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>

Single‐molecule localization microscopy of septin bundles in mammalian cells

Cytoskeleton ◽  
2018 ◽  
Vol 76 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Adriano Vissa ◽  
Maximiliano Giuliani ◽  
Carol D. Froese ◽  
Moshe S. Kim ◽  
Forooz Soroor ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Single-Molecule Localization Microscopy to Study Protein Organization in the Filamentous Fungus Trichoderma atroviride

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3199
Author(s):  
Alexander W.A.F. Reismann ◽  
Lea Atanasova ◽  
Susanne Zeilinger ◽  
Gerhard J. Schütz
Keyword(s):  
Single Molecule ◽  
Filamentous Fungus ◽  
Mammalian Cells ◽  
Hyphal Growth ◽  
Trichoderma Atroviride ◽  
Localization Microscopy ◽  
Family Protein ◽  
Mycoparasitic Fungus ◽  
Microscopy Images ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy has boosted our understanding of biological samples by offering access to subdiffraction resolution using fluorescence microscopy methods. While in standard mammalian cells this approach has found wide-spread use, its application to filamentous fungi has been scarce. This is mainly due to experimental challenges that lead to high amounts of background signal because of ample autofluorescence. Here, we report the optimization of labeling, imaging and data analysis protocols to yield the first single-molecule localization microscopy images of the filamentous fungus Trichoderma atroviride. As an example, we show the spatial distribution of the Sur7 tetraspanin-family protein Sfp2 required for hyphal growth and cell wall stability in this mycoparasitic fungus.

scholarly journals Enhancing the localization precision of single molecule localization microscopy by analyzing Brownian motion of individual fluorescent probe

2021 ◽  
Author(s):  
Jielei Ni ◽  
Bo Cao ◽  
Gang Niu ◽  
Chen Xu ◽  
Yanxiang Ni
Keyword(s):  
Brownian Motion ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Photon Number ◽  
Number System ◽  
Z Score ◽  
Localization Microscopy ◽  
Localization Precision ◽  
Crucial Parameter ◽  
Single Molecule Localization Microscopy

The localization precision is a crucial parameter for single-molecule localization microscopy which directly influences the achievable spatial resolution. Although it can be optimized with photon number, system noise and system drift, we demonstrate that, Brownian motion of the fluorescent probe can be another factor that needs to be taken into consideration. By calculating the z-score of the displacement between adjacent frames, fluorescent probes that are undergoing large amplitude of Brownian motion can be identified. By filtering out these molecules, distinct improvement of localization precision can be achieved.

scholarly journals Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy

2021 ◽  
Vol 8 ◽  
Author(s):  
Sangyoon Ko ◽  
Jiwoong Kwon ◽  
Sang-Hee Shim
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Green Emission ◽  
Switching Behavior ◽  
Light Illumination ◽  
High Concentration ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact.

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.

scholarly journals Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs

2018 ◽  
Vol 148 (12) ◽  
pp. 123311 ◽  
Author(s):  
Koen J. A. Martens ◽  
Arjen N. Bader ◽  
Sander Baas ◽  
Bernd Rieger ◽  
Johannes Hohlbein
Keyword(s):  
Single Molecule ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy
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