1,277 research outputs found
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..
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
Simplified simulation models for control studies of turbojet engines
The essential dynamical characteristics of a simple single spool turbojet engine were determined through simulation of low order system models on an analog computer. An accurate model was studied and system complexity was reduced through various linearizations and approximations. A derivation of a seventh order simplified simulation model is presented with a derivation of an even simpler third order model, and simulation results from each. The control problem studied is one of getting from zero fuel flow equilibrium to a high thrust equilibrium while taking into account surge margin and turbine inlet temperature constraints
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
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