A plasmonic 'antenna-in-box' platform for enhanced single-molecule analysis at micromolar concentrations

International audienceSingle molecule fluorescence techniques [1-3] are key for several applications including DNA sequencing [4, 5], molecular and cell biology [6, 7], and early diagnosis [8]. Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and imperatively requires pico-or nanomolar concentrations, far below the micromolar range where most biological reactions occur [2]. This limitation can be overcome using plasmonic nanostructures, and confinement of light down to nanoscale volumes has been reported [9-13]. While these nanoantennas enhance fluorescence brightness [14-20], large background signals [20-22] and/or unspecific binding to the metallic surface [23-25] has hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel "antenna-in-box" platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single molecule sensitivity (fluorescence enhancement up to 1100 folds and microsecond transit time) at micromolar sample concentrations and zeptolitre-range detection volumes. The antennain-box device can be optimized for single molecule fluorescence studies at physiologicallyrelevant concentrations, as we demonstrate using various biomolecules. Our antenna-in-box design is shown in Figure 1a and b. The rationale behind our design is that in any nanoantenna experiment on molecules in solution, the observed fluorescence signal is a sum of two contributions: the enhanced fluorescence from the few molecules in the nanoantenna gap region (ho

Nano-antenna enhanced two-focus fluorescence correlation spectroscopy

We propose two-focus fluorescence correlation spectroscopy (2fFCS) on basis of plasmonic nanoantennas that provide distinct hot spots that are individually addressable through polarization, yet lie within a single diffraction limited microscope focus. The importance of two-focus FCS is that a calibrated distance between foci provides an intrinsic calibration to derive diffusion constants from measured correlation times. Through electromagnetic modelling we analyze a geometry of perpendicular nanorods, and their inverse, i.e., nanoslits. While we find that nanorods are not suited for nano-antenna enhanced 2fFCS due to substantial background signal, a nanoslit geometry is expected to provide a di tinct cross-correlation between orthogonally polarized detection channels. Furthermore, by utilizing a periodic array of nanoslits instead of a single pair, the amplitude of the cross-correlation can be enhanced. To demonstrate this technique, we present a proof of principle experiment on the basis of a periodic array of nanoslits, applied to lipid diffusion in a supported lipid bilayer

Genetic targeting and anatomical registration of neuronal populations in the zebrafish brain with a new set of BAC transgenic tools

Genetic access to small, reproducible sets of neurons is key to an understanding of the functional wiring of the brain. Here we report the generation of a new Gal4- and Cre-driver resource for zebrafish neurobiology. Candidate genes, including cell type-specific transcription factors, neurotransmitter-synthesizing enzymes and neuropeptides, were selected according to their expression patterns in small and unique subsets of neurons from diverse brain regions. BAC recombineering, followed by Tol2 transgenesis, was used to generate driver lines that label neuronal populations in patterns that, to a large but variable extent, recapitulate the endogenous gene expression. We used image registration to characterize, compare, and digitally superimpose the labeling patterns from our newly generated transgenic lines. This analysis revealed highly restricted and mutually exclusive tissue distributions, with striking resolution of layered brain regions such as the tectum or the rhombencephalon. We further show that a combination of Gal4 and Cre transgenes allows intersectional expression of a fluorescent reporter in regions where the expression of the two drivers overlaps. Taken together, our study offers new tools for functional studies of specific neural circuits in zebrafish