scholarly journals A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

10.3791/55331 ◽  
2017 ◽  
Author(s):  
Charlotte Kirchhelle ◽  
Ian Moore
Keyword(s):  
Confocal Imaging ◽  
Hypocotyl Development ◽  
Simple Chamber

scholarly journals The ontogeny of the endocrine pancreas in the fetal/newborn baboon

2012 ◽  
Vol 214 (3) ◽  
pp. 289-299 ◽  
Author(s):  
Amy R Quinn ◽  
Cynthia L Blanco ◽  
Carla Perego ◽  
Giovanna Finzi ◽  
Stefano La Rosa ◽  
...  
Keyword(s):  
Endocrine Cells ◽  
Endocrine Pancreas ◽  
Fold Increase ◽  
Pancreatic Tissue ◽  
Developmental Differences ◽  
Confocal Imaging ◽  
Cell Type ◽  
Pancreas Islet ◽  
Pancreas Weight

Erratic regulation of glucose metabolism including hyperglycemia is a common condition in premature infants and is associated with increased morbidity and mortality. The objective of this study was to examine histological and ultrastructural differences in the endocrine pancreas in fetal (throughout gestation) and neonatal baboons. Twelve fetal baboons were delivered at 125 days (d) gestational age (GA), 140d GA, or 175d GA. Eight animals were delivered at term (185d GA); half were fed for 5 days. Seventy-three nondiabetic adult baboons were used for comparison. Pancreatic tissue was studied using light microscopy, confocal imaging, and electron microscopy. The fetal and neonatal endocrine pancreas islet architecture became more organized as GA advanced. The percent areas of α-β-δ-cell type were similar within each fetal and newborn GA (NS) but were higher than the adults (P<0.05) regardless of GA. The ratio of β cells within the islet (whole and core) increased with gestation (P<0.01). Neonatal baboons, which survived for 5 days (feeding), had a 2.5-fold increase in pancreas weight compared with their counterparts killed at birth (P=0.01). Endocrine cells were also found in exocrine ductal and acinar cells in 125, 140 and 175d GA fetuses. Subpopulation of tissue that coexpressed trypsin and glucagon/insulin shows the presence of cells with mixed endo–exocrine lineage in fetuses. In summary, the fetal endocrine pancreas has no prevalence of a α-β-δ-cell type with larger endocrine cell percent areas than adults. Cells with mixed endocrine/exocrine phenotype occur during fetal development. Developmental differences may play a role in glucose homeostasis during the neonatal period and may have long-term implications.

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.

Real-time confocal imaging of trafficking of nestin-expressing multipotent stem cells in mouse whiskers in long-term 3-D histoculture

2012 ◽  
Vol 48 (5) ◽  
pp. 301-305 ◽  
Author(s):  
Jennifer Duong ◽  
Sumiyuki Mii ◽  
Aisada Uchugonova ◽  
Fang Liu ◽  
A. R. Moossa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Real Time ◽  
Confocal Imaging ◽  
Multipotent Stem Cells

scholarly journals Intra-Species Interactions in Streptococcus pneumoniae Biofilms

2022 ◽  
Vol 11 ◽  
Author(s):  
Carina Valente ◽  
Ana R. Cruz ◽  
Adriano O. Henriques ◽  
Raquel Sá-Leão
Keyword(s):  
Streptococcus Pneumoniae ◽  
Species Interactions ◽  
Confocal Imaging ◽  
High Morbidity ◽  
Nasopharyngeal Colonization ◽  
Biofilm Model ◽  
Starting Point ◽  
Colonization Ability ◽  
Sequence Types

