The sensitivity and resolution of singlemolecule fluorescence imaging in biology are mainly limited by two known weaknesses of fluorescent proteins: label brightness and photostability. In this work, we use patterned gold substrates to achieve plasmon-enhanced emission from intrinsically fluorescent proteins in living pathogenic bacteria cells. By coupling membrane-bound single fluorescent protein fusions to the virulence regulator TcpP in living Vibrio cholerae bacteria to extracellular gold nanotriangle arrays, we use plasmonics to improve our measurements of this important question in pathogenesis: how does V. cholerae produce its deadly toxin? Based on a simple experimental geometry, we observe a 1.3× enhancement in the rate of emission and a 1.4× enhancement in the number of photons detected prior to photobleaching. Furthermore, by enhancing both the rate of emission and the total number of photons detected from single-molecule fluorescent probes in live cells, we show that plasmon-enhanced fluorescence is a biocompatible, generalizable path to directly improve the resolution and trajectory lengths of single molecules in live cells.

Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane.

Summary Vibrio cholerae causes the human disease cholera by producing a potent toxin. The V. cholerae virulence pathway involves an unusual transcription step: the bitopic inner-membrane proteins TcpP and ToxR activate toxT transcription. As ToxT is the primary direct transcription activator in V. cholerae pathogenicity, its regulation by membrane-localized activators is key in the disease process. However, the molecular mechanisms by which membrane-localized activators engage the transcription process have yet to be uncovered in live cells. Here we report the use of super-resolution microscopy, single-molecule tracking, and gene knockouts to examine the dynamics of individual TcpP proteins in live V. cholerae cells with <40-nm spatial resolution on a 50-ms timescale. Single-molecule trajectory analysis reveals that TcpP diffusion is heterogeneous and can be described by three populations of TcpP motion: one fast, one slow, and one immobile. By comparing TcpP diffusion in wild type V. cholerae to that in mutant strains lacking either toxR or the toxT promoter, we determine that TcpP mobility is greater in the presence of its interaction partners than in their absence. Our findings support a mechanism in which ToxR recruits TcpP to the toxT promoter for transcription activation.

spectroscopy when the fluorophores are excited through pulsed lasers. The large variety of fluorophores requires full spectral coverage through available pulsed lasers. Here, we present a new pulsed laser, filling the existing gap at 560 nm pulsed excitation. This freely triggerable laser source can operate in a wide range of repetition frequencies from 1 MHz up to 80 MHz which makes it easy to adapt the pulse period to different fluorescence lifetimes. Synchronization to other lasers or scanning devices is possible as well as burst operation. The wavelength around 560 nm is appropriate for a wide range of applications, especially in the life sciences where state of the art red fluorescent proteins excited at about 560 nm are essential. A pulsed 559 nm laser enables the long sought Fluorescence Lifetime studies like FLIM (Fluorescence Lifetime Imaging Microscopy) or Fluorescence Lifetime (Cross-)Correlation Spectroscopy (FL(C)CS) involving Red Fluorescent Proteins like mCherry and with that provided access to molecular interaction studies and background reduction. Implemented in a confocal microscope like the MicroTime 200 (PicoQuant) this laser becomes a versatile tool. 2008-Pos Board B738Highly ordered arrays of ryanodine receptor type 2 (RyR2) are believed to be critical for synchronous Ca release and effective, stable excitation-contraction coupling in adult cardiomyocytes. Altered RyR2 distribution and intracellular architectures have been implicated in the genesis of dyssynchronous Ca release often observed in disease hearts. To gain insights into the expression and distribution of RyR2 and their correlation with function in situ in adult cardiomyocytes, we generated a knock-in mouse model in which the green fluorescence protein (GFP) has been inserted into RyR2 after residue T1366. The GFP-tagged RyR2 mice show no gross structural and functional abnormalities. Confocal laser scanning microscopy of isolated cardiomyocytes from the GFP-tagged RyR2 mice revealed discrete clusters of GFP-RyR2 located along Z-lines. Confocal Ca imaging analysis of GFP-tagged RyR2 cardiomyocytes loaded with Rhod-2 AM showed that Ca sparks originate from GFP-RyR2 clusters and rarely occur in non-Z-line region. These observations suggest that the production of Ca sparks may require clustering of RyR2. To further define the distribution of GFP-RyR2 clusters, we employed a camelid single-domain antibody against GFP (GFP-nanobody) conjugated with the Alexa Fluor (AF) 647 fluorescent dye. The distribution of the GFP-nanobody staining in GFP-tagged RyR2 cardiomyocytes was found to be identical to that of GFP-RyR2 clusters. Further super-resolution imaging using the AF647-labelled GFP-nanobody should provide new and detailed insights into the nano-distribution of RyR2 in cardiomyocytes (Supported by CFI, CIHR, and LCIA). 2009-Pos Board B739 Super Resolution Microscopy with Low Power CW LasersWe demonstrate a new and experimentally straightforward method for obtaining sub-diffraction limit resolution in fluorescence microscopy with low o...

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