Confocal Imaging Analysis of Mitochondrial Trafficking of Individual Lipid Species in Live Cells

2021 ◽  
Author(s):  
Jun Zhang ◽  
Jia Nie ◽  
Haoran Sun ◽  
John-Paul Andersen ◽  
Yuguang Shi
Keyword(s):  
Confocal Imaging ◽  
Live Cells ◽  
Imaging Analysis ◽  
Lipid Species ◽  
Mitochondrial Trafficking

scholarly journals Monitoring and Determining Mitochondrial Network Parameters in Live Lung Cancer Cells

2019 ◽  
Vol 8 (10) ◽  
pp. 1723
Author(s):  
Tamara Mirzapoiazova ◽  
Haiqing Li ◽  
Anusha Nathan ◽  
Saumya Srivstava ◽  
Mohd W. Nasser ◽  
...  
Keyword(s):  
Lung Cancer ◽  
3D Reconstruction ◽  
Cellular Response ◽  
Mitochondrial Dynamics ◽  
Confocal Imaging ◽  
Live Cells ◽  
Mitochondrial Morphology ◽  
Electron Microscopic ◽  
Microscopic Images ◽  
Network Parameters

Mitochondria are dynamic organelles that constantly fuse and divide, forming dynamic tubular networks. Abnormalities in mitochondrial dynamics and morphology are linked to diverse pathological states, including cancer. Thus, alterations in mitochondrial parameters could indicate early events of disease manifestation or progression. However, finding reliable and quantitative tools for monitoring mitochondria and determining the network parameters, particularly in live cells, has proven challenging. Here, we present a 2D confocal imaging-based approach that combines automatic mitochondrial morphology and dynamics analysis with fractal analysis in live small cell lung cancer (SCLC) cells. We chose SCLC cells as a test case since they typically have very little cytoplasm, but an abundance of smaller mitochondria compared to many of the commonly used cell types. The 2D confocal images provide a robust approach to quantitatively measure mitochondrial dynamics and morphology in live cells. Furthermore, we performed 3D reconstruction of electron microscopic images and show that the 3D reconstruction of the electron microscopic images complements this approach to yield better resolution. The data also suggest that the parameters of mitochondrial dynamics and fractal dimensions are sensitive indicators of cellular response to subtle perturbations, and hence, may serve as potential markers of drug response in lung cancer.

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 Autofluorescence in freshly isolated adult human liver sinusoidal cells

2021 ◽  
Vol 65 (4) ◽  
Author(s):  
Anett Kristin Larsen ◽  
Jaione Simón-Santamaría ◽  
Kjetil Elvevold ◽  
Bo Göran Ericzon ◽  
Kim Erlend Mortensen ◽  
...  
Keyword(s):  
Human Liver ◽  
Cultured Cells ◽  
Confocal Imaging ◽  
Live Cells ◽  
Sinusoidal Cells ◽  
Imaging Flow Cytometry ◽  
Wide Range ◽  
Liver Sinusoidal Cells ◽  
Liver Endothelial Cells ◽  
Selection Of

Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of fluorophores, especially when labelling of live cells where the quencher is not compatible.

Confocal imaging analysis of ATP-induced Ca2+ response in individual endothelial cells of the artery in situ

1997 ◽  
Vol 272 (6) ◽  
pp. C1980-C1987 ◽  
Author(s):  
H. Ohata ◽  
Y. Ujike ◽  
K. Momose
Keyword(s):  
Endothelial Cells ◽  
Laser Scanning ◽  
Cultured Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Confocal Imaging ◽  
Transient Increase ◽  
Similar Response ◽  
Imaging Analysis ◽  
Acetoxymethyl Ester

The mechanisms for mobilization of intracellular free Ca2+ have been studied in various types of isolated and cultured cells, but little is known about Ca2+ mobilization in individual cells in situ. We tried to establish imaging analysis of intracellular free Ca2+ concentration ([Ca2+]i) in individual cells loaded with the acetoxymethyl ester of fluo 3 in situ, using laser scanning confocal microscopy. The method permitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a transient increase in endothelial [Ca2+]i. It was concluded that the response was induced via P2Y purinoceptors, because adenosine 5'-O-(2-thiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1-10 microM) and the peak amplitude of the transient increase in [Ca2+]i were dose dependently increased. Using rapid xy-scanning and line-scanning modes, we confirmed that 10 microM ATP induced Ca2+ waves, at a rate of 10-30 microns/s, after a lag time of approximately 3 s. These results show that [Ca2+]i waves within endothelial cells are physiologically induced by ATP via P2Y purinoceptor, but not P2U purinoceptor, in aortic strips in situ. The method should be of use in the study of vascular physiology and pathophysiology.

