scholarly journals Time‐Lapse Confocal Imaging of Development ofBacillus anthracisin Macrophages

2004 ◽  
Vol 189 (7) ◽  
pp. 1313-1316 ◽  
Author(s):  
Gordon Ruthel ◽  
Wilson J. Ribot ◽  
Sina Bavari ◽  
Timothy A. Hoover
Keyword(s):  
Time Lapse ◽  
Confocal Imaging

Ex vivo time-lapse confocal imaging of the mouse embryo aorta

2011 ◽  
Vol 6 (11) ◽  
pp. 1792-1805 ◽  
Author(s):  
Jean-Charles Boisset ◽  
Charlotte Andrieu-Soler ◽  
Wiggert A van Cappellen ◽  
Thomas Clapes ◽  
Catherine Robin
Keyword(s):  
Mouse Embryo ◽  
Ex Vivo ◽  
Time Lapse ◽  
Confocal Imaging

scholarly journals Four-color, 4-D time-lapse confocal imaging of chick embryos

BioTechniques ◽  
2005 ◽  
Vol 39 (5) ◽  
pp. 703-710 ◽  
Author(s):  
Jessica M. Teddy ◽  
Rusty Lansford ◽  
Paul M. Kulesa
Keyword(s):  
Time Lapse ◽  
Confocal Imaging ◽  
Chick Embryos

Time-lapse confocal imaging of clock gene expression and calcium in neuronal networks of suprachiasmatic nucleus

2011 ◽  
Vol 71 ◽  
pp. e53
Author(s):  
Ryosuke Enoki ◽  
Shigeru Kuroda ◽  
Daisuke Ono ◽  
Tetsuo Ueda ◽  
Hasan Mazhir ◽  
...  
Keyword(s):  
Gene Expression ◽  
Suprachiasmatic Nucleus ◽  
Neuronal Networks ◽  
Clock Gene ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Clock Gene Expression

6 ANEUPLOIDY TOLERANCE IN RHESUS MACAQUE PRE-IMPLANTATION EMBRYOS VIA MICRONUCLEI FORMATION, CELLULAR FRAGMENTATION, AND BLASTOMERE EXCLUSION

2017 ◽  
Vol 29 (1) ◽  
pp. 110 ◽  
Author(s):  
B. L. Daughtry ◽  
J. L. Rosenkrantz ◽  
N. Lazar ◽  
N. Redmayne ◽  
K. A. Nevonen ◽  
...  
Keyword(s):  
Rhesus Macaque ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Chromosomal Mosaicism ◽  
Human Embryos ◽  
Cleavage Stage ◽  
Chromosomal Breakage

A primary contributor to in vitro fertilization (IVF) failure is the presence of unbalanced chromosomes in pre-implantation embryos. Previous array-based and next-generation sequencing (NGS) studies determined that ~50 to 80% of human embryos are aneuploid at the cleavage stage. During early mitotic divisions, many human embryos also sequester mis-segregated chromosomes into micronuclei and concurrently undergo cellular fragmentation. We hypothesised that cellular fragmentation represents a response to mis-segregated chromosomes that are encapsulated into micronuclei. Here, we utilised the rhesus macaque pre-implantation embryo as a model to study human embryonic aneuploidy using a combination of EevaTM time-lapse imaging for evaluating cell divisions, single-cell/-fragment DNA-Sequencing (DNA-Seq), and confocal microscopy of nuclear structures. Results from our time-lapse image analysis demonstrated that there are considerable differences in the timing of the first and third mitotic divisions between rhesus blastocysts and those that arrested before this stage in development (P < 0.01; ANOVA). By examining the chromosome content of each blastomere from cleavage stage embryos via DNA-Seq, we determined that rhesus embryos have an aneuploidy frequency up to ~62% (N = 26) with several embryos exhibiting chromosomal mosaicism between blastomeres (N = 6). Certain blastomeres also exhibited reciprocal whole chromosomal gains or losses, indicating that these embryos had undergone mitotic non-disjunction early in development. In addition, findings of reciprocal sub-chromosomal deletions/duplications among blastomeres suggest that chromosomal breakage had occurred in some embryos as well. Embryo immunostaining for the nuclear envelope protein, LAMIN-B1, demonstrated that fragmented cleavage-stage rhesus embryos often contain micronuclei and that cellular fragments can enclose DNA. Our DNA-Seq analysis confirmed that cellular fragments might encapsulate whole and/or partial chromosomes lost from blastomeres. When embryos were immunostained with gamma-H2AX, a marker of chromatin fragility, we observed distinct foci solely in micronuclei and DNA-containing cellular fragments. This suggests that micronuclei may be ejected from blastomeres through the process of cellular fragmentation and, once sequestered, these mis-segregated chromosomes become highly unstable and undergo DNA degradation. Finally, we also observed that ~10% of embryos prevented cellular fragments or large blastomeres from incorporating into the inner cell mass or trophectoderm at the blastocyst stage (n = 5). Upon confocal imaging, multiple nuclei and intense gamma-H2AX foci were found in a large unincorporated blastomere in one of the blastocysts. Altogether, our findings demonstrate that the rhesus embryo responds to segregation errors by eliminating chromosome-containing micronuclei via cellular fragmentation and/or selecting against aneuploid blastomeres that fail to divide during pre-implantation development with significant implications for human IVF.

