scholarly journals On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

2019 ◽  
Vol 21 (7) ◽  
pp. 3721-3733 ◽  
Author(s):  
Jochem H. Smit ◽  
Jasper H. M. van der Velde ◽  
Jingyi Huang ◽  
Vanessa Trauschke ◽  
Sarah S. Henrikus ◽  
...  
Keyword(s):  
Triplet State ◽  
Single Molecule ◽  
Photophysical Properties ◽  
Super Resolution ◽  
Organic Fluorophores ◽  
Resolution Imaging ◽  
The Impact ◽  
Kinetics Of

How photostabilizer molecules influence the photophysical properties of various organic fluorophores used for single-molecule and super-resolution imaging.

scholarly journals On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

2018 ◽  
Author(s):  
Jochem H. Smit ◽  
Jasper H. M. van der Velde ◽  
Jingyi Huang ◽  
Vanessa Trauschke ◽  
Sarah S. Henrikus ◽  
...  
Keyword(s):  
Triplet State ◽  
Aromatic Amino Acids ◽  
Super Resolution ◽  
Self Healing ◽  
Fluorescent Markers ◽  
Spatio Temporal ◽  
Covalently Linked ◽  
Super Resolution Microscopy ◽  
The Impact ◽  
Kinetics Of

AbstractWhile buffer cocktails remain the gold-standard for photostabilization and photoswitching of fluorescent markers, intramolecular triplet-state quenchers emerge as an alternative strategy to impart fluorophores with ‘self-healing’ or even functional properties such as photoswitching. In this contribution, we evaluated various combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other rather than being additive or even synergistic. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. In this context we identified a new function of intramolecular photostabilizers, i.e., protection of fluorophores from reversible off-switching events caused by solution-additives, which were previously misinterpreted as photobleaching. Our studies also provide practical guidance for usage of photostabilizer-dye conjugates for STORM-type super-resolution microscopy permitting the exploitation of their improved photophysics for increased spatio-temporal resolution. Finally, we provide evidence that the biochemical environment, e.g., proximity of aromatic amino-acids such as tryptophan, reduces the photostabilization efficiency of commonly used buffer cocktails. Not only have our results important implications for a deeper mechanistic understanding of self-healing dyes, but they will provide a general framework to select label positions for optimal and reproducible photostability or photoswitching kinetics.

scholarly journals A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Jasper H. M. van der Velde ◽  
Jens Oelerich ◽  
Jingyi Huang ◽  
Jochem H. Smit ◽  
Atieh Aminian Jazi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Unnatural Amino Acids ◽  
Photophysical Properties ◽  
Super Resolution ◽  
Single Step ◽  
General Strategy ◽  
Self Healing ◽  
Chemical Transformations ◽  
Scaffolding Strategy ◽  
Organic Fluorophore

Abstract Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.

scholarly journals Super-Resolution Imaging and Single Molecule Tracking of the Nuclear Protein Emerin

2014 ◽  
Vol 106 (2) ◽  
pp. 201a
Author(s):  
Anthony M. Fernandez ◽  
Fabien F. Pinaud
Keyword(s):  
Single Molecule ◽  
Nuclear Protein ◽  
Super Resolution ◽  
Single Molecule Tracking ◽  
Resolution Imaging

scholarly journals Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246138
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

Influence of the lifetime of a metastable triplet state of photostable organic fluorophores on the kinetics of fluorescence fading and recovery

2013 ◽  
Vol 80 (3) ◽  
pp. 357-360 ◽  
Author(s):  
I. V. Stanishevsky ◽  
K. N. Solovyov ◽  
S. M. Arabei ◽  
V. A. Chernyavsky
Keyword(s):  
Triplet State ◽  
Organic Fluorophores ◽  
Kinetics Of ◽  
Metastable Triplet State

Single molecule Coordinate and Height super-resolution Imaging with Dithering and Orientation (CHIDO)

2020 ◽  
Author(s):  
Luis A. Alemán-Castañeda ◽  
Valentina Curcio ◽  
Thomas G. Brown ◽  
Sophie Brasselet ◽  
Miguel A. Alonso
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Fast, Simultaneous Multiple Fluorophore Fitting in Single Molecule Super-Resolution Imaging

2011 ◽  
Vol 100 (3) ◽  
pp. 349a
Author(s):  
Fang Huang ◽  
Samantha L. Schwartz ◽  
Jason M. Byars ◽  
Keith A. Lidke
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Resolution Imaging ◽  
Fitting In

Super-resolution fluorescent materials: an insight into design and bioimaging applications

2016 ◽  
Vol 45 (17) ◽  
pp. 4651-4667 ◽  
Author(s):  
Zhigang Yang ◽  
Amit Sharma ◽  
Jing Qi ◽  
Xiao Peng ◽  
Dong Yeop Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Fluorescent Imaging ◽  
Organic Fluorophores ◽  
Biological Targets ◽  
Fluorescent Materials ◽  
Microscopy Techniques ◽  
Insight Into

With the emerging of super-resolution fluorescent imaging microscopy techniques, biological targets below 200 nm in size are successful to be localized clearly and precisely with unprecedented details. In this tutorial review, the fluorescent materials, including organic fluorophores and nanomaterials, utilized in STED, single molecule localized microscopy (PALM/STORM) and SOFI microscopies, together with their working principles are mainly discussed.

scholarly journals Titelbild: SLAP: Small Labeling Pair for Single-Molecule Super-Resolution Imaging (Angew. Chem. 35/2015)

2015 ◽  
Vol 127 (35) ◽  
pp. 10175-10175
Author(s):  
Ralph Wieneke ◽  
Anika Raulf ◽  
Alina Kollmannsperger ◽  
Mike Heilemann ◽  
Robert Tampé
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Resolution Imaging
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