De Novo Labeling and Trafficking of Individual Lipid Species in Live Cells

2021 ◽  
Author(s):  
Jun Zhang ◽  
Jia Nie ◽  
Haoran Sun ◽  
John-Paul Andersen ◽  
Yuguang Shi
Keyword(s):  
De Novo ◽  
Live Cells ◽  
Lipid Species

scholarly journals Localization of Human Immunodeficiency Virus Type 1 Gag and Env at the Plasma Membrane by Confocal Imaging

2000 ◽  
Vol 74 (18) ◽  
pp. 8670-8679 ◽  
Author(s):  
Luz Hermida-Matsumoto ◽  
Marilyn D. Resh
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Surface Expression ◽  
Confocal Imaging ◽  
Live Cells ◽  
Basic Residue ◽  
Particle Assembly ◽  
Gag Protein ◽  
The Matrix ◽  
Punctate Pattern

ABSTRACT Budding of lentiviruses occurs at the plasma membrane, but the preceding steps involved in particle assembly are poorly understood. Since the Gag polyprotein mediates virion assembly and budding, studies on the localization of Gag within the cell should provide insight into the mechanism of particle assembly. Here, we utilize biochemical fractionation techniques as well as high-resolution confocal imaging of live cells to demonstrate that Gag is localized at the plasma membrane in a striking punctate pattern. Mutation of the N-terminal myristoylation site results in the formation of large cytosolic complexes, whereas mutation of the N-terminal basic residue cluster in the matrix domain redirects the Gag protein to a region partially overlapping the Golgi apparatus. In addition, we show that Gag and Env colocalize at the plasma membrane and that mistargeting of a mutant Gag to the Golgi apparatus alters the pattern of surface expression of Env.

scholarly journals Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging

2012 ◽  
Vol 18 (4) ◽  
pp. 753-760 ◽  
Author(s):  
Manuel Castellano-Muñoz ◽  
Anthony Wei Peng ◽  
Felipe T. Salles ◽  
Anthony J. Ricci
Keyword(s):  
Temporal Resolution ◽  
High Speed ◽  
Signal To Noise Ratio ◽  
Image Plane ◽  
Live Cell ◽  
Confocal Imaging ◽  
Live Cells ◽  
Tissue Viability ◽  
Fixed Tissue ◽  
Charge Coupled Device

AbstractConfocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality.

Pathway and control of sucrose import into initiating cotton fibre cells

2000 ◽  
Vol 27 (9) ◽  
pp. 795 ◽  
Author(s):  
Yong-Ling Ruan ◽  
Danny J. Llewellyn ◽  
Robert T. Furbank
Keyword(s):  
Confocal Imaging ◽  
Assimilate Transport ◽  
Imaging Analysis ◽  
Sucrose Transport ◽  
Trichome Development ◽  
Fibre Cell ◽  
Key Enzyme ◽  
And Control ◽  
Symplastic Pathway ◽  
Unique Mechanism

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 Our aim is to unravel the mechanisms controlling fibre cell initiation from the epidermis of cotton (Gossypium hirsutum L.) ovules. We compared the development of fibres and trichomes in wild type cotton and a fibreless seed (fls) mutant, and determined the cellular pathway of sucrose transport into fibre initials on the day of anthesis. Although fibre initiation is inhibited in the fls mutant, leading to the fibreless phenotype, trichome development in other parts of the plant is normal. Confocal imaging analysis revealed that the fluorescent molecule, 5(6)-carboxyfluorescein, which is transported symplastically, moved readily from the integument phloem into initiating fibres. Plasmolysis studies showed that the fibre initials and adjacent non-initiating ovule epidermal cells have similar osmotic potential. Immunolocalisation analysis showed the absence of sucrose transporter proteins in the initiating fibre, but their abundance in the transfer cell precursors at the innermost integument. These results (i) demonstrate that fibre cell initiation is controlled by unique mechanism(s) that differ from that for normal trichome development; (ii) show a symplastic pathway of sucrose import into initiating fibres and strengthen the current opinion that sucrose synthase is likely to be the key enzyme mobilising sucrose into initiating fibres; and (iii) suggest that the initial protrusion of the fibre cells above the ovule surface is largely achieved by increased cell wall extensibility rather than higher turgor as is commonly thought.

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