Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

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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Nanoparticles for super-resolution microscopy and single-molecule tracking

Nature Methods ◽

10.1038/s41592-018-0012-4 ◽

2018 ◽

Vol 15 (6) ◽

pp. 415-423 ◽

Cited By ~ 86

Author(s):

Dayong Jin ◽

Peng Xi ◽

Baoming Wang ◽

Le Zhang ◽

Jörg Enderlein ◽

...

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Single Molecule Tracking ◽

Super Resolution Microscopy

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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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Super-Beacons: open-source probes with spontaneous tuneable blinking compatible with live-cell super-resolution microscopy

10.1101/2020.01.20.912311 ◽

2020 ◽

Author(s):

Pedro M. Pereira ◽

Nils Gustafsson ◽

Mark Marsh ◽

Musa M. Mhlanga ◽

Ricardo Henriques

Keyword(s):

Open Source ◽

Single Molecule ◽

High Intensity ◽

Transmembrane Proteins ◽

Super Resolution ◽

Live Cell ◽

New Class ◽

High Illumination ◽

Super Resolution Microscopy

Localization based super-resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non-fluorescent states. These transitions are commonly regulated by high-intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single-molecule self-quenching as a mechanism to generate spontaneous photoswitching independent of illumination. To demonstrate this principle, we developed a new class of DNA-based open-source Super-Resolution probes named Super-Beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live-cell friendly Super-Resolution Microscopy without high-illumination or toxic imaging buffers is revealed by imaging Interferon Inducible Transmembrane proteins (IFITMs) at sub-100nm resolutions.

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Super-Resolution Microscopy and Single-Molecule Tracking Reveal Distinct Adaptive Dynamics of MreB and of Cell Wall-Synthesis Enzymes

Frontiers in Microbiology ◽

10.3389/fmicb.2020.01946 ◽

2020 ◽

Vol 11 ◽

Author(s):

Simon Dersch ◽

Johanna Mehl ◽

Lisa Stuckenschneider ◽

Benjamin Mayer ◽

Julian Roth ◽

...

Keyword(s):

Cell Wall ◽

Single Molecule ◽

Adaptive Dynamics ◽

Super Resolution ◽

Cell Wall Synthesis ◽

Single Molecule Tracking ◽

Super Resolution Microscopy

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Photoactivation of silicon rhodamines via a light-induced protonation

10.1101/626853 ◽

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.

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Optimized localization analysis for single-molecule tracking and super-resolution microscopy

Nature Methods ◽

10.1038/nmeth.1447 ◽

2010 ◽

Vol 7 (5) ◽

pp. 377-381 ◽

Cited By ~ 537

Author(s):

Kim I Mortensen ◽

L Stirling Churchman ◽

James A Spudich ◽

Henrik Flyvbjerg

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Single Molecule Tracking ◽

Localization Analysis ◽

Super Resolution Microscopy

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Deep-Learning Super-Resolution Microscopy Reveals Nanometer-Scale Intracellular Dynamics at the Millisecond Temporal Resolution

10.1101/2021.10.08.463746 ◽

2021 ◽

Author(s):

Rong Chen ◽

Xiao Tang ◽

Zeyu Shen ◽

Yusheng Shen ◽

Tiantian Li ◽

...

Keyword(s):

Deep Learning ◽

Spatial Resolution ◽

Temporal Resolution ◽

Single Molecule ◽

Super Resolution ◽

Live Cell ◽

Acquisition Time ◽

Microtubule Network ◽

Single Frame ◽

Super Resolution Microscopy

AbstractSingle-molecule localization microscopy (SMLM) can be used to resolve subcellular structures and achieve a tenfold improvement in spatial resolution compared to that obtained by conventional fluorescence microscopy. However, the separation of single-molecule fluorescence events in thousands of frames dramatically increases the image acquisition time and phototoxicity, impeding the observation of instantaneous intracellular dynamics. Based on deep learning networks, we develop a single-frame super-resolution microscopy (SFSRM) approach that reconstructs a super-resolution image from a single frame of a diffraction-limited image to support live-cell super-resolution imaging at a ∼20 nm spatial resolution and a temporal resolution of up to 10 ms over thousands of time points. We demonstrate that our SFSRM method enables the visualization of the dynamics of vesicle transport at a millisecond temporal resolution in the dense and vibrant microtubule network in live cells. Moreover, the well-trained network model can be used with different live-cell imaging systems, such as confocal and light-sheet microscopes, making super-resolution microscopy accessible to nonexperts.

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