Scattering of Exciting Light by Live Cells in Fluorescence Confocal Imaging: Phototoxic Effects and Relevance for FRAP Studies

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.

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

10.1101/2020.02.11.943696 ◽

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.

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Autofluorescence in freshly isolated adult human liver sinusoidal cells

European Journal of Histochemistry ◽

10.4081/ejh.2021.3337 ◽

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.

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

SSRN Electronic Journal ◽

10.2139/ssrn.3860388 ◽

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

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Fast attenuation correction in fluorescence confocal imaging: A recursive approach

Bioimaging ◽

10.1002/1361-6374(199406)2:2<78::aid-bio2>3.0.co;2-d ◽

1994 ◽

Vol 2 (2) ◽

pp. 78-92

Author(s):

Karel C Strasters ◽

Hans T M van der Voort ◽

J M Geusebroek ◽

Arnold W M Smeulders

Keyword(s):

Attenuation Correction ◽

Confocal Imaging ◽

Fluorescence Confocal Imaging

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

Journal of Virology ◽

10.1128/jvi.74.18.8670-8679.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8670-8679 ◽

Cited By ~ 213

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.

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Topology of Schwann cells and sympathetic innervation along preglomerular vessels: a confocal microscopic study in protein S100B/EGFP transgenic mice

AJP Renal Physiology ◽

10.1152/ajprenal.00599.2007 ◽

2008 ◽

Vol 295 (4) ◽

pp. F1142-F1148 ◽

Cited By ~ 7

Author(s):

Fannie Darlot ◽

Annie Artuso ◽

Nicole Lautredou-Audouy ◽

Daniel Casellas

Keyword(s):

Schwann Cells ◽

Sympathetic Innervation ◽

Three Dimensional ◽

Cell Types ◽

Confocal Imaging ◽

Egfp Expression ◽

Cell Processes ◽

Fluorescence Confocal Imaging ◽

Afferent Arterioles ◽

Protein S100b

Schwann cells (Sc), associated axons, and nearby vascular endothelium constitute a functional trilogy of major importance during the development and regrowth of peripheral vascular nerves. The goal of the present study is to provide a technique of triple fluorescence confocal imaging of these cell types along renal preglomerular vessels. We took advantage of a protein S100B/EGFP transgenic mouse to visualize Sc. The endothelium was labeled with an intravenous injection of fluorescently tagged lectin, and after tissue processing, adrenergic nerves were revealed with an antibody against the marker protein synaptophysin. As a validation step, we found that EGFP-positive perivascular cells with prominent cell bodies and extensive, multidirectional cell processes were protein S100B positive. They were identified as Sc and indirectly assumed to be unmyelinated Sc. By contrast, we found strong EGFP expression in proximal epithelial cells and in the epithelium lining thin limbs of Henle. This epithelial fluorescence was not associated with immunoreactive protein S100B and thus corresponded to ectopic EGFP expressions in this mouse strain. Sc were organized in bundles or as a meshwork surrounding the preglomerular vasculature from arcuate arteries to afferent arterioles. No Sc were detected in the medulla. Although most Sc were closely apposed to adrenergic varicosities, many varicosities were not associated with detectable Sc processes. The present technique, and the capacity of confocal microscopy to yield three-dimensional imaging, allow the study of the microtopology of Sc and related sympathetic axons in the renal perivascular interstitium.

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

Microscopy and Microanalysis ◽

10.1017/s1431927612000542 ◽

2012 ◽

Vol 18 (4) ◽

pp. 753-760 ◽

Cited By ~ 18

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.

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