scholarly journals Image artifacts in Single Molecule Localization Microscopy: why optimization of sample preparation protocols matters

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Donna R. Whelan ◽  
Toby D. M. Bell
Keyword(s):  
Sample Preparation ◽  
Single Molecule ◽  
Image Artifacts ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Artifact Formation in Single Molecule Localization Microscopy

2019 ◽  
Author(s):  
Jochen M. Reichel ◽  
Thomas Vomhof ◽  
Jens Michaelis
Keyword(s):  
Single Molecule ◽  
Ground Truth ◽  
Image Artifacts ◽  
Ground Truth Data ◽  
Localization Accuracy ◽  
Localization Microscopy ◽  
Trade Offs ◽  
Artifact Formation ◽  
Localization Behavior ◽  
Single Molecule Localization Microscopy

AbstractWe investigate the influence of different accuracy-detection rate trade-offs on image reconstruction in single molecule localization microscopy. Our main focus is the investigation of image artifacts experienced when using low localization accuracy, especially in the presence of sample drift and inhomogeneous background. In this context we present a newly developed SMLM software termed FIRESTORM which is optimized for high accuracy reconstruction. For our analysis we used in silico SMLM data and compared the reconstructed images to the ground truth data. We observe two discriminable reconstruction populations of which only one shows the desired localization behavior.

scholarly journals Beginner’s guide to producing super-resolved images on a widefield fluorescence microscope

2020 ◽  
Vol 42 (4) ◽  
pp. 52-56
Author(s):  
Ilijana Vojnovic ◽  
Ulrike Endesfelder
Keyword(s):  
Fluorescence Microscopy ◽  
Sample Preparation ◽  
Single Molecule ◽  
Life Sciences ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Specialized Hardware ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

The development of super-resolution microscopy techniques, which are able to achieve resolutions in the nanometre range and as such allow the visualization of subcellular structures and dynamics, has considerably expanded the possibilities of fluorescence microscopy in the life sciences. While a majority of these techniques require highly specialized hardware, single-molecule localization microscopy (SMLM) can be implemented on conventional widefield fluorescence microscopes. Here, we describe what technical upgrades are necessary and discuss some of the difficulties that can be encountered during sample preparation and imaging.

Sample preparation for single molecule localization microscopy

2013 ◽  
Vol 15 (43) ◽  
pp. 18771 ◽  
Author(s):  
John R. Allen ◽  
Stephen T. Ross ◽  
Michael W. Davidson
Keyword(s):  
Sample Preparation ◽  
Single Molecule ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

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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