Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

We present a single-molecule tool called the CoPro (Concentration of Proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrates its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a 4-fold shift towards higher values in concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, which is almost exclusively localized in the nucleus under high and..

Rapid rotation of micron and submicron dielectric particles measured using optical tweezers

We demonstrate the use of a laser trap (‘optical tweezers’) and back-focal-plane position detector to measure rapid rotation in aqueous solution of single particles with sizes in the vicinity of 1 μm. Two types of rotation were measured: electrorotation of polystyrene microspheres and rotation of the flagellar motor of the bacterium Vibrio alginolyticus. In both cases, speeds in excess of 1000 Hz (rev s−1) were measured. Polystyrene beads of diameter about 1 μm labelled with smaller beads were held at the centre of a microelectrode array by the optical tweezers. Electrorotation of the labelled beads was induced by applying a rotating electric field to the solution using microelectrodes. Electrorotation spectra were obtained by varying the frequency of the applied field and analysed to obtain the surface conductance of the beads. Single cells of V. alginolyticus were trapped and rotation of the polar sodium-driven flagellar motor was measured. Cells rotated more rapidly in media containing higher concentrations of Na+, and photodamage caused by the trap was considerably less when the suspending medium did not contain oxygen. The technique allows single-speed measurements to be made in less than a second and separate particles can be measured at a rate of several per minute

An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells

Analytical tools for single-molecule fluorescence imaging in cellulo

Recent technological advances in cutting-edge ultrasensitive fluorescence microscopy have allowed single-molecule imaging experiments in living cells across all three domains of life to become commonplace. Single-molecule live-cell data is typically obtained in a low signal-to-noise ratio (SNR) regime sometimes only marginally in excess of 1, in which a combination of detector shot noise, sub-optimal probe photophysics, native cell autofluorescence and intrinsically underlying stochasticity of molecules result in highly noisy datasets for which underlying true molecular behaviour is non-trivial to discern. The ability to elucidate real molecular phenomena is essential in relating experimental single-molecule observations to both the biological system under study as well as offering insight into the fine details of the physical and chemical environments of the living cell. To confront this problem of faithful signal extraction and analysis in a noise-dominated regime, the 'needle in a haystack' challenge, such experiments benefit enormously from a suite of objective, automated, high-throughput analysis tools that can home in on the underlying 'molecular signature' and generate meaningful statistics across a large population of individual cells and molecules. Here, I discuss the development and application of several analytical methods applied to real case studies, including objective methods of segmenting cellular images from light microscopy data, tools to robustly localize and track single fluorescently-labelled molecules, algorithms to objectively interpret molecular mobility, analysis protocols to reliably estimate molecular stoichiometry and turnover, and methods to objectively render distributions of molecular parameters

Evolution of the interfacial structure of LaAlO3 on SrTiO3

The evolution of the atomic structure of LaAlO3 grown on SrTiO3 was investigated using surface x-ray diffraction in conjunction with model-independent, phase-retrieval algorithms between two and five monolayers film thickness. A depolarizing buckling is observed between cation and oxygen positions in response to the electric field of polar LaAlO3, which decreases with increasing film thickness. We explain this in terms of competition between elastic strain energy, electrostatic energy, and electronic reconstructions. The findings are qualitatively reproduced by density-functional theory calculations. Significant cationic intermixing across the interface extends approximately three monolayers for all film thicknesses. The interfaces of films thinner than four monolayers therefore extend to the surface, which might affect conductivity

Structural Examination of Au/Ge(001) by Surface X-Ray Diffraction and Scanning Tunneling Microscopy

The one-dimensional reconstruction of Au/Ge(001) was investigated by means of autocorrelation functions from surface x-ray diffraction (SXRD) and scanning tunneling microscopy (STM). Interatomic distances found in the SXRD-Patterson map are substantiated by results from STM. The Au coverage, recently determined to be 3/4 of a monolayer of gold, together with SXRD leads to three non-equivalent positions for Au within the c(8x2) unit cell. Combined with structural information from STM topography and line profiling, two building blocks are identified: Au-Ge hetero-dimers within the top wire architecture and Au homo-dimers within the trenches. The incorporation of both components is discussed using density functional theory and model based Patterson maps by substituting Germanium atoms of the reconstructed Ge(001) surface.Comment: 5 pages, 3 figure