A general design of caging-group free photoactivatable fluorophores for live-cell nanoscopy

2021 ◽  
Author(s):  
Richard Lincoln ◽  
Mariano L. Bossi ◽  
Michael Remmel ◽  
Elisa D'Este ◽  
Alexey N. Butkevich ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Visible Spectrum ◽  
Live Cell ◽  
Stimulated Emission ◽  
Protecting Groups ◽  
Superresolution Microscopy ◽  
Controlled Switching ◽  
New Family ◽  
General Method

The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every superresolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging superresolution methods. Photoactivatable dyes offer significant improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogs, yielding a new family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell fluorescence microscopy, and both coordinate-targeted stimulated emission depletion (STED) and coordinate-stochastic single-molecule localization superresolution microscopy (SMLM).

scholarly journals A general method to fine-tune fluorophores for live-cell and in vivo imaging

2017 ◽  
Author(s):  
Jonathan B. Grimm ◽  
Anand K. Muthusamy ◽  
Yajie Liang ◽  
Timothy A. Brown ◽  
William C. Lemon ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
In Vivo Imaging ◽  
Chemical Properties ◽  
Live Cell ◽  
Live Cells ◽  
Fine Tune ◽  
General Method ◽  
And Chemical Properties

ABSTRACTPushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the ‘Janelia Fluor’ (JF) series of dyes. Here, we refine and extend this strategy, showing that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties with unprecedented precision. This strategy yields a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red with utility in live cells, tissue, and animals.

scholarly journals Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

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.

scholarly journals A general method to improve fluorophores for live-cell and single-molecule microscopy

2015 ◽  
Vol 12 (3) ◽  
pp. 244-250 ◽  
Author(s):  
Jonathan B Grimm ◽  
Brian P English ◽  
Jiji Chen ◽  
Joel P Slaughter ◽  
Zhengjian Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell ◽  
Single Molecule Microscopy ◽  
General Method

scholarly journals Revealing the nanoscale morphology of the primary cilium using super-resolution fluorescence microscopy

2018 ◽  
Author(s):  
Joshua Yoon ◽  
Colin J. Comerci ◽  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Tim Stearns ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Underlying Structure ◽  
Nanoscale Morphology

ABSTRACTSuper-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ~250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (~2-6 μm long and 200-300 nm diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened (SMO), with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction (PSR) algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, 2-color, 2D STimulated Emission Depletion (STED) microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the SMO-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses STED to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

scholarly journals Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

2021 ◽  
Author(s):  
Hela Benaissa ◽  
Karim Ounoughi ◽  
Isabelle Aujard ◽  
Evelyne Fischer ◽  
Rosette Goïame ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Spectral Properties ◽  
Rational Design ◽  
Imaging Techniques ◽  
Visible Spectrum ◽  
Live Cell ◽  
Stimulated Emission ◽  
Live Cells ◽  
Fluorescent Reporters ◽  
Entire Visible Spectrum

AbstractBiocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we present the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of live-cell compatible fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. We showed that the ability to tune the fluorescence color and properties through simple molecular modulation provides an unprecedent experimental versatility for imaging proteins in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms. The ability to tune the spectral properties and fluorescence performance enables to match the spectral specifications and requirements of the most advanced imaging techniques, and allowed us to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

scholarly journals PBAF regulates compartmentalization of actively transcribing chromatin hubs

2017 ◽  
Author(s):  
Charles A. Kenworthy ◽  
Shu-Hao Liou ◽  
Panagiotos Chandris ◽  
Vincent Wong ◽  
Patrycja Dziuba ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Genomic Organization ◽  
Live Cell ◽  
Single Molecule Fluorescence ◽  
Chromatin Remodelers ◽  
Nuclear Compartments ◽  
Inactive Genes ◽  
Active Genes ◽  
Single Molecule Fluorescence Microscopy

AbstractTranscriptionally active genes contain acetyl-rich chromatin and are organized in distinct nuclear compartments that are spatially separated from transcriptionally inactive genes. It is not known how this compartmentalized acetylated chromatin is targeted and regulated by chromatin remodelers such as PBAF. Thus, we sought to understand how PBAF targets chromatin and modulates compartmentalization of transcriptionally active genes using live-cell single molecule fluorescence microscopy. Our work reveals chromatin hubs throughout the nucleus where PBAF cycles on and off the genome. Deletion of PBAF’s bromodomains impairs recognition of hubs and cycling on chromatin. Interestingly, markers for transcriptionally active and inactive genes can be found in compartments harboring acetylated chromatin at the periphery that is selectively recognized by PBAF via bromodomains. Defects in PBAF’s peripheral targeting lead to a select reduction in the size and number of compartments containing transcriptionally active genes. Our data, combined with previous work in Yeast and Drosophila, suggest that PBAF activity serves as a barrier to heterochromatin spreading. Overall, our findings suggest that chromatin compartments are highly structured with unique peripherally associated acetylation marks. PBAF utilizes these marks to help shape nuclear compartments containing transcriptionally active genes, thereby aiding genomic organization.

scholarly journals Bright photoactivatable fluorophores for single-molecule imaging

2016 ◽  
Author(s):  
Jonathan B. Grimm ◽  
Brian P. English ◽  
Anand K. Muthusamy ◽  
Brian P. Mehl ◽  
Peng Dong ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Particle Tracking ◽  
Live Cell ◽  
Cell Labeling ◽  
Advanced Imaging ◽  
Live Cell Microscopy ◽  
Protein Tags ◽  
Live Cell Labeling ◽  
Cell Microscopy ◽  
General Method

ABSTRACTSmall molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the azetidine-containing “Janelia Fluor” (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with established live-cell labeling strategies. These compounds retain the superior brightness of the JF dyes but their facile photoactivation enables improved single-particle tracking and localization microscopy experiments.

Sign in / Sign up

Export Citation Format

Share Document