Automated profiling of individual cell–cell interactions from high-throughput time-lapse imaging microscopy in nanowell grids (TIMING)

Merouane, Amine; Rey-Villamizar, Nicolas; Lu, Yanbin; Liadi, Ivan; Romain, Gabrielle; Lu, Jennifer; Singh, Harjeet; Cooper, Laurence J.N.; Varadarajan, Navin; Roysam, Badrinath

doi:10.1093/bioinformatics/btv355

Cited by 50 publications

(43 citation statements)

References 52 publications

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“…Annexin V Alexa Fluor 647 (Life Technologies) was added to dynamically track APC apoptosis. Raw images were analyzed using scripts for automated image pre-processing and segmentations [22]. Resulting data were then analyzed using EXCEL, ACCESS, and Graphpad PRISM to identify and record serial killing events of interest.…”

Section: Methodsmentioning

confidence: 99%

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets

Moogk

Shi

et al. 2016

The Journal of Immunology

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CD8+ T cells develop increased sensitivity following antigen experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that more than 50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with less than 20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM. Further, we observed superior cytotoxic effector function in TEM compared with TCM, and provide evidence that this results from a lower probability of TCM reaching threshold signaling due to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8+ TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase (Csk), and use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM. Together, this work demonstrates a role for constitutive Lck activity in controlling antigen sensitivity, and suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications.

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Section: Methodsmentioning

confidence: 99%

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets

Moogk

Shi

et al. 2016

The Journal of Immunology

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“…This approach is computationally efficient but makes global optimality elusive by decoupling events that are closely related. The approach of [46], however, focuses on cell segmentation and tracking in the confined nanowell environment. Despite its high throughput, it assumes that the cells do not divide and is therefore not suitable for general-purpose cell tracking.…”

Section: Tracking By Detectionmentioning

confidence: 99%

Network Flow Integer Programming to Track Elliptical Cells in Time-Lapse Sequences

Türetken

Wang

Becker

et al. 2017

IEEE Trans. Med. Imaging

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Abstract-We propose a novel approach to automatically tracking elliptical cell populations in time-lapse image sequences. Given an initial segmentation, we account for partial occlusions and overlaps by generating an over-complete set of competing detection hypotheses. To this end, we fit ellipses to portions of the initial regions and build a hierarchy of ellipses, which are then treated as cell candidates. We then select temporally consistent ones by solving to optimality an integer program with only one type of flow variables. This eliminates the need for heuristics to handle missed detections due to partial occlusions and complex morphology. We demonstrate the effectiveness of our approach on a range of challenging sequences consisting of clumped cells and show that it outperforms state-of-the-art techniques.

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“…In recent years, the imaging technologies have provided new tools in microscopy, sample handling, and hardware and software for live imaging of individual cells. There are several examples of single cell [19][20][21][22] and single molecule monitoring in living cells, using both marker-based and marker-free approaches.…”

Section: The Role Of Live Imaging In Embryologymentioning

confidence: 99%

Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro

Mölder¹,

Fierro-González²,

Khan³

2017

Embryo Cleavage

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Automated or semiautomated time-lapse analysis of early stage embryo images during the cleavage stage can give insight into the timing of mitosis, regularity of both division timing and patern, as well as cell lineage. Simultaneous monitoring of molecular processes enables the study of connections between genetic expression and cell physiology and development. The study of live embryos poses not only new requirements on the hardware and embryo-holding equipment but also indirectly on analytical software and data analysis as four-dimensional video sequencing of embryos easily creates high quantities of data. The ability to continuously ilm and automatically analyze growing embryos gives new insights into temporal embryo development by studying morphokinetics as well as morphology. Until recently, this was not possible unless by a tedious manual process. In recent years, several methods have been developed that enable this dynamic monitoring of live embryos. Here we describe three methods with variations in hardware and software analysis and give examples of the outcomes. Together, these methods open a window to new information in developmental embryology, as embryo division patern and lineage are studied in vivo.

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Automated profiling of individual cell–cell interactions from high-throughput time-lapse imaging microscopy in nanowell grids (TIMING)

Abstract: Supplementary data are available at Bioinformatics online.

Cited by 50 publications

References 52 publications

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets

Network Flow Integer Programming to Track Elliptical Cells in Time-Lapse Sequences

Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro

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