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..

The geology and petrology of the pre-camhrian basement "between Sirdal and Åseral, Vest Agder, Norway

The field relations and petrography of the rocks of the PreCambrian basement complex between Sirdal and Aseral, comprising two series of high-grade metamorphic gneisses separated by a structural discontinuity, syntectonic granites, intrusive quartz monzonites with thermal metamorphic aureoles and basic dykes, are described. During orogeny the gneisses were subjected to intense poly-phase deformation, three regional and two localised phases of which have been recognised. Minor fold relics within augen gneiss in the lower gneiss sequence suggest that this rock was involved in earlier deformation. the climax of metamorphic crystallisation occurred at the low-pressure granulite facies-amphibolite facies boundary with mineral parageneses corresponding closely with the sillimanite-cordierite-orthoclase subfacies of the Abukuma-type cordierite amphibolite facies except for the additional occurrence of orthopyroxene. Major and trace elements X.R.F. analyses of gneissic and some igneous rocks are presented. These data reveal significant differences between basic rocks of the two gneiss series, basic gneisses with different mineral assemblages and to a lesser extent different lithostatigraphical units in the upper gneiss series. Electron microprobe analyses of alkali feldspar, plagioclase, biotite, hornblende, clinopyroxenes, orthopyroxene, sphene, magnetite, ilmenite, chlorite, and garnet from several rock types are presented. With the exceptions of alkali feldspar, magnetite and ilmenite all minerals are chemically homogeneous and represent original equilibrium compositions. The chemical inhomogeneity of alkali feldspar resulted from post-crystallisation leaching and redistribution of alkalies, resistance to which is related to grain size. Equilibrium during original feldspar crystallisation is indicated by the restricted composition of plagioclase coexisting with alkali feldspar. The distribution of titanium and magnesium between coexisting silicates indicates equilibrium compositions, influenced by oxygen fugacity, the nature of the coexisting iron oxides and the tetrahedral aluminium content of the hydrous phases in addition to the rock composition. The application of several means of multicomponent paragenesis analysis reveals that the various mineral assemblages can be interpreted in terms of variations in major element rock composition and oxygen fugacity. The widespread molybdenite mineralisation is considered to have been transported from depth in siliceous hydrothermal solutions into the gneisses, especially where the strike of the gneissic layering coincided with deep fractures. Fixing of the metal as sulphide occurred particularly in the vicinity of pre-existing fahlband sulphide due to release of sulphur in the local environment of increased oxygen fugacity

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

Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision

In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences