scholarly journals Robust and Bright Genetically Encoded Fluorescent Markers for Highlighting Structures and Compartments in Mammalian Cells

2017 ◽  
Author(s):  
Anna O Chertkova ◽  
Marieke Mastop ◽  
Marten Postma ◽  
Nikki van Bommel ◽  
Sanne van der Niet ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Mammalian Cells ◽  
Protein Domains ◽  
Spectroscopic Properties ◽  
Living Cells ◽  
Cellular Structures ◽  
Signal Sequences ◽  
Fluorescent Marker ◽  
Fluorescent Markers

To increase our understanding of the inner working of cells, there is a need for specific markers to identify biomolecules, cellular structures and compartments. One type of markers comprises genetically encoded fluorescent probes that are linked with protein domains, peptides and/or signal sequences. These markers are encoded on a plasmid and they allow straightforward, convenient labeling of cultured mammalian cells by introducing the plasmid into the cells. Ideally, the fluorescent marker combines favorable spectroscopic properties (brightness, photostability) with specific labeling of the structure or compartment of interest. Here, we report our ongoing efforts to generate robust and bright genetically encoded fluorescent markers for highlighting structures and compartments in living cells. The plasmids are distributed by addgene: https://www.addgene.org/browse/article/28189953/

scholarly journals A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae

2019 ◽  
Vol 116 (32) ◽  
pp. 15817-15822 ◽  
Author(s):  
Chenguang Wang ◽  
Masayasu Taki ◽  
Yoshikatsu Sato ◽  
Yasushi Tamura ◽  
Hideyuki Yaginuma ◽  
...  
Keyword(s):  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Fluorescent Labeling ◽  
Spatiotemporal Resolution ◽  
Fluorescent Marker ◽  
Sted Microscopy ◽  
Fluorescent Markers ◽  
Enhanced Photostability ◽  
Single Mitochondrion

Stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. For the visualization of the mitochondrial membrane dynamics in STED microscopy, rationally designed mitochondrial fluorescent markers with enhanced photostability are required. Herein, we report the development of a superphotostable fluorescent labeling reagent with long fluorescence lifetime, whose design is based on a structurally reinforced naphthophosphole fluorophore that is conjugated with an electron-donating diphenylamino group. The combination of long-lived fluorescence and superphotostable features of the fluorophore allowed us to selectively capture the ultrastructures of the mitochondrial cristae with a resolution of ∼60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the intermembrane fusion in different mitochondria as well as the intercristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.

scholarly journals Detection and quantification of single mRNA dynamics with the Riboglow fluorescent RNA tag

2019 ◽  
Author(s):  
Esther Braselmann ◽  
Timothy J. Stasevich ◽  
Kenneth Lyon ◽  
Robert T. Batey ◽  
Amy E. Palmer
Keyword(s):  
Single Molecule ◽  
Fluorescent Probes ◽  
Mammalian Cells ◽  
Fluorescent Antibody ◽  
Living Cells ◽  
Rna Molecules ◽  
Single Molecule Level ◽  
Rna Biology ◽  
Tagging System ◽  
Detection And Quantification

AbstractLabeling and tracking biomolecules with fluorescent probes on the single molecule level enables quantitative insights into their dynamics in living cells. We previously developed Riboglow, a platform to label RNAs in live mammalian cells, consisting of a short RNA tag and a small organic probe that increases fluorescence upon binding RNA. Here, we demonstrate that Riboglow is capable of detecting and tracking single RNA molecules. We benchmark RNA tracking by comparing results with the established MS2 RNA tagging system. To demonstrate versatility of Riboglow, we assay translation on the single molecule level, where the translated mRNA is tagged with Riboglow and the nascent polypeptide is labeled with a fluorescent antibody. The growing effort to investigate RNA biology on the single molecule level requires sophisticated and diverse fluorescent probes for multiplexed, multi-color labeling of biomolecules of interest, and we present Riboglow as a new member in this toolbox.

scholarly journals Enhancing biocompatibility of rhodamine fluorescent probes by a neighbouring group effect

2020 ◽  
Author(s):  
Jonas Bucevičius ◽  
Georgij Kostiuk ◽  
Rūta Gerasimaitė ◽  
Tanja Gilat ◽  
Gražvydas Lukinavičius
Keyword(s):  
Fluorescent Probes ◽  
Staining Intensity ◽  
Living Cells ◽  
Amide Group ◽  
Cell Permeability ◽  
Cellular Structures ◽  
High Contrast ◽  
Group Effect ◽  
First Time

