Transient Fluorescence Labeling: Low Affinity—High Benefits

Fluorescent labeling is an established method for visualizing cellular structures and dynamics. The fundamental diffraction limit in image resolution was recently bypassed with the development of super-resolution microscopy. Notably, both localization microscopy and stimulated emission depletion (STED) microscopy impose tight restrictions on the physico-chemical properties of labels. One of them—the requirement for high photostability—can be satisfied by transiently interacting labels: a constant supply of transient labels from a medium replenishes the loss in the signal caused by photobleaching. Moreover, exchangeable tags are less likely to hinder the intrinsic dynamics and cellular functions of labeled molecules. Low-affinity labels may be used both for fixed and living cells in a range of nanoscopy modalities. Nevertheless, the design of optimal labeling and imaging protocols with these novel tags remains tricky. In this review, we highlight the pros and cons of a wide variety of transiently interacting labels. We further discuss the state of the art and future perspectives of low-affinity labeling methods.

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Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures

International Journal of Molecular Sciences ◽

10.3390/ijms221910194 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10194

Author(s):

Merel Stiekema ◽

Frans C. S. Ramaekers ◽

Dimitrios Kapsokalyvas ◽

Marc A. M. J. van Zandvoort ◽

Rogier J. A. Veltrop ◽

...

Keyword(s):

Layer Thickness ◽

Super Resolution ◽

Dermal Fibroblasts ◽

Stimulated Emission ◽

Immunofluorescence Staining ◽

Fluorescence Labeling ◽

Confocal Scanning Laser Microscopy ◽

Lamin A ◽

Intermediate Filament Proteins ◽

Sted Microscopy

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.

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Intracellular production of hydrogels and synthetic RNA granules by multivalent enhancers

10.1101/117572 ◽

2017 ◽

Author(s):

Hideki Nakamura ◽

Albert A. Lee ◽

Ali Sobhi Afshar ◽

Shigeki Watanabe ◽

Elmer Rho ◽

...

Keyword(s):

Rna Binding ◽

Polymer Network ◽

Chemical Properties ◽

Cellular Functions ◽

Intact Cells ◽

Binding Motifs ◽

Rna Granules ◽

Rapid Induction ◽

Physico Chemical ◽

Powerful Approach

AbstractNon-membrane bound, hydrogel-like entities, such as RNA granules, nucleate essential cellular functions through their unique physico-chemical properties. However, these intracellular hydrogels have not been as extensively studied as their extracellular counterparts, primarily due to technical challenges in probing these materials in situ. Here, by taking advantage of a chemically inducible dimerization paradigm, we developed iPOLYMER, a strategy for rapid induction of protein-based hydrogels inside living cells. A series of biochemical and biophysical characterizations, in conjunction with computational modeling, revealed that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that behaves as a size-dependent molecular sieve. We studied several properties of the gel and functionalized it with RNA binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. Therefore, we here demonstrate that iPOLYMER presents a unique and powerful approach to synthetically reconstitute hydrogel-like structures including RNA granules in intact cells.

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Boating- and Shipping-Related Environmental Impacts and Example Management Measures: A Review

Journal of Marine Science and Engineering ◽

10.3390/jmse8110908 ◽

2020 ◽

Vol 8 (11) ◽

pp. 908

Author(s):

Troy A. Byrnes ◽

Ryan J. K. Dunn

Keyword(s):

Environmental Impacts ◽

Full Range ◽

Chemical Properties ◽

Positive Outcomes ◽

Antifouling Paints ◽

Management Measures ◽

Physico Chemical ◽

Pros And Cons ◽

The Government ◽

Physical Changes

Boating and shipping operations, their associated activities and supporting infrastructure present a potential for environmental impacts. Such impacts include physical changes to bottom substrate and habitats from sources such as anchoring and mooring and vessel groundings, alterations to the physico-chemical properties of the water column and aquatic biota through the application of antifouling paints, operational and accidental discharges (ballast and bilge water, hydrocarbons, garbage and sewage), fauna collisions, and various other disturbances. Various measures exist to sustainably manage these impacts. In addition to a review of associated boating- and shipping-related environmental impacts, this paper provides an outline of the government- and industry-related measures relevant to achieving positive outcomes in an Australian context. Historically, direct regulations have been used to cover various environmental impacts associated with commercial, industrial, and recreational boating and shipping operations (e.g., MARPOL). The effectiveness of this approach is the degree to which compliance can be effectively monitored and enforced. To be effective, environmental managers require a comprehensive understanding of the full range of instruments available, and the respective roles they play in helping achieve positive environmental outcomes, including the pros and cons of the various regulatory alternatives.

