A new detection algorithm for image analysis of single, fluorescence‐labeled proteins in living cells

Michel, René; Steinmeyer, Ralf; Falk, Michael; Harms, Gregory S.

doi:10.1002/jemt.20485

Cited by 16 publications

(15 citation statements)

References 28 publications

(40 reference statements)

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“…The experimental arrangement for single-molecule imaging has been described in detail previously (35,36). Essentially, the samples were mounted onto an inverted microscope (Axiovert 200; Zeiss, Gttingen, Germany) equipped with a 100 objective (PlanNeofluor 100, N.A.…”

Section: Methodsmentioning

confidence: 99%

Covalent Quantum dot Receptor Linkage via the acyl Carrier Protein for Single-Molecule Tracking, Internalization, and Trafficking Studies

et al. 2010

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Here we describe a labeling technique for the covalent linkage of quantum dots to transmembrane receptors for single-molecule tracking. Our method combines the acyl carrier protein (ACP) technique with coenzyme A (CoA)-functionalized quantum dots to covalently attach quantum dots to ACP fusions of receptor proteins. The advantages of this approach include: (i) the use of a smaller attachment linker than in many other quantum dot-labeling systems; (ii) the ability to achieve a reliable 1:1 fluorophore-to-receptor labeling stoichiometry; (iii) the specificity of the method; and (iv) the covalent nature of the quantum dot linkage. We demonstrate the general suitability of this technique in single-molecule tracking, internalization, and trafficking studies by imaging two different transmembrane receptors in living cells.

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Section: Methodsmentioning

confidence: 99%

Covalent Quantum dot Receptor Linkage via the acyl Carrier Protein for Single-Molecule Tracking, Internalization, and Trafficking Studies

et al. 2010

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“…Excursion distance is measured as the maximum separation between any two points on the organelle track. The image denoising uses an approach proposed for protein image denoising (Michel et al, 2007) and subsequently applied by us for 2-D organelle images (Chenouard et al, 2014) and 3-D stem cell time lapse images (Wait et al, 2014). This approach models the noise as slow varying background combined with high-frequency shot noise.…”

Section: Methodsmentioning

confidence: 99%

A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells

et al. 2016

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Summary Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo”) occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins; the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naïve Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes, and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels and signaling proteins having a role in lysosome motility regulation, and find an unexpected relationship between the dynein motor, Rab7a and lysosome motility regulation.

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“…Here the HSCs typically appear as dark circular regions within the stromal layer. To detect these circular regions we used Michel's technique to enhance the contrast in the image region [10], segmented the image into foreground and background using Otsu thresholding, and then looked for pixels on the edges of circles using the Circle Hough Transform (CHT) [11]. …”

Section: Methodsmentioning

confidence: 99%

Segmentation of occluded hematopoietic stem cells from tracking

Mankowski

Winter

Wait

et al. 2014

2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society

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Image sequences of live proliferating cells often contain visual ambiguities that are difficult even for human domain experts to resolve. Here we present a new approach to analyzing image sequences that capture the development of clones of hematopoietic stem cells (HSCs) from live cell time lapse microscopy. The HSCs cannot survive long term imaging unless they are cultured together with a secondary cell type, OP9 stromal cells. The HSCs frequently disappear under the OP9 cell layer, making segmentation difficult or impossible from a single image frame, even for a human domain expert. We have developed a new approach to the segmentation of HSCs that captures these occluded cells. Starting with an a priori segmentation that uses a Monte Carlo technique to estimate the number of cells in a clump of touching cells, we proceed to track and lineage the image data. Following user validation of the lineage information, an a posteriori resegmentation step utilizing tracking results delineates the HSCs occluded by the OP9 layer. Resegmentation has been applied to 3031 occluded segmentations from 77 tracks, correctly recovering over 84% of the occluded segmentations.

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A new detection algorithm for image analysis of single, fluorescence‐labeled proteins in living cells

Cited by 16 publications

References 28 publications

Covalent Quantum dot Receptor Linkage via the acyl Carrier Protein for Single-Molecule Tracking, Internalization, and Trafficking Studies

Covalent Quantum dot Receptor Linkage via the acyl Carrier Protein for Single-Molecule Tracking, Internalization, and Trafficking Studies

A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells

Segmentation of occluded hematopoietic stem cells from tracking

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