoylsn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (l,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-l-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 mu m is found to be free. In the case of functionalization with gold and 6-mercapto-l-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.

We show that a broadband Fabry Perot microcavity can assist an emitter coupled to an off-resonant plasmonic nanoantenna to inhibit the nonradiative channels that affect the quenching of fluorescence. We identify the interference mechanism that creates the necessary enhanced couplings and bandwidth narrowing of the hybrid resonance and show that it can assist entering into the strong coupling regime. Our results provide new possibilities for improving the efficiency of solid-state emitters and accessing diverse realms of photophysics with hybrid structures that can be fabricated using existing technologies.

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.