scholarly journals Counting fluorescently labeled proteins in tissues in the spinning disk microscope using single-molecule calibrations

2022 ◽  
Author(s):  
Maijia Liao ◽  
Yin-Wei Kuo ◽  
Jonathon Howard
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Imaging Techniques ◽  
Calibration Method ◽  
Absolute Number ◽  
Single Step ◽  
Isolated Cells ◽  
Spinning Disk ◽  
Biological Phenomena ◽  
Wide Range

Quantification of molecular numbers and concentrations in living cells is critical for testing models of complex biological phenomena. Counting molecules in cells requires estimation of the fluorescence intensity of single molecules, which is generally limited to imaging near cell surfaces, in isolated cells, or where motions are diffusive. To circumvent this difficulty, we have devised a calibration technique for spinning-disk confocal (SDC) microscopy, commonly used for imaging in tissues, that uses single-step bleaching kinetics to estimate the single-fluorophore intensity. To cross-check our calibrations, we compared the brightness of fluorophores in the SDC microscope to those in the total-internal-reflection (TIRF) and epifluorescence microscopes. We applied this calibration method to quantify the number of EB1-eGFP proteins in the comets of growing microtubule ends and to measure the cytoplasmic concentration of EB1-eGFP in sensory neurons in fly larvae. These measurements allowed us to estimate the dissociation constant of EB1-eGFP from the microtubules as wells as the GTP-tubulin cap size. Our results show the unexplored potential of single-molecule imaging using spinning disk confocal microscopy and provide a straight-forward method to count the absolute number of fluorophores in tissues which can be applied to a wide range of biological systems and imaging techniques.

scholarly journals Introduction: Modern Imaging in Biology and Medicine: Papers from the Seventh Omaha Imaging Symposium, April 2011

2012 ◽  
Vol 18 (4) ◽  
pp. 728-729
Author(s):  
Richard Hallworth ◽  
Michael G. Nichols
Keyword(s):  
Confocal Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Imaging Techniques ◽  
Special Section ◽  
Biological Imaging ◽  
Internal Reflection ◽  
Biological Research ◽  
Student Life ◽  
Multiphoton Excitation

The ability to see, or visualize, a phenomenon is an essential tool of modern biological research. Our ability to create static and dynamic images has grown exponentially in the 25 or so years since confocal microscopy became readily available. The ingenious and energetic application of insights from optical physics to biological imaging in recent years has brought us far-reaching extensions of simple imaging, including nonlinear (or multiphoton) excitation, total internal reflection imaging, and even single molecule counting techniques. The annual Omaha Imaging Symposium has since 2003 brought together experts in advanced biological imaging techniques for a one-day exposition of how these techniques help move biological science forward. The seventh iteration of the series was held Friday, April 8, 2011, at the Harper Student Life Center of Creighton University, in Omaha, Nebraska. This special section of Microscopy and Micronanalysis consists of papers from speakers at the symposium.

Synthesis and Biological Evaluation of Novel 4,5,6,7-Tetrahydrobenzo[D]-Thiazol-2- Yl Derivatives Derived from Dimedone with Anti-Tumor, C-Met, Tyrosine Kinase and Pim-1 Inhibitions

2019 ◽  
Vol 19 (12) ◽  
pp. 1438-1453 ◽  
Author(s):  
Rafat M. Mohareb ◽  
Amr S. Abouzied ◽  
Nermeen S. Abbas
Keyword(s):  
Tyrosine Kinase ◽  
Tumor Cell ◽  
Cell Lines ◽  
Single Molecule ◽  
Anticancer Agents ◽  
Biological Evaluation ◽  
New Drugs ◽  
Tumor Cell Lines ◽  
Thiazole Derivatives ◽  
Wide Range

