Live‐Cell Localization Microscopy with a Fluorogenic and Self‐Blinking Tetrazine Probe

Fluorophores with photo-modulatory fluorescence properties are valuable for cutting-edge localization microscopy. The existing probes are either photo-activatable, or photo-switchable, but not both. We report a probe (DH-SiR), a leuco-dye obtained...

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Live‐Cell Localization Microscopy with a Fluorogenic and Self‐Blinking Tetrazine Probe

Angewandte Chemie ◽

10.1002/ange.201906806 ◽

2019 ◽

Vol 132 (2) ◽

pp. 814-820 ◽

Cited By ~ 1

Author(s):

Philipp Werther ◽

Klaus Yserentant ◽

Felix Braun ◽

Nicolai Kaltwasser ◽

Christoph Popp ◽

...

Keyword(s):

Live Cell ◽

Localization Microscopy ◽

Cell Localization

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Dictyostelium Discoideum: Live Cell Imaging in Changing Perspective

Protocols used in Molecular Biology ◽

10.2174/9789811439315120010016 ◽

2020 ◽

pp. 126-145

Keyword(s):

Dictyostelium Discoideum ◽

Protein Localization ◽

Red Light ◽

Live Cell ◽

Cell Organelle ◽

Light Spectrum ◽

Localization Study ◽

Cell Localization ◽

Uv Visible ◽

Fluorescent Tags

The advent of advanced microscopes; during microscope evolution from simple microscopes to confocal and live cell microscope; having digital imaging facility revolutionized our view for the living cells. In the protein localization study, fluorescent proteins are tagged at amino or carboxyl (preferably) terminal of desired protein for live cell study. These live cell studies improved our understanding of protein dynamics and understanding its role in biological regulation. The mutational variants of fluorescent tags (GFP, RFP); can be used with different protein; which will efficiently use UV-Visible to Far Red light spectrum; without overlapping of excitation and emission spectrum. Further, various cell organelle (Lysosome, Golgi bodies, Endoplasmic Reticulum, Mitochondria, Nucleus) trackers; improved our live cell localization studies in the wide non-overlapping UV-Visible spectrum.This chapter gives an overview for live cell protein localization study in mitotically active, unicellular stage of Dictyostelium discoideum. This evolutionary cutting edge organism had both unicellular as well as multicellular stages during its life cycle. This chapter will provide the design of fusion of fluorescent tag to the specific gene and its live cell localization. Further, it will cover; transformation of the unicellular organism; drug based selection; sample preparation with nuclear, mitochondrial localization markers (trackers) and live cell localization study on live cell-confocal microscope setup. It will also have a glimpse of the design of fusion protein with an aspect of advantage and disadvantages.

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Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics

Nature Methods ◽

10.1038/nmeth.1202 ◽

2008 ◽

Vol 5 (5) ◽

pp. 417-423 ◽

Cited By ~ 598

Author(s):

Hari Shroff ◽

Catherine G Galbraith ◽

James A Galbraith ◽

Eric Betzig

Keyword(s):

Live Cell ◽

Localization Microscopy ◽

Photoactivated Localization Microscopy

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Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

10.1101/2021.05.20.444797 ◽

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.

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Photoactivation of silicon rhodamines via a light-induced protonation

Nature Communications ◽

10.1038/s41467-019-12480-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

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.

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Correction: Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

Nanoscale ◽

10.1039/d0nr90195d ◽

2020 ◽

Vol 12 (35) ◽

pp. 18476-18477

Author(s):

Andres I. König ◽

Raya Sorkin ◽

Ariel Alon ◽

Dikla Nachmias ◽

Kalyan Dhara ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Single Molecule ◽

Live Cell ◽

Plasma Membrane Proteins ◽

Single Molecule Tracking ◽

Localization Microscopy

Correction for ‘Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins’ by Andres I. König et al., Nanoscale, 2020, 12, 3236–3248, DOI: 10.1039/C9NR08594G.

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