scholarly journals Robust nonparametric descriptors for clustering quantification in single-molecule localization microscopy

2016 ◽  
Author(s):  
Shenghang Jiang ◽  
Sai Divya Challapalli ◽  
Yong Wang
Keyword(s):  
Single Molecule ◽  
Spatial Organization ◽  
Nearest Neighbor ◽  
Distribution Functions ◽  
Localization Microscopy ◽  
Point Patterns ◽  
Direct Measurements ◽  
Single Molecule Localization Microscopy

ABSTRACTWe report a robust nonparametric descriptor,J′(r), for quantifying the spatial organization of molecules in singlemolecule localization microscopy.J′(r), based on nearest neighbor distribution functions, does not require any parameter as an input for analyzing point patterns. We show thatJ′(r) displays a valley shape in the presence of clusters of molecules, and the characteristics of the valley reliably report the clustering features in the data. More importantly, the position of theJ′(r) valley (rJ′m) depends exclusively on the density of clustering molecules (ρc). Therefore, it is ideal for direct measurements of clustering density of molecules in single-molecule localization microscopy.

scholarly journals Quantification of fibrous spatial point patterns from single-molecule localization microscopy (SMLM) data

2017 ◽  
pp. btx026 ◽  
Author(s):  
Ruby Peters ◽  
Marta Benthem Muñiz ◽  
Juliette Griffié ◽  
David J. Williamson ◽  
George W. Ashdown ◽  
...  
Keyword(s):  
Single Molecule ◽  
Localization Microscopy ◽  
Point Patterns ◽  
Spatial Point ◽  
Spatial Point Patterns ◽  
Single Molecule Localization Microscopy

scholarly journals A cross–nearest neighbor/Monte Carlo algorithm for single molecule localization microscopy defines interactions between p53, Mdm2, and MEG3

2019 ◽  
Author(s):  
Nicholas C. Bauer ◽  
Anli Yang ◽  
Xin Wang ◽  
Yunli Zhou ◽  
Anne Klibanski ◽  
...  
Keyword(s):  
Monte Carlo ◽  
Single Molecule ◽  
Nearest Neighbor ◽  
Monte Carlo Algorithm ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Cellular Context ◽  
Optical Reconstruction ◽  
Suppress Cell Proliferation ◽  
Single Molecule Localization Microscopy

AbstractThe functions of long noncoding (lnc)RNAs such as MEG3 are defined by their interactions with other RNAs and proteins. These interactions, in turn, are shaped by their subcellular localization and temporal context. Therefore, it is important to be able to analyze the relationships of lncRNAs while preserving cellular architecture. The ability of MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has been proposed to activate p53 by disrupting the interaction of p53 with Mdm2. To test this mechanism in the native cellular context, we employed two-color direct stochastic optical reconstruction microscopy (dSTORM), a single-molecule localization microscopy (SMLM) technique to detect and quantify the localizations of p53, Mdm2, and MEG3 in U2OS cells. We developed a new cross–nearest neighbor/Monte Carlo algorithm to quantify the association of these molecules. Proof of concept for our method was obtained by examining the binding between MEG3 and p53, and Mdm2 and p53. In contrast to previous models, our data support a model in which MEG3 modulates p53 independently of the interaction with Mdm2.

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

scholarly journals Single-molecule localization microscopy

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Mickaël Lelek ◽  
Melina T. Gyparaki ◽  
Gerti Beliu ◽  
Florian Schueder ◽  
Juliette Griffié ◽  
...  
Keyword(s):  
Single Molecule ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

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.

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