scholarly journals Evaluation of direct grafting strategies in Expansion Microscopy

2019 ◽  
Author(s):  
Gang Wen ◽  
Marisa Vanheusden ◽  
Aline Acke ◽  
Donato Vali ◽  
Simon Finn Mayer ◽  
...  
Keyword(s):  
Fine Structure ◽  
Lipid Membranes ◽  
Signal Amplification ◽  
Polymerization Reaction ◽  
Nanometer Scale ◽  
Biomolecular Systems ◽  
Covalent Grafting ◽  
Two Factors ◽  
Secondary Antibodies ◽  
Polymerizable Group

AbstractHigh resolution fluorescence microscopy is a key tool in the elucidation of biological fine-structure, providing insights into the distribution and interactions of biomolecular systems down to the nanometer scale. Expansion microscopy is a recently developed approach to achieving nanoscale resolution in optical imaging. In the experiment, biological samples are embedded in a hydrogel, which is isotropicaly swollen. This physically pulls labels apart, allowing more of them to be resolved. However, in the gelation and swelling process, two factors combine to reduce the signal in the final image; signal dilution and the polymerization reaction, which can damage some fluorophores. Here, we show a chemical linking approach that allows covalent grafting of biomolecular target and reporter in expansion microscopy. Through the combination of a targeting ligand, a reporter moiety and a polymerizable group in a single linker, complex constructs can be prepared in a single, labelling step. We show application of this new series of molecules in the targeting of the cell cytoskeleton, a first example of lipid membranes in expansion microscopy; direct immunostaining with primary and secondary antibodies, and direct grafting of ISH probes and signal amplification initiators (HCR and RollFISH). Our probes allow direct, multiplexed targeting of the cellular blueprint and enable a range of novel imaging approaches in combination with expansion microscopy.

Analysis of Extended Energy-Loss Fine Structure of Nanometerscale Clusters

1999 ◽  
Vol 5 (S2) ◽  
pp. 708-709
Author(s):  
Y. Ito ◽  
H. Jain ◽  
D.B. Williams
Keyword(s):  
Fine Structure ◽  
Energy Loss ◽  
Atomic Clusters ◽  
Nanometer Scale ◽  
X Ray ◽  
Small Clusters ◽  
Electron Energy Loss ◽  
X Ray Absorption ◽  
Ionization Edge

Small atomic clusters are of great importance for applications such as catalysts whose activity depends on the surface of the cluster. Attempts to determine the atomic short-range order and size of clusters have been made by analyzing the extended X-ray absorption fine structure (EXAFS). However, the analysis was made on an average of many small clusters. Analysis of extended energy-loss fine structure (EXELFS) in an electron energy-loss spectrum (EELS) has developed to the point where in some cases, the quality of the results is comparable to its X-ray analogue, EXAFS. No other technique provides nanometer-scale spatial resolution of the analyzed area for determining the atomic structure. Most EXELFS analysis has been performed on the K-ionization edge of lighter elements. For heavier elements, a more complex ionization edge such as the L-edge has to be used, due to the inefficiency of collecting high quality EEL spectra at higher energy-losses (Z > 18).

Feedback Control of Biomolecular Systems Formed From Droplet-Interface Bilayers

2008 ◽  
Author(s):  
Stephen A. Sarles ◽  
Donald J. Leo
Keyword(s):  
Feedback Control ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Large Scale ◽  
Control Strategies ◽  
Current Control ◽  
New Paradigm ◽  
Biomolecular Systems ◽  
Integral Current ◽  
Feedback Voltage

Applying feedback control strategies to biological materials establishes a new paradigm for creating controlled biomolecular systems. Specifically, current tracking and feedback voltage amplification are demonstrated separately on bilayer lipid membranes (BLMs) formed via the droplet-interface bilayer (DIB) method. Ion channel induced degradation of the bilayer is studied in order to provide a convenient method for causing changes to the bilayer which can be monitored using proportional-integral (PI) feedback voltage control. Alpha-hemolysin (αHL) from Staphylococcus aureus was shown to cause large scale reductions (+90%) to the resistance of the lipid bilayers formed at the interface of connected water droplets within 90 minutes of bilayer formation. Feedback integral current control was demonstrated on pure 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) DIBs not containing αHL and provided accurate current tracking of a 100pA desired current signal driven at a rate of 10mHz and less. Voltage amplification monitoring was achieved on DPhPC DIBs containing αHL, providing a way to detect decreasing resistance and capacitance of the bilayer and nonlinear current-voltage relationship.

