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Single metallic bowtie nanoantennas provide a controllable environment for surface-enhanced Raman scattering (SERS) of adsorbed molecules. Bowties have experimentally measured electromagnetic enhancements, enabling estimation of chemical enhancement for both the bulk and the few-molecule regime. Strong fluctuations of selected Raman lines imply that a small number of p-mercaptoaniline molecules on a single bowtie show chemical enhancement >10 7 , much larger than previously believed, likely due to charge transfer between the Au surface and the molecule. This chemical sensitivity of SERS has significant implications for ultra-sensitive detection of single molecules.Rapid and accurate detection and identification of trace amounts of chemical species is of the utmost importance in biology, chemistry, medicine, and defense. Recent advances in fluorescence spectroscopy methods offer exquisite sensitivity, enabling the ultimate in analytical detection: single molecules. 1 However, fluorescence studies require specially engineered labels, a limitation that places constraints on potential applications. Raman spectroscopy does not suffer from this limitation, since most molecules display a unique set of molecular vibrations that give rise to a distinctive chemical fingerprint, especially attractive for ultra-selective analysis.Because Raman transitions are incredibly weak, this technique was not generally believed to offer the potential sensitivity afforded by fluorescence. However, over 30 years ago, it was first observed that the Raman signal of pyridine dramatically increases when adsorbed on a roughened Ag electrode, 2,3 and the detailed origins of surface-enhanced Raman scattering (SERS) arising from nanostructured metals have remained a topic of debate. Researchers linked SERS signals to a combination of two effects, 4 electromagnetic (EM) enhancement, where illumination intensity is enhanced due to sharp metal edges or plasmon effects, and chemical enhancement (CE), where the Raman cross-section of adsorbed molecules is increased above the solution value, 5 with EM enhancement dominant. Detailed SERS experiments performed on roughened metal films in electrochemical cells revealed the importance of CE due to the applied potential dependence of SERS spectra, 6,7 but measured values of either enhancement were not available.Interest in the SERS mechanism blossomed with the recent observation of Raman lines apparently arising from single molecules adsorbed onto colloidal Ag and Au particles. 4,8,9 To obtain the 14-order of magnitude enhancement required to make Raman signals competitive

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The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.bacteria ͉ cytoskeleton ͉ single-molecule fluorescence I n both eukaryotic and prokaryotic cells, actin mediates essential cellular processes. A quantitative understanding of the kinetic dynamics and ultrastructural architecture of actin's polymerized filaments has helped elucidate the mechanisms by which eukaryotic actin functions. For example, high-resolution imaging and the in vivo and in vitro dissection of the kinetics of its assembly have demonstrated how actin polymerization at the tips of a rigid, crosslinked actin meshwork can drive cell motility at the leading edge of Dictyostelium (1, 2). In budding yeast, the polarized assembly of actin cables provides both a road and direction signs for the directed transport of proteins to the tip of growing buds (3).There are two known bacterial actin homologs, the widely conserved, chromosomally encoded MreB family of proteins and the plasmid-specific ParM family of proteins. ParM functions to partition plasmid DNA by polymerizing in between two sister plasmids, thereby generating a tension rod that physically pushes them apart (4). MreB is essential in most bacteria and has been shown to form a lengthwise spiral that contributes to cell shape, chromosome segregation, and polar protein localization in multiple species, including Caulobacter crescentus, Escherichia coli, and Bacillus subtilis (5-10). The mechanism by which MreB executes its functions remains largely unknown (11).In vitro studies of the dynamics of eukaryotic actin filament assembly have demonstrated th...

Cytosolic fluid dynamics have been implicated in cell motility 1-5 because of the hydrodynamic forces they induce and because of their influence on transport of components of the actin machinery to the leading edge. To investigate the existence and the direction of fluid flow in rapidly moving cells, we introduced inert quantum dots into the lamellipodia of fish epithelial keratocytes and analysed their distribution and motion. Our results indicate that fluid flow is directed from the cell body towards the leading edge in the cell frame of reference, at about 40% of cell speed. We propose that this forward-directed flow is driven by increased hydrostatic pressure generated at the rear of the cell by myosin contraction, and show that inhibition of myosin II activity by blebbistatin reverses the direction of fluid flow and leads to a decrease in keratocyte speed. We present a physical model for fluid pressure and flow in moving cells that quantitatively accounts for our experimental data.Various indirect results 4,6 suggest that fluid influx at the leading edge could play an active part in actin-based cell motility by generating hydrodynamic forces that oppose the membrane load and thus increase the rate of actin polymerization at a protruding edge. Notably, the expression of aquaporins, which are highly enriched at the leading edge and increase water permeability of the membrane, accelerates motility and increases the metastatic potential of melanoma cells4 , 7. Alternatively, it has been suggested that intracellular fluid flow towards the leading edge might contribute to motility by expediting transport of actin and other soluble proteins to the leading edge8. Direct measurements of fluid flow in the lamellipodia of moving cells are © 2009 Macmillan Publishers Limited. All rights reserved.7Correspondence should be addressed to K. K. (kinneret@ph.technion.ac.il). AUTHOR CONTRIBUTIONS K.K. and J.A.T. conceived and designed the experiments; K.K. performed the experiments and analysed the data; A.M. and K.K. developed the model; P.T.Y. made the initial observation of enhancement of large probes at the leading edge; A.K. contributed to the single-particle tracking experiments; K.K., A.M., P.T.Y. and J.A.T. discussed the results and wrote the paper.Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING INTERESTSThe authors declare that they have no competing financial interest. Fish keratocytes are a simple and widely used model system for studying the dynamics of the motility process. They are among the fastest moving animal cells, with average speeds of about 0.3 μm s -1 , yet their motion is extremely persistent, with hardly any change in cell shape, speed or direction over many minutes12 ,13 . In these cells, any intracellular fluid flow associated with motility should be persistent because of the steady-state nature of their motion. Furthermore, the broad, flat and extremely thin (~100-200 nm) 10 lamellipodia of these cells alone are sufficient for persistent motility12...