Oblique plane microscope plate reader for time lapse 3D imaging of live cells in collagen (Conference Presentation)

2020 ◽  
Author(s):  
Nathan Curry ◽  
Hugh Sparks ◽  
Lucas Dent ◽  
Vicky Bousgouni ◽  
Vincent Maioli ◽  
...  
Keyword(s):  
3D Imaging ◽  
Time Lapse ◽  
Live Cells ◽  
Plate Reader

scholarly journals Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination

2012 ◽  
Vol 109 (14) ◽  
pp. 5311-5315 ◽  
Author(s):  
R. Fiolka ◽  
L. Shao ◽  
E. H. Rego ◽  
M. W. Davidson ◽  
M. G. L. Gustafsson
Keyword(s):  
3D Imaging ◽  
Time Lapse ◽  
Live Cells ◽  
Structured Illumination

scholarly journals Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

10.3791/57566 ◽  
2018 ◽  
Author(s):  
Markus Horsthemke ◽  
Janine Wilden ◽  
Anne C. Bachg ◽  
Peter J. Hanley
Keyword(s):  
3D Imaging ◽  
Time Lapse ◽  
Mouse Macrophages

scholarly journals Time Lapse 3D Imaging of Mineral Dissolution

2021 ◽  
Author(s):  
Chandra Winardhi ◽  
Jose Godinho ◽  
Jens Gutzmer ◽  
Gero Frisch
Keyword(s):  
3D Imaging ◽  
Time Lapse ◽  
Mineral Dissolution

Comparison study of live cells by atomic force microscopy, confocal microscopy, and scanning electrochemical microscopy

2007 ◽  
Vol 85 (3) ◽  
pp. 175-183 ◽  
Author(s):  
Xiaocui Zhao ◽  
Nils O Petersen ◽  
Zhifeng Ding
Keyword(s):  
Atomic Force Microscopy ◽  
Confocal Microscopy ◽  
Scanning Electrochemical Microscopy ◽  
Time Lapse ◽  
Live Cells ◽  
Tapping Mode ◽  
Force Microscopy ◽  
Atomic Force ◽  
Tapping Mode Afm ◽  
Electrochemical Microscopy

In this report, three kinds of scanning probe microscopy techniques, atomic force microscopy (AFM), confocal microscopy (CM), and scanning electrochemical microscopy (SECM), were used to study live cells in the physiological environment. Two model cell lines, CV-1 and COS-7, were studied. Time-lapse images were obtained with both contact and tapping mode AFM techniques. Cells were more easily scratched or moved by contact mode AFM than by tapping mode AFM. Detailed surface structures such as filamentous structures on the cell membrane can be obtained and easily discerned with tapping mode AFM. The toxicity of ferrocenemethanol (Fc) on live cells was studied by CM in reflection mode by recording the time-lapse images of controlled live cells and live cells with different Fc concentrations. No significant change in the morphology of cells was caused by Fc. Cells were imaged by SECM with Fc as the mediator at a biased potential of 0.35 V (vs. Ag/AgCl with a saturated KCl solution). Cells did not change visibly within 1 h, which indicated that SECM was a noninvasive technique and thus has a unique advantage for the study of soft cells, since the electrode scanned above the cells instead of in contact with them. Reactive oxygen species (ROS) generated by the cells were detected and images based on these chemical species were obtained. It is demonstrated that SECM can provide not only the topographical images but also the images related to the chemical or biochemical species released by the live cells.Key words: live cells, atomic force microscopy, confocal microscopy, scanning electrochemical microscopy.

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 PGE2 upregulates the Na+/K+ ATPase in HepG2 cells via EP4 receptors and intracellular calcium

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245400
Author(s):  
Rawad Hodeify ◽  
Mohamed Chakkour ◽  
Reem Rida ◽  
Sawsan Kreydiyyeh
Keyword(s):  
Intracellular Calcium ◽  
Hepg2 Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Live Cells ◽  
Ionic Homeostasis ◽  
Significant Stimulation ◽  
Time Lapse Imaging ◽  
Stimulation Of

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP–Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

scholarly journals A non-fluorescent HaloTag blocker for improved measurement and visualization of protein synthesis in living cells

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 302
Author(s):  
Laurie D. Cohen ◽  
Ayub Boulos ◽  
Noam E. Ziv
Keyword(s):  
Protein Synthesis ◽  
Fusion Protein ◽  
Protein Trafficking ◽  
Binding Sites ◽  
Cortical Neurons ◽  
Protein Detection ◽  
Time Lapse ◽  
Live Cells ◽  
Fluorescent Cell ◽  
Bacterial Enzyme