Memcapacitive Devices in Neuromorphic Circuits via Polymeric Biomimetic Membranes

2019 ◽  
Author(s):  
Colin Basham ◽  
Megan Pitz ◽  
Joseph Najem ◽  
Stephen Sarles ◽  
Md Sakib Hasan
Keyword(s):  
Lipid Membranes ◽  
Soft Matter ◽  
Electrical Response ◽  
Material System ◽  
Biomolecular Systems ◽  
Neuromorphic Circuits ◽  
Computing Systems ◽  
Generation Computing ◽  
Voltage Dependent ◽  
Composition Structure

Abstract Two-terminal adaptive materials and circuit elements that mimic the signal processing, learning, and computing capabilities of biological synapses are essential for next-generation computing systems. To this end, we have recently developed resistive (ion channel) and capacitive (lipid bilayer) memory elements that mimic the composition, structure, and plasticity of biological synapses. Unlike solid-state counterparts, these biomolecular systems are low-power, analog, less noisy, biocompatible, and capable of exhibiting multiple timescales of short-term synaptic plasticity. However, lipid membranes lack structural stability and modularity necessary for a long-lasting adaptive material system. To address this issue, we propose the replacement of phospholipids with amphiphilic polymers to create artificial membranes, which have been demonstrated to be more durable than phospholipids. With the focus on memory capacitors, we demonstrate that polymeric bilayers can exhibit pinched hysteresis in the Q-v plane because of voltage-induced geometrical changes. Further, we demonstrate that the memcapacitive response is altered based on the surrounding oil medium; smaller oil molecules are retained at higher volume in the membrane, so that thicker bilayers have lower nominal capacitance but can vary this value by over 400%. Finally, we present a physics-based model that enables us to predict the device’s areal voltage-dependent response. Polymeric bilayers represent a significant enhancement in the field of soft-matter, geometrically-reconfigurable memcapacitors, and their highly customizable compositions will allow for a finely tuned electrical response that has a future in brain-inspired materials and circuits.

A ratiometric photoelectrochemical immunosensor based on g-C3N4@TiO2 NTs amplified by signal antibodies–Co3O4 nanoparticle conjugates

The Analyst ◽  
2018 ◽  
Vol 143 (20) ◽  
pp. 5030-5037 ◽  
Author(s):  
Qiong Wu ◽  
Fengxia Zhang ◽  
Huijuan Li ◽  
Zhihua Li ◽  
Qi Kang ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Tio2 Nts ◽  
Secondary Antibodies

Herein, we report a ratiometric photoelectrochemical (PEC) immunosensor coupled with secondary antibodies–Co3O4 nanoparticle conjugates (Ab2–Co3O4 NPs) for signal amplification.

scholarly journals Three-Dimensional Fine Structure of Nanometer-Scale Nafion Thin Films

2021 ◽  
Vol 3 (2) ◽  
pp. 1078-1086
Author(s):  
A. Peltonen ◽  
J. Etula ◽  
J. Seitsonen ◽  
P. Engelhardt ◽  
T. Laurila
Keyword(s):  
Thin Films ◽  
Fine Structure ◽  
Three Dimensional ◽  
Nanometer Scale

scholarly journals Studying biological membranes with extended range high-speed atomic force microscopy

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Adrian P. Nievergelt ◽  
Blake W. Erickson ◽  
Nahid Hosseini ◽  
Jonathan D. Adams ◽  
Georg E. Fantner
Keyword(s):  
Atomic Force Microscopy ◽  
Lipid Membranes ◽  
High Speed ◽  
Open Loop ◽  
Peak Force ◽  
High Speed Imaging ◽  
Biomolecular Systems ◽  
Biologically Relevant ◽  
Force Microscopy ◽  
Atomic Force

