scholarly journals Nanotomography of Cell Surfaces with Evanescent Fields

2008 ◽  
Vol 2008 ◽  
pp. 1-7 ◽  
Author(s):  
Michael Wagner ◽  
Petra Weber ◽  
Wolfgang S. L. Strauss ◽  
Henri-Pierre Lassalle ◽  
Herbert Schneckenburger
Keyword(s):  
Total Internal Reflection ◽  
3D Imaging ◽  
Cell Swelling ◽  
Cholesterol Depletion ◽  
In Vitro Diagnostics ◽  
Axial Resolution ◽  
Cell Surfaces ◽  
Cell Substrate ◽  
Cell Topology

The technique of variable-angle total internal reflection fluorescence microscopy (TIRFM) and its application to nanotomography of cell surfaces are described. Present applications include (1) 3D imaging of chromosomes in their metaphase to demonstrate axial resolution in the nanometre range, (2) measurements of cell-substrate topology, which upon cholesterol depletion shows some loosening of cell-substrate contacts, and (3) measurements of cell topology upon photodynamic therapy (PDT), which demonstrate cell swelling and maintenance of focal contacts. The potential of the method for in vitro diagnostics, but also some requirements and limitations are discussed.

scholarly journals Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching

2016 ◽  
Vol 113 (16) ◽  
pp. 4368-4373 ◽  
Author(s):  
Yan Fu ◽  
Peter W. Winter ◽  
Raul Rojas ◽  
Victor Wang ◽  
Matthew McAuliffe ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
3D Imaging ◽  
Incident Angle ◽  
Axial Resolution ◽  
Optical Sectioning ◽  
Excitation Light ◽  
Epidermal Growth ◽  
20 Nm ◽  
Fluorescent Molecules ◽  
Axial Superresolution

We report superresolution optical sectioning using a multiangle total internal reflection fluorescence (TIRF) microscope. TIRF images were constructed from several layers within a normal TIRF excitation zone by sequentially imaging and photobleaching the fluorescent molecules. The depth of the evanescent wave at different layers was altered by tuning the excitation light incident angle. The angle was tuned from the highest (the smallest TIRF depth) toward the critical angle (the largest TIRF depth) to preferentially photobleach fluorescence from the lower layers and allow straightforward observation of deeper structures without masking by the brighter signals closer to the coverglass. Reconstruction of the TIRF images enabled 3D imaging of biological samples with 20-nm axial resolution. Two-color imaging of epidermal growth factor (EGF) ligand and clathrin revealed the dynamics of EGF-activated clathrin-mediated endocytosis during internalization. Furthermore, Bayesian analysis of images collected during the photobleaching step of each plane enabled lateral superresolution (<100 nm) within each of the sections.

scholarly journals Nanoimprinted multifunctional nanoprobes for a homogeneous immunoassay in a top-down fabrication approach

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hubert Brueckl ◽  
Astrit Shoshi ◽  
Stefan Schrittwieser ◽  
Barbara Schmid ◽  
Pia Schneeweiss ◽  
...  
Keyword(s):  
Thin Film ◽  
Nanoimprint Lithography ◽  
Thin Film Deposition ◽  
Film Deposition ◽  
In Vitro Diagnostics ◽  
Top Down ◽  
Highly Sensitive ◽  
Optical And Magnetic Properties ◽  
Lift Off

AbstractMultifunctional nanoparticles are discussed as versatile probes for homogeneous immunoassays for in-vitro diagnostics. Top-down fabrication allows to combine and tailor magnetic and plasmonic anisotropic properties. The combination of nanoimprint lithography, thin film deposition, and lift-off processing provides a top-down fabrication platform, which is both flexible and reliable. Here, we discuss the material compositions and geometrical designs of monodisperse multicomponent nanoparticles and their consequences on optical and magnetic properties. The rotational hydrodynamics of nanoparticles is measured and considered under the influence of magnetic shape anisotropy in the framework of the Stoner-Wohlfarth theory. The plasmon-optical properties are explained by discrete-dipole finite-element simulations. Rotational dynamical measurements of imprinted nanoprobes for two test proteins demonstrate the applicability as highly sensitive biomolecular nanoprobes.

