scholarly journals Automated hiPSC culture and sample preparation for 3D live cell microscopy

2020 ◽  
Author(s):  
Mackenzie E. Coston ◽  
Benjamin W. Gregor ◽  
Joy Arakaki ◽  
Antoine Borensztejn ◽  
Thao P. Do ◽  
...  
Keyword(s):  
High Resolution ◽  
Plate Movement ◽  
3D Microscopy ◽  
Large Numbers ◽  
Cell Organization ◽  
Cellular Behaviors ◽  
Live Cell Microscopy ◽  
Cell Microscopy ◽  
Automated Methods ◽  
Culture Procedure

Our goal is to identify and understand cellular behaviors using 3D live imaging of cell organization. To do this, we image human inducible pluripotent stem cell (hiPSC) lines expressing fluorescently tagged protein representing specific cellular organelles and structures. To produce large numbers of standardized cell images, we developed an automated hiPSC culture procedure, to maintain, passage and Matrigel coat 6-well plastic plates and 96-well glass plates compatible with high-resolution 3D microscopy. Here we describe this system including optimization procedures and specific values for plate movement, angle of tips, speed of aspiration and dispense, seeding strategies and timing of every step. We validated this approach through a side-by-side comparison of quality control results obtained from manual and automated methods. Additionally, we developed an automated image-based colony segmentation and feature extraction pipeline to predict cell count and select wells with consistent morphology for high resolution 3D microscopy.

scholarly journals Dynamic regulation of subcellular mitochondrial position for localized metabolite levels

2019 ◽  
Author(s):  
Haya Alshaabi ◽  
Meara Heininger ◽  
Brian Cunniff
Keyword(s):  
Reactive Oxygen Species ◽  
High Resolution ◽  
Cell Function ◽  
Supporting Cell ◽  
Research Area ◽  
Oxygen Species ◽  
Dynamic Nature ◽  
Dynamic Regulation ◽  
Live Cell Microscopy ◽  
Cell Microscopy

Abstract Mitochondria are not passive bystanders aimlessly floating throughout our cell’s cytoplasm. Instead, mitochondria actively move, anchor, divide, fuse, self-destruct and transfer between cells in a coordinated fashion, all to ensure proper structure and position supporting cell function. The existence of the mitochondria in our cells has long been appreciated, but their dynamic nature and interaction with other subcellular compartments has only recently been fully realized with the advancement of high-resolution live-cell microscopy and improved fractionization techniques. The how and why that dictates positioning of mitochondria to specific subcellular sites is an ever-expanding research area. Furthermore, the advent of new and improved functional probes, sensitive to changes in subcellular metabolite levels has increased our understanding of local mitochondrial populations. In this review, we will address the evidence for intentional mitochondrial positioning in supporting subcellular mitochondrial metabolite levels, including calcium, adenosine triphosphate and reactive oxygen species and the role mitochondrial metabolites play in dictating cell outcomes.

scholarly journals Dissecting T-cell activation with high-resolution live-cell microscopy

Immunology ◽  
2012 ◽  
Vol 135 (3) ◽  
pp. 198-206 ◽  
Author(s):  
Joseph J. Illingworth ◽  
P. Anton van der Merwe
Keyword(s):  
T Cell ◽  
High Resolution ◽  
T Cell Activation ◽  
Cell Activation ◽  
Live Cell ◽  
Live Cell Microscopy ◽  
Cell Microscopy

scholarly journals Cooperative 4Pi Excitation and Detection Yields Sevenfold Sharper Optical Sections in Live-Cell Microscopy

2004 ◽  
Vol 87 (6) ◽  
pp. 4146-4152 ◽  
Author(s):  
Hilmar Gugel ◽  
Jörg Bewersdorf ◽  
Stefan Jakobs ◽  
Johann Engelhardt ◽  
Rafael Storz ◽  
...  
Keyword(s):  
Live Cell ◽  
Live Cell Microscopy ◽  
Cell Microscopy

scholarly journals Live-cell microscopy - tips and tools

2009 ◽  
Vol 122 (6) ◽  
pp. 753-767 ◽  
Author(s):  
M. M. Frigault ◽  
J. Lacoste ◽  
J. L. Swift ◽  
C. M. Brown
Keyword(s):  
Live Cell ◽  
Live Cell Microscopy ◽  
Cell Microscopy

scholarly journals Klp2 and Ase1 synergize to maintain meiotic spindle stability during metaphase I

2020 ◽  
Author(s):  
Fan Zheng ◽  
Fenfen Dong ◽  
Shuo Yu ◽  
Tianpeng Li ◽  
Yanze Jian ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Yeast Cells ◽  
Meiotic Spindle ◽  
Homologous Chromosomes ◽  
Spindle Dynamics ◽  
Live Cell Microscopy ◽  
Spindle Stability ◽  
Cell Microscopy ◽  
Mitosis And Meiosis ◽  
Meiosis I