Abstract High—speed atomic force microscopy has proven to be a valuable tool for the study of biomolecular systems at the nanoscale. Expanding its application to larger biological specimens such as membranes or cells has, however, proven difficult, often requiring fundamental changes in the AFM instrument. Here we show a way to utilize conventional AFM instrumentation with minor alterations to perform high-speed AFM imaging with a large scan range. Using a two—actuator design with adapted control systems, a 130 × 130 × 5 μm scanner with nearly 100 kHz open—loop small-signal Z—bandwidth is implemented. This allows for high-speed imaging of biologically relevant samples as well as high-speed measurements of nanomechanical surface properties. We demonstrate the system performance by real-time imaging of the effect of charged polymer nanoparticles on the integrity of lipid membranes at high imaging speeds and peak force tapping measurements at 32 kHz peak force rate.

Exploring the temperature–pressure configurational landscape of biomolecules: from lipid membranes to proteins

2004 ◽  
Vol 363 (1827) ◽  
pp. 537-563 ◽  
Author(s):  
R. Winter ◽  
W. Dzwolak
Keyword(s):  
High Pressure ◽  
Lipid Membranes ◽  
Phase Behaviour ◽  
Pressure Jump ◽  
Spectroscopic Techniques ◽  
Biomolecular Systems ◽  
Time Resolved ◽  
Temperature And Pressure ◽  
Pressure Jump Relaxation ◽  
The Stability

Hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of biomolecular systems, such as lipid mesophases and proteins, but also because high pressure is an important feature of certain natural membrane environments and because the high–pressure phase behaviour of biomolecules is of biotechnological interest. By using spectroscopic and scattering techniques, the temperature– and pressure–dependent structure and phase behaviour of lipid systems, differing in chain configuration, headgroup structure and concentration, and proteins have been studied and are discussed. A thermodynamic approach is presented for studying the stability of proteins as a function of both temperature and pressure. The results demonstrate that combined temperature–pressure dependent studies can help delineate the free–energy landscape of proteins and hence help elucidate which features and thermodynamic parameters are essential in determining the stability of the native conformational state of proteins. We also introduce pressure as a kinetic variable. Applying the pressure jump relaxation technique in combination with time–resolved synchrotron X–ray diffraction and spectroscopic techniques, the kinetics of un/refolding of proteins has been studied. Finally, recent advances in using pressure for studying misfolding and aggregation of proteins will be discussed.

scholarly journals Tyramide Signal Amplification coupled with multiple immunolabeling and RNAscope in situ hybridization in formaldehyde-fixed paraffin-embedded human fetal brain.

2021 ◽  
Author(s):  
Ayman Alzu'bi ◽  
Niveditha Sankar ◽  
Gavin Clowry
Keyword(s):  
Signal Amplification ◽  
Fetal Brain ◽  
Morphological Characteristics ◽  
High Sensitivity ◽  
Tyramide Signal Amplification ◽  
Human Fetal Brain ◽  
Immunofluorescence Labelling ◽  
Complex Detection ◽  
Secondary Antibodies

Abstract Several strategies have been recently introduced to improve the practicality of multiple immunolabelling and RNA in situ hybridization methods. We present a modified hybrid protocol of recently described complex detection strategies: (1) elution of antibodies prior to second round of staining (2) use of integrated polymers of HRP with secondary antibodies, and (3) tyramide signal amplification of multiple immunofluorescence labelling, to achieve a high sensitivity sequential multiple labeling using antibodies from the same species. A modified protocol of the novel RNAscope in situ hybridization method, including coupling with immunofluorescence on sections of early human fetal brain, has also been developed. These two techniques, when properly optimized, were highly compatible with routine formaldehyde-fixed paraffin-embedded tissue that preserves the best morphological characteristics of delicate fetal brain samples, allowing high power signal amplification for detection of protein and mRNA of genes that are sparsely expressed in the human fetal telencephalon.

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