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Utilization of Plant Biomass for the Production of Renewable and Sustainable Biofuels With Zero Carbon Emission

Recent Technologies for Enhancing Performance and Reducing Emissions in Diesel Engines - Advances in Mechatronics and Mechanical Engineering ◽

10.4018/978-1-7998-2539-5.ch002 ◽

2020 ◽

pp. 26-43

Author(s):

Sandip Kumar Singh

Keyword(s):

Carbon Emission ◽

Plant Biomass ◽

Chemical Properties ◽

Value Added ◽

Product Portfolio ◽

Physico Chemical ◽

Biomass Resources ◽

Zero Carbon ◽

Pros And Cons ◽

Conversion Technologies

Biofuels for use of transport and industrial purposes have been synthesised on a substantial scale since 1970s, using a set of technologies. Today, biofuels are widely available using sugar, grains, starch-based bioethanol, and oil seeds-based biodiesel. For enhancing the anticipations of product portfolio of plant biomass-to-biofuels formation, it is vital to develop effective conversion technologies for upgradation of abundantly available lignocellulosic biomass resources into value-added co-products particularly biofuels and chemicals. In this chapter, brief synthesis processes and utilization of synthesised biofuels such as methanol, ethanol, butanol, gasoline, diesel, and jet fuel have been outlined for their use in transport sectors either as a neat or blended with gasoline. Biofuels' physico-chemical properties, performances, gas emissions, pros, and cons of various synthesised biofuels' neat and blend are compared with non-renewable fuels. Thenceforth, discussion gradually focuses towards the zero-carbon emission upon the utilization of biofuels derived from plant biomass.

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Deep learning achieves super-resolution in fluorescence microscopy

10.1101/309641 ◽

2018 ◽

Cited By ~ 4

Author(s):

Hongda Wang ◽

Yair Rivenson ◽

Yiyin Jin ◽

Zhensong Wei ◽

Ronald Gao ◽

...

Keyword(s):

Deep Learning ◽

Fluorescence Microscopy ◽

Numerical Models ◽

Numerical Aperture ◽

Super Resolution ◽

Image Resolution ◽

Stimulated Emission ◽

Wide Field ◽

Generative Adversarial Network ◽

Adversarial Network

AbtsractWe present a deep learning-based method for achieving super-resolution in fluorescence microscopy. This data-driven approach does not require any numerical models of the imaging process or the estimation of a point spread function, and is solely based on training a generative adversarial network, which statistically learns to transform low resolution input images into super-resolved ones. Using this method, we super-resolve wide-field images acquired with low numerical aperture objective lenses, matching the resolution that is acquired using high numerical aperture objectives. We also demonstrate that diffraction-limited confocal microscopy images can be transformed by the same framework into super-resolved fluorescence images, matching the image resolution acquired with a stimulated emission depletion (STED) microscope. The deep network rapidly outputs these super-resolution images, without any iterations or parameter search, and even works for types of samples that it was not trained for.

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Decoration of specific sites on freeze-fractured membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081565 ◽

1978 ◽

Vol 36 (2) ◽

pp. 140-141

Author(s):

H. Gross ◽

H. Moor

Keyword(s):

Pure Water ◽

Freeze Fracture ◽

Chemical Properties ◽

Surface Forces ◽

Sulfate Pentahydrate ◽

Copper Sulfate Pentahydrate ◽

Physico Chemical ◽

Water Of Hydration ◽

Small Container ◽

Binding Forces

Fracturing under ultrahigh vacuum (UHV, p ≤ 10-9 Torr) produces membrane fracture faces devoid of contamination. Such clean surfaces are a prerequisite foe studies of interactions between condensing molecules is possible and surface forces are unequally distributed, the condensate will accumulate at places with high binding forces; crystallites will arise which may be useful a probes for surface sites with specific physico-chemical properties. Specific “decoration” with crystallites can be achieved nby exposing membrane fracture faces to water vopour. A device was developed which enables the production of pure water vapour and the controlled variation of its partial pressure in an UHV freeze-fracture apparatus (Fig.1a). Under vaccum (≤ 10-3 Torr), small container filled with copper-sulfate-pentahydrate is heated with a heating coil, with the temperature controlled by means of a thermocouple. The water of hydration thereby released enters a storage vessel.

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PHYSICO CHEMICAL PROPERTIES OF NICKEL OXIDE AT HIGH TEMPERATURE

Le Journal de Physique Colloques ◽

10.1051/jphyscol:1976798 ◽

1976 ◽

Vol 37 (C7) ◽

pp. C7-438-C7-442 ◽

Cited By ~ 2

Author(s):

R. FARHI ◽

G. PETOT-ERVAS

Keyword(s):

High Temperature ◽

Nickel Oxide ◽

Chemical Properties ◽

Physico Chemical

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Physico-Chemical Properties of Recombinant Desulphatohirudin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645073 ◽

1990 ◽

Vol 63 (03) ◽

pp. 499-504 ◽

Cited By ~ 4

Author(s):

A Electricwala ◽

L Irons ◽

R Wait ◽

R J G Carr ◽

R J Ling ◽

...

Keyword(s):

Molecular Weight ◽

Gel Filtration ◽

Chemical Properties ◽

Alpha Helix ◽

Apparent Molecular Weight ◽

Correlation Spectroscopy ◽

Nmr Studies ◽

Recombinant Hirudin ◽

Physico Chemical ◽

Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

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