scholarly journals Large domain movements upon UvrD dimerization and helicase activation

2017 ◽  
Vol 114 (46) ◽  
pp. 12178-12183 ◽  
Author(s):  
Binh Nguyen ◽  
Yerdos Ordabayev ◽  
Joshua E. Sokoloski ◽  
Elizabeth Weiland ◽  
Timothy M. Lohman
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Self Assembly ◽  
Total Internal Reflection ◽  
Dna Helicase ◽  
Conformational State ◽  
Helicase Activity ◽  
Multiple Conformations ◽  
Dna Substrate

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

scholarly journals Simulated diabetic ketoacidosis therapy in vitro elicits brain cell swelling via sodium-hydrogen exchange and anion transport

2015 ◽  
Vol 309 (4) ◽  
pp. E370-E379 ◽  
Author(s):  
Keeley L. Rose ◽  
Andrew J. Watson ◽  
Thomas A. Drysdale ◽  
Gediminas Cepinskas ◽  
Melissa Chan ◽  
...  
Keyword(s):  
Diabetic Ketoacidosis ◽  
Hydrogen Exchange ◽  
Brain Slices ◽  
Anion Transport ◽  
Brain Cell ◽  
Cell Swelling ◽  
Sodium Hydrogen ◽  
Disulphonic Acid

A common complication of type 1 diabetes mellitus is diabetic ketoacidosis (DKA), a state of severe insulin deficiency. A potentially harmful consequence of DKA therapy in children is cerebral edema (DKA-CE); however, the mechanisms of therapy-induced DKA-CE are unknown. Our aims were to identify the DKA treatment factors and membrane mechanisms that might contribute specifically to brain cell swelling. To this end, DKA was induced in juvenile mice with the administration of the pancreatic toxins streptozocin and alloxan. Brain slices were prepared and exposed to DKA-like conditions in vitro. Cell volume changes were imaged in response to simulated DKA therapy. Our experiments showed that cell swelling was elicited with isolated DKA treatment components, including alkalinization, insulin/alkalinization, and rapid reductions in osmolality. Methyl-isobutyl-amiloride, a nonselective inhibitor of sodium-hydrogen exchangers (NHEs), reduced cell swelling in brain slices elicited with simulated DKA therapy (in vitro) and decreased brain water content in juvenile DKA mice administered insulin and rehydration therapy (in vivo). Specific pharmacological inhibition of the NHE1 isoform with cariporide also inhibited cell swelling, but only in the presence of the anion transport (AT) inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid. DKA did not alter brain NHE1 isoform expression, suggesting that the cell swelling attributed to the NHE1 was activity dependent. In conclusion, our data raise the possibility that brain cell swelling can be elicited by DKA treatment factors and that it is mediated by NHEs and/or coactivation of NHE1 and AT.

Design and Construction of Three-Dimensional Physiologically-Based Vascular Branching Networks for Respiratory Assist Devices

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Jose Santos ◽  
Alla A Gimbel ◽  
Athanasios Peppas ◽  
James G Truslow ◽  
Daniel Lang ◽  
...  
Keyword(s):  
Drug Development ◽  
Three Dimensional ◽  
Lab On A Chip ◽  
In Vitro Diagnostics ◽  
Assist Devices ◽  
Branching Networks ◽  
Physiologically Based ◽  
Vascular Branching ◽  
Design And Construction

Microfluidic lab-on-a-chip devices are changing the way that in vitro diagnostics and drug development are conducted, based on the increased precision, miniaturization and efficiency of these systems relative to prior...

