scholarly journals Counting numbers of synaptic proteins: absolute quantification and single molecule imaging techniques

2016 ◽  
Vol 3 (4) ◽  
pp. 041805 ◽  
Author(s):  
Angela Patrizio ◽  
Christian G. Specht
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Absolute Quantification ◽  
Synaptic Proteins ◽  
Single Molecule Imaging

Stochastic signal inputs for chemotactic response in Dictyostelium cells revealed by single molecule imaging techniques

Biosystems ◽  
2007 ◽  
Vol 88 (3) ◽  
pp. 251-260 ◽  
Author(s):  
Yukihiro Miyanaga ◽  
Satomi Matsuoka ◽  
Toshio Yanagida ◽  
Masahiro Ueda
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chemotactic Response ◽  
Single Molecule Imaging ◽  
Stochastic Signal

scholarly journals Dynamics and Signal Transduction of EGFR Clusters Visualized by Single-molecule Imaging Techniques

2001 ◽  
Vol 41 (supplement) ◽  
pp. S119
Author(s):  
Y. Sako ◽  
S. Minoguchi ◽  
A. Yoshimura ◽  
T. Yanagida
Keyword(s):  
Signal Transduction ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Single Molecule Imaging

Combining Single Molecule Imaging Techniques to Unravel the Nanoscale Organization of the Presynapse

2020 ◽  
pp. 265-286
Author(s):  
Christopher Small ◽  
Ramon Martínez-Mármol ◽  
Rumelo Amor ◽  
Frederic A. Meunier ◽  
Merja Joensuu
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Single Molecule Imaging

scholarly journals Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lisa S. Fischer ◽  
Christoph Klingner ◽  
Thomas Schlichthaerle ◽  
Maximilian T. Strauss ◽  
Ralph Böttcher ◽  
...  
Keyword(s):  
Complex Formation ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Detection Efficiency ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Absolute Quantification ◽  
Molecular Probes ◽  
Single Protein ◽  
In Cells

AbstractSingle-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex—consisting of talin, kindlin and active β1-integrin—that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells.

scholarly journals Accurate EMCCD Photoelectron Calibration for Single Molecule Imaging Techniques

2013 ◽  
Vol 104 (2) ◽  
pp. 666a
Author(s):  
Alexander Song ◽  
Han Gao ◽  
Avtar Singh ◽  
Warren R. Zipfel
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Single Molecule Imaging

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

2019 ◽  
Author(s):  
Adam Eördögh ◽  
Carolina Paganini ◽  
Dorothea Pinotsi ◽  
Paolo Arosio ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Logic Gate ◽  
Living Cells ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Molecular Logic Gate ◽  
Molecular Logic ◽  
Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

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