scholarly journals High-density mapping of single-molecule trajectories with photoactivated localization microscopy

2008 ◽  
Vol 5 (2) ◽  
pp. 155-157 ◽  
Author(s):  
Suliana Manley ◽  
Jennifer M Gillette ◽  
George H Patterson ◽  
Hari Shroff ◽  
Harald F Hess ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Density ◽  
Localization Microscopy ◽  
Density Mapping ◽  
Photoactivated Localization Microscopy

scholarly journals Unscrambling Fluorophore Blinking for Comprehensive Cluster Detection via Photoactivated Localization Microscopy

2019 ◽  
Author(s):  
Rene Platzer ◽  
Benedikt K. Rossboth ◽  
Magdalena C. Schneider ◽  
Eva Sevcsik ◽  
Florian Baumgart ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cluster Detection ◽  
Protein Distribution ◽  
Localization Microscopy ◽  
Organic Fluorophores ◽  
Photoactivated Localization Microscopy ◽  
Precise Knowledge ◽  
Simulation Based ◽  
Analysis Package ◽  
Molecular Clustering

ABSTRACTDetermining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform we revealed complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allowed for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.GRAPHICAL ABSTRACT

scholarly journals Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
René Platzer ◽  
Benedikt K. Rossboth ◽  
Magdalena C. Schneider ◽  
Eva Sevcsik ◽  
Florian Baumgart ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cluster Detection ◽  
Protein Distribution ◽  
Localization Microscopy ◽  
Organic Fluorophores ◽  
Photoactivated Localization Microscopy ◽  
Precise Knowledge ◽  
Simulation Based ◽  
Analysis Package ◽  
Molecular Clustering

Abstract Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

scholarly journals ULK1 forms distinct oligomeric states and nanoscopic morphologies during autophagy initiation

2020 ◽  
Author(s):  
Chiranjib Banerjee ◽  
Dushyant Mehra ◽  
Daihyun Song ◽  
Angel Mancebo ◽  
Do-Hyung Kim ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Single Molecule ◽  
Amino Acid Starvation ◽  
Autophagosome Formation ◽  
Localization Microscopy ◽  
Photoactivated Localization Microscopy ◽  
Autophagy Induction ◽  
Evolutionarily Conserved ◽  
Spherical Structures ◽  
Insight Into

AbstractAutophagy is an evolutionarily conserved process for the degradation and recycling of intracellular components. Although autophagy has been extensively studied, it still remains unclear how autophagosome formation occurs in response to starvation. Here we combined CRISPR-cas9-assisted genome-editing with quantitative Photoactivated Localization Microscopy (qPALM) to analyze the nanoscopic spatial distribution and oligomeric states of endogenous ULK1, the central autophagy induction regulator with single molecule sensitivity. Amino acid starvation induced a small fraction of ULK1 molecules to localize to arc-shaped and spherical structures with radii up to 300 nm and with more than 30 ULK1 molecules. These starvation-induced structures with high ULK1 content occurred only when ULK1 was colocalized with Atg13 and within 100 nm distance to the endoplasmic reticulum. This analysis revealed that a threshold number of ULK1 molecules around 30 is necessary to drive the formation of early autophagic ULK1 structures under starvation, providing an unprecedented quantitative insight into a hierarchical transition of ULK1 states during autophagy initiation.

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>

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