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 3D single molecule localization microscopy reveals the topography of the immunological synapse at isotropic precision below 15 nm

2021 ◽  
Author(s):  
Lukas Velas ◽  
Mario Brameshuber ◽  
Johannes B. Huppa ◽  
Elke Kurz ◽  
Michael Dustin ◽  
...  
Keyword(s):  
T Cells ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Protein Interactions ◽  
Immunological Synapse ◽  
Size Exclusion ◽  
Antigenic Peptides ◽  
Protein Distribution ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. A variety of protein-protein interactions in trans-configuration defines the topography of the synapse and orchestrates the antigen-recognition process. In turn, the synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. For better understanding it is hence critical to map the 3D topography of the immunological synapse at high precision. Current methods, however, provide only rather coarse images of the protein distribution within the synapse, which do not reach the dimension of the protein ectodomains. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows 3-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and non-activating bilayers, respectively.

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 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.

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