Platinum-Decorated Gold Nanoparticles with Dual Functionalities for Ultrasensitive Colorimetric in Vitro Diagnostics

Nano Letters ◽  
2017 ◽  
Vol 17 (9) ◽  
pp. 5572-5579 ◽  
Author(s):  
Zhuangqiang Gao ◽  
Haihang Ye ◽  
Dianyong Tang ◽  
Jing Tao ◽  
Sanaz Habibi ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
In Vitro Diagnostics

Volume-responsive sodium movements in dog red blood cells

1983 ◽  
Vol 244 (5) ◽  
pp. C324-C330 ◽  
Author(s):  
J. C. Parker
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Cell Swelling ◽  
Cell Shrinkage ◽  
Ph Change ◽  
Sodium Permeability ◽  
Sodium Gradient ◽  
Sodium Flux ◽  
Sodium Hydrogen Exchanger

As dog red blood cells are shrunken in vitro, their sodium permeability increases progressively. Some new features of this volume-responsive transport process are described. Retardation of sodium movements in shrunken cells occurs when chloride is replaced by the more conductive anions: nitrate or thiocyanate. Micromolar concentrations of amiloride or quinidine inhibit the increment of sodium flux associated with a reduction in cell volume. In the presence of a large outwardly directed sodium gradient, dog red blood cells can progressively alkalinize the medium in which they are suspended. This pH change is stimulated by cell shrinkage, reversed by cell swelling, retarded when chloride is replaced by nitrate or thiocyanate, and inhibited by micromolar concentrations of amiloride or quinidine. The similarities between the shrinkage-associated sodium flux and the alkalinization phenomenon suggest that the mechanism responsible for increased sodium permeability in shrunken cells can be made to operate as a sodium-hydrogen exchanger.

scholarly journals Combining in Vitro Diagnostics with in Vivo Imaging for Earlier Detection of Pancreatic Ductal Adenocarcinoma: Challenges and Solutions

Radiology ◽  
2015 ◽  
Vol 277 (3) ◽  
pp. 644-661 ◽  
Author(s):  
Paul F. Laeseke ◽  
Ru Chen ◽  
R. Brooke Jeffrey ◽  
Teresa A. Brentnall ◽  
Jürgen K. Willmann
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
In Vivo Imaging ◽  
Ductal Adenocarcinoma ◽  
In Vitro Diagnostics

scholarly journals In silico mechanobiochemical modeling of morphogenesis in cell monolayers

2017 ◽  
Author(s):  
Bahador Marzban ◽  
Xiao Ma ◽  
Xiaoliang Qing ◽  
Hongyan Yuan
Keyword(s):  
Computational Model ◽  
Reaction Diffusion ◽  
Cell Interaction ◽  
Cell Polarization ◽  
Cell Morphogenesis ◽  
Cell Monolayers ◽  
Irregular Shapes ◽  
Cell Substrate ◽  
Living Tissues

Cell morphogenesis is a fundamental process involved in tissue formation. One of the challenges in the fabrication of living tissues in vitro is to recapitulate the complex morphologies of individual cells. Despite tremendous progress in understanding biophysical principles underlying tissue/organ morphogenesis at the organ level, little work has been done to understand morphogenesis at the cellular and microtissue level. In this work, we developed a 2D computational model for studying cell morphogenesis in monolayer tissues. The model is mainly composed of four modules: mechanics of cytoskeleton, cell motility, cell-substrate interaction, and cell-cell interaction. The model integrates the biochemical and mechanical activities within individual cells spatiotemporally. Finite element method (FEM) is used to model the irregular shapes of cells and to solve the resulting system of reaction-diffusion-stress equations. Automated mesh generation is used to handle the element distortion in FEM due to the large shape changes of the cells. The computer program can simulate tens to hundreds of cells interacting with each other and with the elastic substrate on desktop workstations efficiently. The simulations demonstrated that our computational model can be used to study cell polarization, single cell migration, durotaxis, and morphogenesis in cell monolayers.

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