Differential Localization of RAC1 and RAC2 Reflects Their Specific Functions in Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2302-2302
Author(s):  
Marta Ewa Capala ◽  
Henny Maat ◽  
Francesco Bonardi ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Hematopoietic Cells ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Interaction Partners

Abstract Abstract 2302 Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38−/Lin− HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic and involved in processes such as cell cycle progression, mitosis and regulation of apoptosis. Consistently with these findings, confocal time-lapse imaging of living hematopoietic cells revealed that pharmacological inhibition of RAC2 activity resulted in greatly decreased frequency of cell division. Moreover, the average division time was significantly extended upon RAC2 inhibition. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In Vivo Imaging of Cerebral Microvascular Plasticity from Birth to Death

2012 ◽  
Vol 33 (1) ◽  
pp. 146-156 ◽  
Author(s):  
Roa Harb ◽  
Christina Whiteus ◽  
Catarina Freitas ◽  
Jaime Grutzendler
Keyword(s):  
Large Scale ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Adult Brain ◽  
Vessel Formation ◽  
Adult Mice ◽  
Age Related ◽  
Vascular Stability

Cerebral function and viability are critically dependent on efficient delivery of oxygen and glucose through the microvasculature. Here, we studied individual microvessels in the intact brain using high-resolution confocal imaging and long-term time-lapse two-photon microscopy across the lifetime of a mouse. In the first postnatal month, we found large-scale sprouting but to our surprise the majority of sprouts underwent pruning and only a small fraction became perfused capillaries. After the first month, microvessel formation and elimination decreased and the net number of vessels stabilized. Although vascular stability was the hallmark of the adult brain, some vessel formation and elimination continued throughout life. In young adult mice, vessel formation was markedly increased after exposure to hypoxia; however, upon return to normoxia, no vessel elimination was observed, suggesting that new vessels constitute a long-term adaptive response to metabolic challenges. This plasticity was markedly reduced in older adults and aging where hypoxia-induced angiogenesis was absent. Our study describes, for the first time in vivo patterns of cerebral microvascular remodeling throughout life. Disruption of the observed balance between baseline turnover and vascular stability may underlie a variety of developmental and age-related degenerative neurological disorders.

Resting Microglial Motility Is Independent of Synaptic Plasticity in Mammalian Brain

2008 ◽  
Vol 99 (4) ◽  
pp. 2026-2032 ◽  
Author(s):  
Long-Jun Wu ◽  
Min Zhuo
Keyword(s):  
Synaptic Plasticity ◽  
Brain Injuries ◽  
Fluorescent Protein ◽  
Hippocampal Slices ◽  
Time Lapse ◽  
Long Term Potentiation ◽  
Confocal Imaging ◽  
Mammalian Brain ◽  
Protective Function ◽  
Green Fluorescent

Microglia are well known for their roles in brain injuries and infections. However, there is no function attributes to resting microglia thus far. Here we performed a combination of simultaneous electrophysiology and time-lapse confocal imaging in green fluorescent protein–labeled microglia in acute hippocampal slices. In contrast to CA1 neurons, microglia showed no spontaneous or evoked synaptic currents. Neither glutamate- nor GABA-induced current/chemotaxis of microglia was detected. Strong tetanic stimulation of Schaffer-collateral pathways that induce CA1 long-term potentiation did not affect microglial motilities. Our results suggest that microglia are highly reserved for neuronal protective function but not synaptic plasticity in the brain.

scholarly journals Agent-based model of angiogenesis simulates capillary sprout initiation in multicellular networks

2015 ◽  
Vol 7 (9) ◽  
pp. 987-997 ◽  
Author(s):  
J. Walpole ◽  
J. C. Chappell ◽  
J. G. Cluceru ◽  
F. Mac Gabhann ◽  
V. L. Bautch ◽  
...  
Keyword(s):  
Monte Carlo ◽  
Monte Carlo Simulations ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Agent Based Model ◽  
Agent Based

We developed an agent-based model of endothelial sprout initiations based on time-lapse confocal imaging in vitro that outperforms Monte Carlo simulations, suggesting that sprout location and frequency are not purely stochastic behaviors.

scholarly journals Fibronectin-Based Nanomechanical Biosensors to Map 3D Strains in Live Cells and Tissues

2020 ◽  
Author(s):  
Daniel J. Shiwarski ◽  
Joshua W. Tashman ◽  
Alkiviadis Tsamis ◽  
Jacqueline M. Bliley ◽  
Malachi A. Blundon ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Cells ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Square Lattice ◽  
Live Cells ◽  
3D Analysis ◽  
Wide Range ◽  
And Function ◽  
Analysis Platform

AbstractMechanical forces are integral to a wide range of cellular processes including migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution and with minimal tissue perturbation. Here, we fabricated, calibrated, and tested a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to cells and tissues to measure the magnitude, direction, and dynamics of strain from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultrathin square lattice FN mesh with spatial resolution tailored by adjusting the width and spacing of the lattice fibers from 2-100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrated strain tracking in 2D and 3D following mechanical deformation of known materials and was validated with finite element modeling. Imaging and 3D analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles demonstrated the ability to dynamically track microscopic tensile and compressive strains in various biological applications with minimal tissue perturbation. This fabrication and analysis platform serves as a novel tool for studying cells, tissues, and more complex systems where forces guide structure and function.

scholarly journals Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

2021 ◽  
Author(s):  
Cassio Pedroso ◽  
Victor Mann ◽  
Kathrin Zuberbühler ◽  
Markus-Frederik Bohn ◽  
Jessica Yu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
Time Lapse ◽  
Live Cell ◽  
Confocal Imaging ◽  
Cellular Targets ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Isopeptide Bonds ◽  
Time Lapse Imaging

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibod-ies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrys-tal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human can-cers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding caus-es uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal com-partments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

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