1,344 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..
The Filament Sensor for Near Real-Time Detection of Cytoskeletal Fiber Structures
A reliable extraction of filament data from microscopic images is of high
interest in the analysis of acto-myosin structures as early morphological
markers in mechanically guided differentiation of human mesenchymal stem cells
and the understanding of the underlying fiber arrangement processes. In this
paper, we propose the filament sensor (FS), a fast and robust processing
sequence which detects and records location, orientation, length and width for
each single filament of an image, and thus allows for the above described
analysis. The extraction of these features has previously not been possible
with existing methods. We evaluate the performance of the proposed FS in terms
of accuracy and speed in comparison to three existing methods with respect to
their limited output. Further, we provide a benchmark dataset of real cell
images along with filaments manually marked by a human expert as well as
simulated benchmark images. The FS clearly outperforms existing methods in
terms of computational runtime and filament extraction accuracy. The
implementation of the FS and the benchmark database are available as open
source.Comment: 32 pages, 21 figure
Segmentation, tracking and cell cycle analysis of live-cell imaging data with Cell-ACDC
Background: High-throughput live-cell imaging is a powerful tool to study dynamic cellular processes in single cells but creates a bottleneck at the stage of data analysis, due to the large amount of data generated and limitations of analytical pipelines. Recent progress on deep learning dramatically improved cell segmentation and tracking. Nevertheless, manual data validation and correction is typically still required and tools spanning the complete range of image analysis are still needed. Results: We present Cell-ACDC, an open-source user-friendly GUI-based framework written in Python, for segmentation, tracking and cell cycle annotations. We included state-of-the-art deep learning models for single-cell segmentation of mammalian and yeast cells alongside cell tracking methods and an intuitive, semi-automated workflow for cell cycle annotation of single cells. Using Cell-ACDC, we found that mTOR activity in hematopoietic stem cells is largely independent of cell volume. By contrast, smaller cells exhibit higher p38 activity, consistent with a role of p38 in regulation of cell size. Additionally, we show that, in S. cerevisiae, histone Htbl concentrations decrease with replicative age. Conclusions: Cell-ACDC provides a framework for the application of state-of-the-art deep learning models to the analysis of live cell imaging data without programming knowledge. Furthermore, it allows for visualization and correction of segmentation and tracking errors as well as annotation of cell cycle stages. We embedded several smart algorithms that make the correction and annotation process fast and intuitive. Finally, the open-source and modularized nature of Cell-ACDC will enable simple and fast integration of new deep learning-based and traditional methods for cell segmentation, tracking, and downstream image analysis.Peer reviewe
Fuzzy-based Propagation of Prior Knowledge to Improve Large-Scale Image Analysis Pipelines
Many automatically analyzable scientific questions are well-posed and offer a
variety of information about the expected outcome a priori. Although often
being neglected, this prior knowledge can be systematically exploited to make
automated analysis operations sensitive to a desired phenomenon or to evaluate
extracted content with respect to this prior knowledge. For instance, the
performance of processing operators can be greatly enhanced by a more focused
detection strategy and the direct information about the ambiguity inherent in
the extracted data. We present a new concept for the estimation and propagation
of uncertainty involved in image analysis operators. This allows using simple
processing operators that are suitable for analyzing large-scale 3D+t
microscopy images without compromising the result quality. On the foundation of
fuzzy set theory, we transform available prior knowledge into a mathematical
representation and extensively use it enhance the result quality of various
processing operators. All presented concepts are illustrated on a typical
bioimage analysis pipeline comprised of seed point detection, segmentation,
multiview fusion and tracking. Furthermore, the functionality of the proposed
approach is validated on a comprehensive simulated 3D+t benchmark data set that
mimics embryonic development and on large-scale light-sheet microscopy data of
a zebrafish embryo. The general concept introduced in this contribution
represents a new approach to efficiently exploit prior knowledge to improve the
result quality of image analysis pipelines. Especially, the automated analysis
of terabyte-scale microscopy data will benefit from sophisticated and efficient
algorithms that enable a quantitative and fast readout. The generality of the
concept, however, makes it also applicable to practically any other field with
processing strategies that are arranged as linear pipelines.Comment: 39 pages, 12 figure
Image Processing and Simulation Toolboxes of Microscopy Images of Bacterial Cells
Recent advances in microscopy imaging technology have allowed the characterization of the dynamics of cellular processes at the single-cell and single-molecule level. Particularly in bacterial cell studies, and using the E. coli as a case study, these techniques have been used to detect and track internal cell structures such as the Nucleoid and the Cell Wall and fluorescently tagged molecular aggregates such as FtsZ proteins, Min system proteins, inclusion bodies and all the different types of RNA molecules. These studies have been performed with using multi-modal, multi-process, time-lapse microscopy, producing both morphological and functional images.
To facilitate the finding of relationships between cellular processes, from small-scale, such as gene expression, to large-scale, such as cell division, an image processing toolbox was implemented with several automatic and/or manual features such as, cell segmentation and tracking, intra-modal and intra-modal image registration, as well as the detection, counting and characterization of several cellular components.
Two segmentation algorithms of cellular component were implemented, the first one based on the Gaussian Distribution and the second based on Thresholding and morphological structuring functions. These algorithms were used to perform the segmentation of Nucleoids and to identify the different stages of FtsZ Ring formation (allied with the use of machine learning algorithms), which allowed to understand how the temperature influences the physical properties of the Nucleoid and correlated those properties with the exclusion of protein aggregates from the center of the cell. Another study used the segmentation algorithms to study how the temperature affects the formation of the FtsZ Ring.
The validation of the developed image processing methods and techniques has been based on benchmark databases manually produced and curated by experts. When dealing with thousands of cells and hundreds of images, these manually generated datasets can become the biggest cost in a research project. To expedite these studies in terms of time and lower the cost of the manual labour, an image simulation was implemented to generate realistic artificial images.
The proposed image simulation toolbox can generate biologically inspired objects that mimic the spatial and temporal organization of bacterial cells and their processes, such as cell growth and division and cell motility, and cell morphology (shape, size and cluster organization). The image simulation toolbox was shown to be useful in the validation of three cell tracking algorithms: Simple Nearest-Neighbour, Nearest-Neighbour with Morphology and DBSCAN cluster identification algorithm. It was shown that the Simple Nearest-Neighbour still performed with great reliability when simulating objects with small velocities, while the other algorithms performed better for higher velocities and when there were larger clusters present
Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms
The zip archive contains real images showing macrophages. (ZIP 28979 kb
New Methods to Improve Large-Scale Microscopy Image Analysis with Prior Knowledge and Uncertainty
Multidimensional imaging techniques provide powerful ways to examine various kinds of scientific questions. The routinely produced data sets in the terabyte-range, however, can hardly be analyzed manually and require an extensive use of automated image analysis. The present work introduces a new concept for the estimation and propagation of uncertainty involved in image analysis operators and new segmentation algorithms that are suitable for terabyte-scale analyses of 3D+t microscopy images
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