Super-resolution fluorescence microscopy for investigating bacterial macromolecular complexes DOI Open Access
Alexander Carsten, Manuel Wolters, Martin Aepfelbacher

et al.

Authorea (Authorea), Journal Year: 2023, Volume and Issue: unknown

Published: Aug. 5, 2023

Super-resolution fluorescence microscopy techniques developed over the past two decades have pushed resolution limit for fluorescently labeled molecules into nanometer range. These potential to study bacterial macromolecular complexes such as secretion systems with single-molecule on a millisecond time scale. Here we review recent applications of super-resolution in molecular bacteriology focus systems. We also describe MINFLUX nanoscopy, relatively new technique that promises one day produce movies machines action.

Language: Английский

HaloTag‐Based Reporters for Fluorescence Imaging and Biosensing DOI Creative Commons
Alexander Cook, Franziska Walterspiel, Claire Deo

et al.

ChemBioChem, Journal Year: 2023, Volume and Issue: 24(12)

Published: Feb. 23, 2023

Abstract Visualizing the structure and dynamics of biomolecules is critical to understand biological function, requires methods fluorescently label targets interest in their cellular context. Self‐labelling proteins, which combine a genetically encoded tag with small‐molecule fluorophore, have attracted considerable attention for this purpose, as they can overcome limitations fluorescent proteins. Among them, HaloTag protein most broadly used, showing fast specific labelling small, easy functionalize cell‐permeant ligand. Synthetic chemistry engineering provided portfolio powerful imaging tools exploiting HaloTag, along general optimize adapt them applications. Here, we provide an overview reporters based on biosensing, highlighting strategies

Language: Английский

Citations

36
Spontaneously Blinking Rhodamine Dyes for Single‐Molecule Localization Microscopy DOI Open Access
Weijie Chi, Davin Tan, Qinglong Qiao

et al.

Angewandte Chemie International Edition, Journal Year: 2023, Volume and Issue: 62(39)

Published: May 29, 2023

Abstract Single‐molecule localization microscopy (SMLM) has found extensive applications in various fields of biology and chemistry. As a vital component SMLM, fluorophores play an essential role obtaining super‐resolution fluorescence images. Recent research on spontaneously blinking greatly simplified the experimental setups extended imaging duration SMLM. To support this crucial development, review provides comprehensive overview development rhodamines from 2014 to 2023, as well key mechanistic aspects intramolecular spirocyclization reactions. We hope that by offering insightful design guidelines, will contribute accelerating advancement technologies.

Language: Английский

Citations

27
Bioorthogonal Caging-Group-Free Photoactivatable Probes for Minimal-Linkage-Error Nanoscopy DOI Creative Commons
Ayse Aktalay, Richard Lincoln,

Lukas Heynck

et al.

ACS Central Science, Journal Year: 2023, Volume and Issue: 9(8), P. 1581 - 1590

Published: July 26, 2023

Here we describe highly compact, click compatible, and photoactivatable dyes for super-resolution fluorescence microscopy (nanoscopy). By combining the xanthone (PaX) core with a tetrazine group, achieve minimally sized sensitive molecular dyads selective labeling of unnatural amino acids introduced by genetic code expansion. We exploit excited state quenching properties group to attenuate photoactivation rates PaX, further reduce overall emission photogenerated fluorophore, providing two mechanisms selectivity off-target signal. Coupled MINFLUX nanoscopy, employ our in minimal-linkage-error imaging vimentin filaments, demonstrating molecular-scale precision fluorophore positioning.

Language: Английский

Citations

13
Guidelines for MINFLUX Excitation Pattern Design DOI

Nahuel Tarkowski,

Fernando D. Stefani

ACS Photonics, Journal Year: 2025, Volume and Issue: unknown

Published: March 9, 2025

Language: Английский

Citations

0
Enhancing precision for simultaneous 3D localization and 3D orientation with structured illumination DOI
Zhengyi Zhan,

Xiaofan Sun,

Chenying He

et al.