ABSTRACTThe spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during meiosis I, but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis I requires the synergism between Klp2 and the conserved microtubule crosslinker Ase1 as the absence of both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and mitotic events.

scholarly journals Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy

Biochemistry ◽  
2018 ◽  
Vol 57 (39) ◽  
pp. 5648-5653 ◽  
Author(s):  
Alison G. Tebo ◽  
Frederico M. Pimenta ◽  
Yu Zhang ◽  
Arnaud Gautier
Keyword(s):  
Live Cell ◽  
Chemical Genetic ◽  
Fluorescent Markers ◽  
Live Cell Microscopy ◽  
Cell Microscopy

Dynamics and Traffic for Transfecting Exogenous MicroRNA as Studied by Live-Cell Microscopy

2021 ◽  
Vol 17 (8) ◽  
pp. 1647-1653
Author(s):  
Ke Yang ◽  
Yuanyuan Wang ◽  
Bo Sun ◽  
Tian Tian ◽  
Zhu Dai ◽  
...  
Keyword(s):  
Dynamic Properties ◽  
Mean Square Displacement ◽  
Small Region ◽  
Single Particle Tracking ◽  
Live Cell ◽  
Calculation Results ◽  
Long Time ◽  
Live Cell Microscopy ◽  
Cell Microscopy

MicroRNA (miRNA) has emerged as an important gene-regulator that shows great potential in gene therapy because of its unique roles in gene-regulation. However, the knowledge on their function and transportation in vivo is still lacking, and there are limited obvious evidences to define intracellular transportation of miRNA. In this study, the dynamics of exogenous miR-21 transfected into HeLa cells was traced by live-cell microscopy. Their transportation at key time points was recorded and dynamic properties were analyzed by single particle tracking (SPT) and mean square displacement (MSD) calculation. Results showed that the exogenous miRNAs bounded to cells quickly and went through lysosome into cytosol, where they were subsequently recruited into p-body. They finally were degraded, otherwise went back to cytosol in some way. Long time observation and analysis of motion mode showed that the miRNAs were confined in a small region and their motion modes were flexible in different intracellular microenvironment after entering the cells.

scholarly journals Direct lysosome-based autophagy of lipid droplets in hepatocytes

2020 ◽  
Vol 117 (51) ◽  
pp. 32443-32452
Author(s):  
Ryan J. Schulze ◽  
Eugene W. Krueger ◽  
Shaun G. Weller ◽  
Katherine M. Johnson ◽  
Carol A. Casey ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Lipid Droplets ◽  
Critical Role ◽  
Specific Protein ◽  
Lipid Homeostasis ◽  
Dynamic Interactions ◽  
Double Membrane ◽  
Live Cell Microscopy ◽  
Cell Microscopy

Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be “extruded” directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.

scholarly journals Supra-glacial debris cover changes in the Greater Caucasus from 1986 to 2014

2020 ◽  
Vol 14 (2) ◽  
pp. 585-598 ◽  
Author(s):  
Levan G. Tielidze ◽  
Tobias Bolch ◽  
Roger D. Wheate ◽  
Stanislav S. Kutuzov ◽  
Ivan I. Lavrentiev ◽  
...  
Keyword(s):  
High Resolution ◽  
Sea Level ◽  
Google Earth ◽  
Gps Data ◽  
Glacier Area ◽  
Greater Caucasus ◽  
Abstract Knowledge ◽  
Covered Area ◽  
Debris Cover ◽  
Automated Methods

Abstract. Knowledge of supra-glacial debris cover and its changes remain incomplete in the Greater Caucasus, in spite of recent glacier studies. Here we present data of supra-glacial debris cover for 659 glaciers across the Greater Caucasus based on Landsat and SPOT images from the years 1986, 2000 and 2014. We combined semi-automated methods for mapping the clean ice with manual digitization of debris-covered glacier parts and calculated supra-glacial debris-covered area as the residual between these two maps. The accuracy of the results was assessed by using high-resolution Google Earth imagery and GPS data for selected glaciers. From 1986 to 2014, the total glacier area decreased from 691.5±29.0 to 590.0±25.8 km2 (15.8±4.1 %, or ∼0.52 % yr−1), while the clean-ice area reduced from 643.2±25.9 to 511.0±20.9 km2 (20.1±4.0 %, or ∼0.73 % yr−1). In contrast supra-glacial debris cover increased from 7.0±6.4 %, or 48.3±3.1 km2, in 1986 to 13.4±6.2 % (∼0.22 % yr−1), or 79.0±4.9 km2, in 2014. Debris-free glaciers exhibited higher area and length reductions than debris-covered glaciers. The distribution of the supra-glacial debris cover differs between the northern and southern and between the western, central and eastern Greater Caucasus. The observed increase in supra-glacial debris cover is significantly stronger on the northern slopes. Overall, we have observed up-glacier average migration of supra-glacial debris cover from about 3015 to 3130 m a.s.l. (metres above sea level) during the investigated period.

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