scholarly journals Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging

2017 ◽  
Vol 18 (7) ◽  
pp. 1524 ◽  
Author(s):  
◽  
◽  
◽  
Keyword(s):  
Single Molecule ◽  
Live Cell ◽  
Microscopy Imaging ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

2021 ◽  
Author(s):  
Nicolas Lardon ◽  
Lu Wang ◽  
Aline Tschanz ◽  
Philipp Hoess ◽  
Mai Tran ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Range ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Important Class ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

scholarly journals Photoactivation of silicon rhodamines via a light-induced protonation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Michelle S. Frei ◽  
Philipp Hoess ◽  
Marko Lampe ◽  
Bianca Nijmeijer ◽  
Moritz Kueblbeck ◽  
...  
Keyword(s):  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Super Resolution ◽  
Spectroscopic Properties ◽  
Single Particle Tracking ◽  
Live Cell ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Abstract Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

Three-dimensional single-molecule localization microscopy imaging based on compressed sensing

2020 ◽  
Vol 13 (5) ◽  
pp. 1065-1074
Author(s):  
ZHANG Sai-wen ◽  
◽  
LIN Dan-ying ◽  
YU Bin ◽  
LENG Xiao-ling ◽  
...  
Keyword(s):  
Compressed Sensing ◽  
Single Molecule ◽  
Three Dimensional ◽  
Microscopy Imaging ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

Dynamic Bayesian Cluster Analysis of Live-Cell Single Molecule Localization Microscopy Datasets

Small Methods ◽  
2018 ◽  
Vol 2 (9) ◽  
pp. 1800008 ◽  
Author(s):  
Juliette Griffié ◽  
Garth L. Burn ◽  
David J. Williamson ◽  
Ruby Peters ◽  
Patrick Rubin-Delanchy ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Single Molecule ◽  
Live Cell ◽  
Localization Microscopy ◽  
Bayesian Cluster Analysis ◽  
Bayesian Cluster ◽  
Single Molecule Localization Microscopy

scholarly journals Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii

2020 ◽  
Vol 11 ◽  
Author(s):  
Bartosz Turkowyd ◽  
Sandra Schreiber ◽  
Julia Wörtz ◽  
Ella Shtifman Segal ◽  
Moshe Mevarech ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Particle Tracking ◽  
Haloferax Volcanii ◽  
Microscopy Imaging ◽  
Localization Microscopy ◽  
Cellular Dna ◽  
Eukaryotic Organisms ◽  
Single Molecule Localization Microscopy

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here, we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view (FOV) as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA.This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

scholarly journals Photoactivation of silicon rhodamines via a light-induced protonation

2019 ◽  
Author(s):  
Michelle S. Frei ◽  
Philipp Hoess ◽  
Marko Lampe ◽  
Bianca Nijmeijer ◽  
Moritz Kueblbeck ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Spectroscopic Properties ◽  
Single Particle Tracking ◽  
Live Cell ◽  
Localization Microscopy ◽  
Rhodamine Derivative ◽  
New Type ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractWe present a new type of photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent isomerization followed by protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we created probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

scholarly journals Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii

2020 ◽  
Author(s):  
Bartosz Turkowyd ◽  
Sandra Schreiber ◽  
Julia Wörtz ◽  
Ella Shtifman Segal ◽  
Moshe Mevarech ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Particle Tracking ◽  
Haloferax Volcanii ◽  
Microscopy Imaging ◽  
Localization Microscopy ◽  
Cellular Dna ◽  
Eukaryotic Organisms ◽  
Single Molecule Localization Microscopy

AbstractIn recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology.Here we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA. This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

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>

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