Optics Letters, Journal Year: 2025, Volume and Issue: 50(9), P. 2856 - 2856

Published: March 28, 2025

Determining the three-dimensional (3D) position and orientation of molecules from limited fluorescence photons is challenging. Existing modulated illumination localization microscopy partially transfers photon burden to excitation intensity gradients, advancing precision molecular scale. However, most simultaneous methods rely on polarization splitting in detection path are sensitive signal-to-background ratio. This sensitivity further exacerbated by sequential structured (SSI), potentially compromising robustness. Here, we introduce V-SIMFLUX, which integrates SSI Vortex PSF without polarization-splitting detection. Our theoretical analysis shows that V-SIMFLUX improves all estimated parameters, notably achieving a typical 2.9-fold improvement lateral 1.7-fold enhancement azimuthal compared conventional wide-field illumination.

Language: Английский

Citations

0
Advancing Single-Molecule Biophysics: Next-Generation Organic Fluorophores with Tailored Labeling Strategies DOI Creative Commons
Lei Zhang,

Dongwen Shen,

Jia-Zhen Yang

et al.

Chemical & Biomedical Imaging, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

Citations

0
MINFLUX achieves molecular resolution with minimal photons DOI
Lukas Scheiderer, Zach Marin, Jonas Ries

et al.

Nature Photonics, Journal Year: 2025, Volume and Issue: 19(3), P. 238 - 247

Published: March 1, 2025

Language: Английский

Citations

0
MINFLUX nanoscopy enhanced with high-order vortex beams DOI Creative Commons

Xiao-Jie Tan,

Zhiwei Huang

Light Science & Applications, Journal Year: 2025, Volume and Issue: 14(1)

Published: May 6, 2025

Abstract Minimal photon fluxes (MINFLUX) nanoscopy has emerged as a transformative advancement in superresolution imaging, enabling unprecedented nanoscale observations across diverse biological scenarios. In this work, we propose, for the first time, that employing high-order vortex beams can significantly enhance performance of MINFLUX, surpassing limitations conventional MINFLUX using first-order beam. Our theoretical analysis indicates that, standard improve maximum localization precision by factor corresponding to their order, which approach sub-nanometer scale under optimal conditions, and raster scan allow wider field view while maintaining enhanced precision. These findings underscore potential elevate paving way towards ultra-high resolution imaging broad range applications.

Language: Английский

Citations

0
Photoactivatable Carbo‐ and Silicon‐Rhodamines and Their Application in MINFLUX Nanoscopy DOI Creative Commons
Ayse Aktalay, Taukeer A. Khan, Mariano L. Bossi

et al.

Angewandte Chemie International Edition, Journal Year: 2023, Volume and Issue: 62(41)

Published: Aug. 9, 2023

New photoactivatable fluorescent dyes (rhodamine, carbo- and silicon-rhodamines [SiR]) with emission ranging from green to far red have been prepared, their photophysical properties studied. The photocleavable 2-nitrobenzyloxycarbonyl unit an alpha-carboxyl group as a branching point additional functionality was attached polycyclic lipophilic dye. probes having the HaloTagTM amine (O2) ligand bound dye core were obtained applied for live-cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused HaloTag. 2D (VIM, NUP96) 3D MINFLUX nanoscopy, well superresolution fluorescence microscopy single fluorophore activation labeling). Images of VIM NUPs labeled different acquired apparent dimensions shapes assessed on lower single-digit nanometer scale. Applicability performance derivatives evaluated terms photoactivation rate, labeling detection efficiency, number detected photons per molecule other parameters related nanoscopy.

Language: Английский

Citations

8
Molecular mapping of neuronal architecture using STORM microscopy and new fluorescent probes for SMLM imaging DOI Creative Commons
Victor Breton, Paul Nazac, David Boulet

et al.

Neurophotonics, Journal Year: 2024, Volume and Issue: 11(01)

Published: March 8, 2024

Imaging neuronal architecture has been a recurrent challenge over the years, and localization of synaptic proteins is frequent in neuroscience. To quantitatively detect analyze structure synapses, we recently developed free SODA software to association pre postsynaptic proteins. fully take advantage spatial distribution analysis complex cells, such as neurons, also selected some new dyes for plasma membrane labeling. Using Icy plugin, could both conventional single molecule microscopy, giving access molecular map at nanoscale level. replace those distributions within three-dimensional (3D) shape, used MemBright probes 3D STORM decipher entire shape various dendritic spine types single-molecule resolution We report here example mask, but these tools have broader spectrum interest since they can be whatever or cellular type. Altogether with thus provide perfect toolkit nanometric context.

Language: Английский

Citations

2