Streptococcus pneumoniae is a human pathogen responsible for high morbidity and mortality worldwide. Disease is incidental and is preceded by asymptomatic nasopharyngeal colonization in the form of biofilms. Simultaneous colonization by multiple pneumococcal strains is frequent but remains poorly characterized. Previous studies, using mostly laboratory strains, showed that pneumococcal strains can reciprocally affect each other’s colonization ability. Here, we aimed at developing a strategy to investigate pneumococcal intra-species interactions occurring in biofilms. A 72h abiotic biofilm model mimicking long-term colonization was applied to study eight pneumococcal strains encompassing 6 capsular types and 7 multilocus sequence types. Strains were labeled with GFP or RFP, generating two fluorescent variants for each. Intra-species interactions were evaluated in dual-strain biofilms (1:1 ratio) using flow cytometry. Confocal microscopy was used to image representative biofilms. Twenty-eight dual-strain combinations were tested. Interactions of commensalism, competition, amensalism and neutralism were identified. The outcome of an interaction was independent of the capsular and sequence type of the strains involved. Confocal imaging of biofilms confirmed the positive, negative and neutral effects that pneumococci can exert on each other. In conclusion, we developed an experimental approach that successfully discriminates pneumococcal strains growing in mixed biofilms, which enables the identification of intra-species interactions. Several types of interactions occur among pneumococci. These observations are a starting point to study the mechanisms underlying those interactions.

Differential Localization Of RAC1 and RAC2 Reflects Their Specific Functions In Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2892-2892
Author(s):  
Marta Ewa Capala ◽  
Francesco Bonardi ◽  
Henny Maat ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Hematopoietic Cells ◽  
Confocal Imaging ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Interaction Partners

Abstract 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, indicative for reduced self-renewal. Consistently, the frequency of long-term culture initiating leukemic cells was markedly reduced upon RAC2 downregulation. 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 or mitochondria-associated, and involved in processes such as cell cycle progression and regulation of apoptosis. Consistently, the proportion of dividing cells was decreased in RAC2-depleted BCR-ABL leukemic cobblestones coinciding with an increased apoptosis. Finally, a marked decrease in mitochondrial membrane potential was observed upon RAC2 but not RAC1 downregulation pointing to mitochondrial dysfunction as the initiating event of the apoptotic response. Moreover, preliminary electron microscopy data suggest that this functional change may be paralleled by structural aberrations of mitochondria. 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 Age-Dependent Contributions of NMDA Receptors and L-Type Calcium Channels to Long-Term Depression in the Piriform Cortex

2021 ◽  
Vol 22 (24) ◽  
pp. 13551
Author(s):  
Vishaal Rajani ◽  
Aida Maziar ◽  
Kwun Nok Mimi Man ◽  
Johannes W. Hell ◽  
Qi Yuan
Keyword(s):  
Synaptic Plasticity ◽  
Calcium Channels ◽  
Piriform Cortex ◽  
Confocal Imaging ◽  
Receptor Expression ◽  
Sprague Dawley ◽  
Long Term Depression ◽  
Age Dependent ◽  
Fluorescent Immunohistochemistry

In the hippocampus, the contributions of N-methyl-D-aspartate receptors (NMDARs) and L-type calcium channels (LTCCs) to neuronal transmission and synaptic plasticity change with aging, underlying calcium dysregulation and cognitive dysfunction. However, the relative contributions of NMDARs and LTCCs in other learning encoding structures during aging are not known. The piriform cortex (PC) plays a significant role in odor associative memories, and like the hippocampus, exhibits forms of long-term synaptic plasticity. Here, we investigated the expression and contribution of NMDARs and LTCCs in long-term depression (LTD) of the PC associational fiber pathway in three cohorts of Sprague Dawley rats: neonatal (1–2 weeks), young adult (2–3 months) and aged (20–25 months). Using a combination of slice electrophysiology, Western blotting, fluorescent immunohistochemistry and confocal imaging, we observed a shift from an NMDAR to LTCC mediation of LTD in aged rats, despite no difference in the amount of LTD expression. These changes in plasticity are related to age-dependent differential receptor expression in the PC. LTCC Cav1.2 expression relative to postsynaptic density protein 95 is increased in the associational pathway of the aged PC layer Ib. Enhanced LTCC contribution in synaptic depression in the PC may contribute to altered olfactory function and learning with aging.

scholarly journals A novel ex vivo set-up for dynamic long-term characterization of processes on mucosal interfaces by confocal imaging and simultaneous cytokine measurements

2011 ◽  
Vol 13 (5) ◽  
pp. 742-751 ◽  
Author(s):  
A. Wieser ◽  
C. Guggenberger ◽  
M. Pritsch ◽  
J. Heesemann ◽  
S. Schubert
Keyword(s):  
Ex Vivo ◽  
Confocal Imaging ◽  
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