AbstractFluorescence microscopy is an essential tool for understanding dynamic processes in living cells and organisms. However, many fluorescent probes for labelling cellular structures suffer from unspecific interactions and low cell permeability. Herein, we demonstrate that the neighbouring group effect which results from positioning an amide group next to a carboxyl group in the benzene ring of rhodamines dramatically increases cell permeability of the rhodamine-based probes through stabilizing a fluorophore in a hydrophobic spirolactone state. Based on this principle, we create probes targeting tubulin, actin and DNA. Their superb staining intensity, tuned toxicity and specificity allows long-term 3D confocal and STED nanoscopy with sub-30 nm resolution. As a result, the real microtubule diameter of 23 nm was resolved inside a living cell for the first time. Due to their unrestricted cell permeability and efficient accumulation on tubulin, the new probes produce high contrast images at sub-nanomolar concentrations.

A Fused Thiophene-S,S-Dioxide-Based Super-Photostable Fluorescent Marker for Lipid Droplets

2020 ◽  
Author(s):  
Masayasu Taki ◽  
Keiji Kajiwara ◽  
Eriko Yamaguchi ◽  
Yoshikatsu Sato ◽  
Shigehiro Yamaguchi
Keyword(s):  
Small Molecules ◽  
Lipid Droplets ◽  
Trajectory Analysis ◽  
Super Resolution ◽  
Fluorescent Marker ◽  
Selective Staining ◽  
Fluorescent Markers ◽  
Biological Studies ◽  
Particle Level

Lipid droplets (LDs) are essential organelle in most eukaryotes, and tracking intracellular LDs dynamics using synthetic small molecules is crucial for biological studies. However, only a limited number of fluorescent markers that satisfy all requirements, such as the selective staining of LDs, high photostability, and sufficient biocompatibility, have been developed. Herein, we report a series of donor-p-acceptor dyes based on the thiophene-containing fused polycyclic scaffold [1]benzothieno[3,2-<i>b</i>][1]benzothiophene (BTBT), in which either or both thiophene rings are oxidized into thiophene-<i>S</i>,<i>S</i>-dioxide to form an electron-accepting building block. Among these dyes, LAQ1 satisfied all the aforementioned requirements, and allowed us capturing ultra-small LDs on the endoplasmic reticulum (ER) membrane by stimulation emission depletion (STED) microscopy with a super-resolution below the diffraction limit of light. Moreover, the extremely high photostability of LAQ1 enabled recording the lipolysis of LDs and the concomitant lipogenesis as well as long-term trajectory analysis of micro LDs at the single particle level in living cells.

scholarly journals Structure modulation on fluorescent probes for biothiols and the reversible imaging of glutathione in living cells

RSC Advances ◽  
2021 ◽  
Vol 11 (34) ◽  
pp. 21116-21126
Author(s):  
Yu Li ◽  
Li Chen ◽  
Yan Zhu ◽  
Liming Chen ◽  
Xianglin Yu ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Fluorescent Probes ◽  
Living Cells ◽  
Structure Modulation

A reversible fluorescent probe for GSH was obtained through structure modulation, by which the intracellular GSH fluctuation was imaged.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

scholarly journals Pyridazino-1,3a,6a-Triazapentalenes as Versatile Fluorescent Probes: Impact of Their Post-Functionalization and Application for Cellular Imaging

2021 ◽  
Vol 22 (12) ◽  
pp. 6645
Author(s):  
Doina Sirbu ◽  
Nicolas Chopin ◽  
Ivana Martinić ◽  
Moussa Ndiaye ◽  
Svetlana Eliseeva ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Cross Coupling ◽  
Spectroscopic Properties ◽  
Cellular Imaging ◽  
Fluorescent Properties ◽  
Post Functionalization

Pyridazino-1,3a,6a-triazapentalenes (PyTAP) are compact fused 6/5/5 tricyclic scaffolds which exhibit promising fluorescent properties. Chemically stable, they can be post-functionalized using standard Pd-catalyzed cross-coupling chemistry. Several original PyTAP bearing additional unsaturated substituents in positions 2 and 8 were synthetized and their spectroscopic properties analyzed. They have been successfully tested as fluorescent probes for cellular imaging.

Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes

Nano Letters ◽  
2015 ◽  
Vol 15 (2) ◽  
pp. 1374-1381 ◽  
Author(s):  
Simon Hennig ◽  
Sebastian van de Linde ◽  
Martina Lummer ◽  
Matthias Simonis ◽  
Thomas Huser ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Super Resolution ◽  
Live Cell ◽  
Cellular Structures ◽  
Resolution Imaging
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