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 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.

Common fluorescent proteins for single-molecule localization microscopy

2015 ◽  
Author(s):  
Natalia V. Klementieva ◽  
Nina G. Bozhanova ◽  
Natalie M. Mishina ◽  
Elena V. Zagaynova ◽  
Konstantin A. Lukyanov ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

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.

Common fluorescent proteins for single-molecule localization microscopy

2015 ◽  
Author(s):  
Natalia V. Klementieva ◽  
Nina G. Bozhanova ◽  
Natalie M. Mishina ◽  
Elena V. Zagaynova ◽  
Konstantin A. Lukyanov ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals The Benefits of Unnatural Amino Acid Incorporation as Protein Labels for Single Molecule Localization Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Pooja Laxman ◽  
Shirin Ansari ◽  
Katharina Gaus ◽  
Jesse Goyette
Keyword(s):  
Single Molecule ◽  
Unnatural Amino Acids ◽  
Fluorescent Dyes ◽  
Fluorescent Proteins ◽  
Unnatural Amino Acid ◽  
Fluorescent Label ◽  
Imaging Method ◽  
Localization Microscopy ◽  
The Right ◽  
Single Molecule Localization Microscopy

Single Molecule Localization Microscopy (SMLM) is an imaging method that allows for the visualization of structures smaller than the diffraction limit of light (~200 nm). This is achieved through techniques such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM). A large part of obtaining ideal imaging of single molecules is the choice of the right fluorescent label. An upcoming field of protein labeling is incorporating unnatural amino acids (UAAs) with an attached fluorescent dye for precise localization and visualization of individual molecules. For this technique, fluorescent probes are conjugated to UAAs and are introduced into the protein of interest (POI) as a label. Here we contrast this labeling method with other commonly used protein-based labeling methods such as fluorescent proteins (FPs) or self-labeling tags such as Halotag, SNAP-tags, and CLIP-tags, and highlight the benefits and shortcomings of the site-specific incorporation of UAAs coupled with fluorescent dyes in SMLM.

scholarly journals Symmetrically-dispersed spectroscopic single-molecule localization microscopy

2019 ◽  
Author(s):  
K. Song ◽  
Y. Zhang ◽  
B. Brenner ◽  
C. Sun ◽  
H. F. Zhang
Keyword(s):  
Spectral Analysis ◽  
Single Molecule ◽  
Particle Tracking ◽  
Three Dimensional ◽  
Spatial Localization ◽  
Single Particle Tracking ◽  
Localization Microscopy ◽  
First Time ◽  
Spectral Channels ◽  
Single Molecule Localization Microscopy

AbstractSpectroscopic single-molecule localization microscopy (sSMLM) achieved simultaneously imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from reduced photon budget because of dividing photons from individual stochastic emission into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically-dispersed sSMLM or SDsSMLM to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Comparing with existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multi-color imaging in fixed cells and three-dimensional single-particle tracking using SDsSMLM.

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

scholarly journals In cellulo Evaluation of Phototransformation Quantum Yields in Fluorescent Proteins Used As Markers for Single-Molecule Localization Microscopy

PLoS ONE ◽  
2014 ◽  
Vol 9 (6) ◽  
pp. e98362 ◽  
Author(s):  
Sergiy Avilov ◽  
Romain Berardozzi ◽  
Mudalige S. Gunewardene ◽  
Virgile Adam ◽  
Samuel T. Hess ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Quantum Yields ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Resolving Biology Beyond the Diffraction Limit with Single-Molecule Localization Microscopy

2016 ◽  
Author(s):  
Nafiseh Rafiei ◽  
Daniel Nino ◽  
Joshua N. Milstein
Keyword(s):  
Optical Imaging ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Diffraction Limit ◽  
Review Article ◽  
Localization Microscopy ◽  
Short Overview ◽  
The Future ◽  
Microscopic World ◽  
Single Molecule Localization Microscopy

Optical imaging provides a window into the microscopic world, but the level of observable detail is ultimately limited by the wavelength of light being employed. By harnessing the physics of photoswitchable dyes and fluorescent proteins, single-molecule localization microscopy (SMLM) provides a window into the nano-world of biology. This mini-review article provides a short overview of SMLM and discusses some of its prospects for the future.

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