Background: Dimedone and thiazole moieties are privileged scaffolds (acting as primary pharmacophores) in many compounds that are useful to treat several diseases, mainly tropical infectious diseases. Thiazole derivatives are a very important class of compounds due to their wide range of pharmaceutical and therapeutic activities. On the other hand, dimedone is used to synthesize many therapeutically active compounds. Therefore, the combination of both moieties through a single molecule to produce heterocyclic compounds will produce excellent anticancer agents. Objective: The present work reports the synthesis of 47 new substances belonging to two classes of compounds: Dimedone and thiazoles, with the purpose of developing new drugs that present high specificity for tumor cells and low toxicity to the organism. To achieve this goal, our strategy was to synthesize a series of 4,5,6,7-tetrahydrobenzo[d]-thiazol-2-yl derivatives using the reaction of the 2-bromodimedone with cyanothioacetamide. Methods: The reaction of 2-bromodimedone with cyanothioacetamide gave the 4,5,6,7-tetrahydrobenzo[d]- thiazol-2-yl derivative 4. The reactivity of compound 4 towards some chemical reagents was observed to produce different heterocyclic derivatives. Results: A cytotoxic screening was performed to evaluate the performance of the new derivatives in six tumor cell lines. Thirteen compounds were shown to be promising toward the tumor cell lines which were further evaluated toward five tyrosine kinases. Conclusion: The results of antitumor screening showed that many of the tested compounds were of high inhibition towards the tested cell lines. Compounds 6c, 8c, 11b, 11d, 13b, 14b, 15c, 15g, 21b, 21c, 20d and 21d were the most potent compounds toward c-Met kinase and PC-3 cell line. The most promising compounds 6c, 8c, 11b, 11d, 13b, 14b, 15c, 15g, 20c, 20d, 21b, 21c and 21d were further investigated against tyrosine kinase (c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR). Compounds 6c, 11b, 11d, 14b, 15c, and 20d were selected to examine their Pim-1 kinase inhibition activity the results revealed that compounds 11b, 11d and 15c had high activities.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Femtosecond Laser Fabrication of Hybrid Metal-Dielectric Structures with Nonlinear Photoluminescence

Photonics ◽  
2021 ◽  
Vol 8 (4) ◽  
pp. 121
Author(s):  
Ekaterina Ponkratova ◽  
Eduard Ageev ◽  
Filipp Komissarenko ◽  
Sergei Koromyslov ◽  
Dmitry Kudryashov ◽  
...  
Keyword(s):  
Visible Spectral Region ◽  
Single Step ◽  
Hybrid Nanostructures ◽  
Nonlinear Properties ◽  
Nanophotonic Devices ◽  
Sensing Applications ◽  
Structure Morphology ◽  
Wide Range ◽  
Nonlinear Signal ◽  
Dielectric Structures

Fabrication of hybrid micro- and nanostructures with a strong nonlinear response is challenging and represents a great interest due to a wide range of photonic applications. Usually, such structures are produced by quite complicated and time-consuming techniques. This work demonstrates laser-induced hybrid metal-dielectric structures with strong nonlinear properties obtained by a single-step fabrication process. We determine the influence of several incident femtosecond pulses on the Au/Si bi-layer film on produced structure morphology. The created hybrid systems represent isolated nanoparticles with a height of 250–500 nm exceeding the total thickness of the Au-Si bi-layer. It is shown that fabricated hybrid nanostructures demonstrate enhancement of the SHG signal (up to two orders of magnitude) compared to the initial planar sample and a broadband photoluminescence signal (more than 200 nm in width) in the visible spectral region. We establish the correlation between nonlinear signal and phase composition provided by Raman scattering measurements. Such laser-induced structures have significant potential in optical sensing applications and can be used as components for different nanophotonic devices.

scholarly journals A Mixed Numerical-Experimental Method to Characterize Metal-Polymer Interfaces for Crash Applications

Metals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 818
Author(s):  
Jonas Richter ◽  
Moritz Kuhtz ◽  
Andreas Hornig ◽  
Mohamed Harhash ◽  
Heinz Palkowski ◽  
...  
Keyword(s):  
Experimental Parameter ◽  
Dissimilar Materials ◽  
Calibration Method ◽  
Parameter Determination ◽  
Failure Behavior ◽  
Cohesive Elements ◽  
Interface Failure ◽  
Wide Range ◽  
Polymer Metal ◽  
Metal Polymer

