Bacterial replication origins move towards opposite ends of the cell during DNA segregation. We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PopZ interacts directly with the ParB protein bound to specific DNA sequences near the replication origin. As the origin/ParB complex is being replicated and moved across the cell, PopZ accumulates at the cell pole and tethers the origin in place upon arrival. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. High molecular weight oligomers of PopZ assemble in vitro into a filamentous network with trimer junctions, suggesting that the PopZ network and ParB-bound DNA interact in an adhesive complex, fixing the chromosome origin at the cell pole.

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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An interesting property of several yellow-emitting mutants of the green fluorescent protein (GFP) is that they switch between a fluorescent and a nonfluorescent state on a time scale of seconds. This peculiar blinking behavior was observed in single-molecule fluorescence studies of GFP mutants in poly(acrylamide) gels et al. Nature 1997, 388, 355.). Utilizing primarily the yellow-emitting phenolate anion mutant EGFP, we report new single-molecule experiments studying the effect of several parameters on the blinking process: pH, host matrix, and pumping intensity. The primary measurement in these studies is the observed distribution of on-times and off-times. The on-time dynamics of EGFP are independent of pH over the range of 6-10, thus making protonation/deprotonation of the chromophore unlikely as the source of the blinking. The excitation intensity, however, has a considerable effect on the blinking: the on-times are shorter at high intensity. We compare these results to ensemble bleaching measurements which find the bleaching quantum yield of EGFP in agarose gel at pH 8 to be (8 ( 2) × 10 -6 . The probability of termination of single-molecule emission per photon absorbed is in agreement with the bulk bleaching quantum yield, thus suggesting that the two processes are related.

This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules.