scholarly journals Label-free single-instance protein detection in vitrified cells

2020 ◽  
Author(s):  
J. Peter Rickgauer ◽  
Heejun Choi ◽  
Jennifer Lippincott-Schwartz ◽  
Winfried Denk
Keyword(s):  
Cell Biology ◽  
Template Matching ◽  
Electron Tomography ◽  
Protein Detection ◽  
Structural Data ◽  
Small Subunit ◽  
Label Free ◽  
Intact Cells ◽  
Precise Position ◽  
Single Instance

AbstractA general method to map molecular interactions and conformational states in structurally intact cells would find wide application in biochemistry and cell biology. We used a library of images— calculated on the basis of known structural data—as search templates to detect targets as small as the “head” domain (350 kDa) of the ribosome’s small subunit in single-tilt electron cryo-micrographs by cellular high resolution template matching (cHRTM). Atomically precise position and orientation estimates reveal the conformation of individual ribosomes and enable the detection of specifically bound ligands down to 24 kDa. We show that highly head-swivelled states are likely to play a role in mRNA translocation in living cells. cHRTM outperforms cryo-electron tomography three-fold in sensitivity and completely avoids the vicissitudes of exogenous labelling.

scholarly journals Deep Learning Improves Macromolecules Localization and Identification in 3D Cellular Cryo-Electron Tomograms

2020 ◽  
Author(s):  
Emmanuel Moebel ◽  
Antonio Martinez-Sanchez ◽  
Damien Larivière ◽  
Eric Fourmentin ◽  
Julio Ortiz ◽  
...  
Keyword(s):  
Deep Learning ◽  
Template Matching ◽  
Structural Information ◽  
Electron Tomography ◽  
Single Cells ◽  
Computational Procedure ◽  
Label Free ◽  
Small Areas ◽  
Synthetic Datasets ◽  
Subtomogram Averaging

abstractCryo-electron tomography (cryo-ET) allows one to visualize and study the 3D spatial distribution of macromolecules, in their native states and at nanometer resolution in single cells. While this label-free cryogenic imaging technology produces data containing rich structural information, automatic localization and identification of macromolecules are prone to noise and reconstruction artifacts, and to the presence of many molecular species in small areas. Hence, we present a computational procedure that uses artificial neural networks to accurately localize several macromolecular species in cellular cryo-electron tomograms. The DeepFinder algorithm leverages deep learning and outperforms the commonly-used template matching method on synthetic datasets. Meanwhile, DeepFinder is very fast when compared to template matching, and is better capable of localizing and identifying small macro-molecules than other competitive deep learning methods. On experimental cryo-ET data depicting ribosomes, the localization and structure resolution (determined through subtomogram averaging) results obtained with DeepFinder are consistent with those obtained by experts. The DeepFinder algorithm is able to imitate the analysis performed by experts, and is therefore a very promising algorithm to investigate efficiently the contents of cellular tomograms. Furthermore, we show that DeepFinder can be combined with a template matching procedure to localize the missing macromolecules not found by one or the other method. Application of this collaborative strategy allowed us to find additional 20.5% membrane-bound ribosomes that had been missed or discarded during manual template matching-assisted annotation.

scholarly journals Electrokinetic Formation of “Microbridges” for Protein Biomarkers as Sensors

2007 ◽  
Vol 12 (5) ◽  
pp. 311-317 ◽  
Author(s):  
Vindhya Kunduru ◽  
Shalini Prasad
Keyword(s):  
Electric Fields ◽  
Microelectrode Array ◽  
Protein Detection ◽  
High Specificity ◽  
Detection Methods ◽  
Label Free ◽  
Protein Biomarkers ◽  
Detection Scheme ◽  
Polystyrene Beads ◽  
Conducting Path

We demonstrate a technique to detect protein biomarkers contained in vulnerable coronary plaque using a platform-based microelectrode array (MEA). The detection scheme is based on the property of high specificity binding between antibody and antigen similar to most immunoassay techniques. Rapid clinical diagnosis can be achieved by detecting the amount of protein in blood by analyzing the protein's electrical signature. Polystyrene beads which act as transportation agents for the immobile proteins (antigen) are electrically aligned by application of homogenous electric fields. The principle of electrophoresis is used to produce calculated electrokinetic movement among the anti-C-reactive protein (CRP), or in other words antibody funtionalized polystyrene beads. The electrophoretic movement of antibody-functionalized polystyrene beads results in the formation of “Microbridges” between the two electrodes of interest which aid in the amplification of the antigen—antibody binding event. Sensitive electrical equipment is used for capturing the amplified signal from the “Microbridge” which essentially behaves as a conducting path between the two electrodes. The technique circumvents the disadvantages of conventional protein detection methods by being rapid, noninvasive, label-free, repeatable, and inexpensive. The same principle of detection can be applied for any receptor—ligand-based system because the technique is based only on the volume of the analyte of interest. Detection of the inflammatory coronary disease biomarker CRP is achieved at concentration levels spanning over the lower microgram/milliliter to higher order nanogram/milliliter ranges.

scholarly journals Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function

2021 ◽  
Vol 7 (1) ◽  
pp. eabe4310
Author(s):  
Yue Li ◽  
Adam Eshein ◽  
Ranya K.A. Virk ◽  
Aya Eid ◽  
Wenli Wu ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Super Resolution ◽  
Population Level ◽  
Average Diameter ◽  
Live Cells ◽  
Molecular Function ◽  
Label Free ◽  
Multiple Length Scales ◽  
Chromatin Packing ◽  
Analysis Platform

Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells. Using nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nanometers, sub-megabase genomic size, and an internal fractal structure. The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.

scholarly journals Trends in SPR Cytometry: Advances in Label-Free Detection of Cell Parameters

Biosensors ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 102 ◽  
Author(s):  
Richard Schasfoort ◽  
Fikri Abali ◽  
Ivan Stojanovic ◽  
Gestur Vidarsson ◽  
Leon Terstappen
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Mammalian Cells ◽  
Living Cells ◽  
Label Free ◽  
Intact Cells ◽  
Cell Parameters ◽  
Spr Imaging ◽  
Label Free Detection

SPR cytometry entails the measurement of parameters from intact cells using the surface plasmon resonance (SPR) phenomenon. Specific real-time and label-free binding of living cells to sensor surfaces has been made possible through the availability of SPR imaging (SPRi) instruments and researchers have started to explore its potential in the last decade. Here we will discuss the mechanisms of detection and additionally describe the problems and issues of mammalian cells in SPR biosensing, both from our own experience and with information from the literature. Finally, we build on the knowledge and applications that has already materialized in this field to give a forecast of some exciting applications for SPRi cytometry.

scholarly journals Amyloid-like aggregating proteins cause lysosomal defects in neurons via gain-of-function toxicity

2021 ◽  
Vol 5 (3) ◽  
pp. e202101185
Author(s):  
Irene Riera-Tur ◽  
Tillman Schäfer ◽  
Daniel Hornburg ◽  
Archana Mishra ◽  
Miguel da Silva Padilha ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Protein Trafficking ◽  
Cell Biology ◽  
Electron Tomography ◽  
Lysosomal Storage ◽  
Gain Of Function ◽  
Cryo Electron Tomography ◽  
A Subunit ◽  
Late Stages ◽  
Β Sheet

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like β-sheet proteins (β proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, β proteins interact with and sequester AP-3 μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity.

scholarly journals Nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform bridges 4-D chromatin organization with molecular function

2020 ◽  
Author(s):  
Yue Li ◽  
Adam Eshein ◽  
Ranya K.A. Virk ◽  
Aya Eid ◽  
Wenli Wu ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Electron Tomography ◽  
Population Level ◽  
Average Diameter ◽  
Chromatin Organization ◽  
Live Cells ◽  
Molecular Function ◽  
Label Free ◽  
A Cell ◽  
Chromatin Packing

AbstractIn eukaryotic cells, chromatin structure is linked to transcription processes through the regulation of genome organization. Extending across multiple length-scales - from the nucleosome to higher-order three-dimensional structures - chromatin is a dynamic system which evolves throughout the lifetime of a cell. However, no individual technique can fully elucidate the behavior of chromatin organization and its relation to molecular function at all length- and timescales at both a single-cell and a cell population level. Herein, we present a multi-technique nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform that bridges electron tomography and optical superresolution imaging of chromatin conformation and transcriptional processes, with resolution down to the level of individual nucleosomes, with high-throughput, label-free analysis of chromatin packing and its dynamics in live cells. Utilizing nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nm, sub-Mb genomic size, and an internal fractal structure. The chromatin packing behavior of these domains is directly influenced by active gene transcription. Furthermore, we demonstrated that the chromatin packing domain structure is correlated among progenitor cells and all their progeny, indicating that the organization of chromatin into fractal packing domains is heritable across cell division. Further studies employing the nano-ChIA platform have the potential to provide a more coherent picture of chromatin structure and its relation to molecular function.

A Novel Method for Automated Acquisition of Tilt Series for Electron Tomography Based on Pre-Calibration of the Specimen Stage

2000 ◽  
Vol 6 (S2) ◽  
pp. 1148-1149
Author(s):  
U. Ziese ◽  
A.H. Janssen ◽  
T.P. van der Krift ◽  
A.G. van Balen ◽  
W.J. de Ruijter ◽  
...  
Keyword(s):  
High Resolution ◽  
3D Reconstruction ◽  
Cell Biology ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Imaging Method ◽  
3D Images ◽  
Tilt Series ◽  
3D Information ◽  
Structural Arrangements

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. Conventional TEM images are in first approximation mere 2D-projections of a 3D sample under investigation. With electron tomographya series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments (so called tilt-series). For the subsequent 3D-reconstruction, the images of the tilt series are aligned relative to each other and the 3D-reconstruction is computed. Electron tomography is the only technique that can provide true 3D information with nm-scale resolution of individual and unique samples. For (cell) biology and material science applications the availability of high-resolution 3D images of structural arrangements within individual samples provides unique architectural information that cannot be obtained otherwise. Routine application of electron tomography will comprise a major revolutionary step forward in the characterization of complex materials and cellular arrangements.

VEGF Cancer Biomarker Protein Detection in Real Human Serum Using Capacitive Label-Free Aptasensor

2015 ◽  
Vol 357 (1) ◽  
pp. 74-78
Author(s):  
Anjum Qureshi ◽  
Irena Roci ◽  
Yasar Gurbuz ◽  
Javed H. Niazi
Keyword(s):  
Human Serum ◽  
Protein Detection ◽  
Cancer Biomarker ◽  
Label Free
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