Metallic (M) and polymer (P) materials as layered hybrid metal-polymer-metal (MPM) sandwiches offer a wide range of applications by combining the advantages of both material classes. The interfaces between the materials have a considerable impact on the resulting mechanical properties of the composite and its structural performance. Besides the fact that the experimental methods to determine the properties of the single constituents are well established, the characterization of interface failure behavior between dissimilar materials is very challenging. In this study, a mixed numerical–experimental approach for the determination of the mode I energy release rate is investigated. Using the example of an interface between a steel (St) and a thermoplastic polyolefin (PP/PE), the process of specimen development, experimental parameter determination, and numerical calibration is presented. A modified design of the Double Cantilever Beam (DCB) is utilized to characterize the interlaminar properties and a tailored experimental setup is presented. For this, an inverse calibration method is used by employing numerical studies using cohesive elements and the explicit solver of LS-DYNA based on the force-displacement and crack propagation results.

scholarly journals Potential predictors of severe cardiovascular involvement in Marfan syndrome: the emphasized role of genotype–phenotype correlations in improving risk stratification—a literature review

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Roland Stengl ◽  
Bence Ágg ◽  
Miklós Pólos ◽  
Gábor Mátyás ◽  
Gábor Szabó ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Marfan Syndrome ◽  
Current Knowledge ◽  
Imaging Techniques ◽  
Connective Tissue Disorder ◽  
Main Body ◽  
Wide Range ◽  
Stratification System ◽  
Genetically Determined ◽  
Aortic Dissections

Abstract Background Marfan syndrome (MFS) is a genetically determined systemic connective tissue disorder, caused by a mutation in the FBN1 gene. In MFS mainly the cardiovascular, musculoskeletal and ocular systems are affected. The most dangerous manifestation of MFS is aortic dissection, which needs to be prevented by a prophylactic aortic root replacement. Main body The indication criteria for the prophylactic procedure is currently based on aortic diameter, however aortic dissections below the threshold defined in the guidelines have been reported, highlighting the need for a more accurate risk stratification system to predict the occurrence of aortic complications. The aim of this review is to present the current knowledge on the possible predictors of severe cardiovascular manifestations in MFS patients, demonstrating the wide range of molecular and radiological differences between people with MFS and healthy individuals, and more importantly between MFS patients with and without advanced aortic manifestations. These differences originating from the underlying common molecular pathological processes can be assessed by laboratory (e.g. genetic testing) and imaging techniques to serve as biomarkers of severe aortic involvement. In this review we paid special attention to the rapidly expanding field of genotype–phenotype correlations for aortic features as by collecting and presenting the ever growing number of correlations, future perspectives for risk stratification can be outlined. Conclusions Data on promising biomarkers of severe aortic complications of MFS have been accumulating steadily. However, more unifying studies are required to further evaluate the applicability of the discussed predictors with the aim of improving the risk stratification and therefore the life expectancy and quality of life of MFS patients.

scholarly journals Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Fluorescence Microscope ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Axial Position ◽  
Localization Microscopy ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

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 A Structurally Variable Hinged Tetrahedron Framework from DNA Origami

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
David M. Smith ◽  
Verena Schüller ◽  
Carsten Forthmann ◽  
Robert Schreiber ◽  
Philip Tinnefeld ◽  
...  
Keyword(s):  
Self Assembly ◽  
Gel Electrophoresis ◽  
Imaging Techniques ◽  
Building Blocks ◽  
Dna Origami ◽  
Microscopy Technique ◽  
Wire Frame ◽  
Wide Range ◽  
Potential Applications ◽  
Linker Molecules

Nanometer-sized polyhedral wire-frame objects hold a wide range of potential applications both as structural scaffolds as well as a basis for synthetic nanocontainers. The utilization of DNA as basic building blocks for such structures allows the exploitation of bottom-up self-assembly in order to achieve molecular programmability through the pairing of complementary bases. In this work, we report on a hollow but rigid tetrahedron framework of 75 nm strut length constructed with the DNA origami method. Flexible hinges at each of their four joints provide a means for structural variability of the object. Through the opening of gaps along the struts, four variants can be created as confirmed by both gel electrophoresis and direct imaging techniques. The intrinsic site addressability provided by this technique allows the unique targeted attachment of dye and/or linker molecules at any point on the structure's surface, which we prove through the superresolution fluorescence microscopy technique DNA PAINT.

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