Background: HaloTag is a modified bacterial enzyme that binds rapidly and irreversibly to an array of synthetic ligands, including chemical dyes. When expressed in live cells in conjunction with a protein of interest, HaloTag can be used to study protein trafficking, synthesis, and degradation. For instance, sequential HaloTag labeling with spectrally separable dyes can be used to separate preexisting protein pools from proteins newly synthesized following experimental manipulations or the passage of time. Unfortunately, incomplete labeling by the first dye, or labeling by residual, trapped dye pools can confound interpretation. Methods: Labeling specificity of newly synthesized proteins could be improved by blocking residual binding sites. To that end, we synthesized a non-fluorescent, cell permeable blocker (1-chloro-6-(2-propoxyethoxy)hexane; CPXH), essentially the HaloTag ligand backbone without the reactive amine used to attach fluorescent groups. Results: High-content imaging was used to quantify the ability of CPXH to block HaloTag ligand binding in live HEK cells expressing a fusion protein of mTurquoise2 and HaloTag. Full saturation was observed at CPXH concentrations of 5-10 µM at 30 min. No overt effects on cell viability were observed at any concentration or treatment duration. The ability of CPXH to improve the reliability of newly synthesized protein detection was then demonstrated in live cortical neurons expressing the mTurquoise2-HaloTag fusion protein, in both single and dual labeling time lapse experiments. Practically no labeling was observed after blocking HaloTag binding sites with CPXH when protein synthesis was suppressed with cycloheximide, confirming the identification of newly synthesized protein copies as such, while providing estimates of protein synthesis suppression in these experiments. Conclusions: CPXH is a reliable (and inexpensive) non-fluorescent ligand for improving assessment of protein-of-interest metabolism in live cells using HaloTag technology.

scholarly journals Microscopy quantification of microbial birth and death dynamics

2018 ◽  
Author(s):  
Samuel F. M. Hart ◽  
David Skelding ◽  
Adam J. Waite ◽  
Justin Burton ◽  
Li Xie ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Time Lapse ◽  
Microbial Evolution ◽  
Cell Counting ◽  
Live Cells ◽  
Nutrient Concentrations ◽  
Death Rates ◽  
Time Lapse Microscopy ◽  
Wide Range ◽  
Total Fluorescence

AbstractMicrobes live in dynamic environments where nutrient concentrations fluctuate. Quantifying fitness (birth and death) in a wide range of environments is critical for understanding microbial evolution as well as ecological interactions where one species alters the fitness of another. Here, using high-throughput time-lapse microscopy, we have quantified howSaccharomyces cerevisiaemutants incapable of synthesizing an essential metabolite grow or die in various concentrations of the required metabolite. We establish that cells normally expressing fluorescent proteins lose fluorescence upon death and that the total fluorescence in an imaging frame is proportional to the number of live cells even when cells form multiple layers. We validate our microscopy approach of measuring birth and death rates using flow cytometry, cell counting, and chemostat culturing. For lysine-requiring cells, very low concentrations of lysine are not detectably consumed and do not support cell birth, but delay the onset of death phase and reduce the death rate. In contrast, in low hypoxanthine, hypoxanthine-requiring cells can produce new cells, yet also die faster than in the absence of hypoxanthine. For both strains, birth rates under various metabolite concentrations are better described by the sigmoidal-shaped Moser model than the well-known Monod model, while death rates depend on the metabolite concentration and can vary with time. Our work reveals how time-lapse microscopy can be used to discover non-intuitive microbial dynamics and to quantify growth rates in many environments.

scholarly journals Wide-field Surface-Enhanced Coherent Anti-Stokes Raman Scattering Microscopy

2021 ◽  
Author(s):  
Cheng Zong ◽  
Ran Cheng ◽  
Fukai Chen ◽  
Peng Lin ◽  
Meng Zhang ◽  
...  
Keyword(s):  
Raman Scattering ◽  
Large Scale ◽  
Time Lapse ◽  
Live Cells ◽  
Large Field ◽  
Wide Field ◽  
Label Free ◽  
Label Free Detection ◽  
Surface Enhanced ◽  
Anti Stokes

Surface-enhanced Raman scattering (SERS) spectroscopy has been used extensively to study biology, chemistry, and materials. However, a point-by-point SERS mapping is time-consuming, taking minutes to hours for large-scale imaging. Here, we report a wide-field surface-enhanced coherent anti-Stokes Raman scattering (WISE-CARS) microscopy for monitoring nanotags in live cells and label-free detection of metabolic molecules. The WISE-CARS microscope achieves an imaging speed as fast as 120 frames per second for a large field of view of 130 microns X 130 microns. By spectral focusing of femtosecond lasers, a hyperspectral WISE-CARS stack of 120 frames can be acquired with a spectral resolution of 10 cm-1, where over 1 million Raman spectra are parallelly recorded within 0.5 seconds. As applications, we demonstrate time-lapse, 3D WISE-CARS imaging of nanotags in live cells as well as label-free detection of adenine released from S. aureus.

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