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 Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

2018 ◽  
Author(s):  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Michel Kranendonk ◽  
Emmanuel Margeat ◽  
Gilles Truan
Keyword(s):  
Single Molecule ◽  
Conformational Equilibrium ◽  
Fluorescent Dyes ◽  
Photophysical Properties ◽  
Accurate Determination ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

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 Self-healing dyes for super-resolution microscopy

2018 ◽  
Author(s):  
Jasper H. M. van der Velde ◽  
Jochem Smit ◽  
Michiel Punter ◽  
Thorben Cordes
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Superior Performance ◽  
Self Healing ◽  
Sted Microscopy ◽  
Performance Results ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Oligonucleotide Conjugates ◽  
Kinetics Of

AbstractIn recent years optical microscopy techniques have emerged that allow optical imaging at unprecented resolution beyond the diffraction limit. Up to date, photostabilizing buffers are the method of choice to realize either photoswitching and/or to enhance the signal brightness and stability of the employed fluorescent probes. This strategy has, however, restricted applicability and is not suitable for live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores with intramolecular photostabilization in super-resolution microscopy with targeted (STED) and stochastic readout (STORM). The overall goal of the study was to improve the spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. Due to its past superior performance we identified ATTO647N-photostabilizer conjugates as suitable candidates for STED microscopy. We characterize the photostability and resulting performance of NPA-ATTO647N oligonucleotide conjugates in STED microscopy. We find that the superior photophysical performance results in optimal STED imaging and demonstrate the possibility to obtain single-molecule fluorescent transients of individual fluorophores while illuminating with both the excitation- and STED-laser. Secondly, we show an analysis of photoswitching kinetics of self-healing Cy5 dyes (comprising TX, COT and NPA stabilizers) in the presence of TCEP- and cysteamine, which are typically used in STORM microscopy. In line with previous work, we find that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.

Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments

2010 ◽  
Vol 44 (1) ◽  
pp. 123-151 ◽  
Author(s):  
Marcia Levitus ◽  
Suman Ranjit
Keyword(s):  
Single Molecule ◽  
Fluorescent Dyes ◽  
Photophysical Properties ◽  
Super Resolution ◽  
Cyanine Dyes ◽  
Reducing Agents ◽  
Photochemical Reactions ◽  
Polymethine Chain ◽  
Relaxation Processes ◽  
Single Molecule Spectroscopy

AbstractThe breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis–trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.

scholarly journals Quantitative Single-Molecule Imaging with Statistical Machine Learning

2021 ◽  
Author(s):  
Artittaya Boonkird ◽  
Daniel F. Nino ◽  
Joshua N Milstein
Keyword(s):  
Machine Learning ◽  
Single Molecule ◽  
Photophysical Properties ◽  
Super Resolution ◽  
Nanometer Scale ◽  
Dna Nanostructures ◽  
Statistical Machine Learning ◽  
Machine Learning Technique ◽  
Learning Technique ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) is a super-resolution technique capable of rendering nanometer scale images of cellular structures. Recently, much effort has gone into developing SMLM into a quantitative method capable of determining the abundance and stoichiometry of macromolecular complexes. These methods often require knowledge of the complex photophysical properties of photoswitchable flourophores. We previously developed a simpler method built upon the observation that most photswitchable fluorophores emit an exponentially distributed number of blinks before photobleaching, but its utility was limited by the need to calibrate for the blinking distribution. Here we extend this method by incorporating a machine learning technique known as Expectation-Maximization (EM) and apply it to a statistical mixture model of monomers, dimers and trimers. We show that the protomer fractions and the underlying single-fluorophore blinking distributions can be inferred, simultaneously, from SMLM datasets, obviating the need for an additional calibration and greatly expanding the applicability of this technique. To illustrate the utility of our approach, we benchmark the method on both simulated datasets and experimental datasets assembled from dSTORM images of Alexa-647 labeled DNA nanostructures.

scholarly journals Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jagadish Sankaran ◽  
Harikrushnan Balasubramanian ◽  
Wai Hoh Tang ◽  
Xue Wen Ng ◽  
Adrian Röllin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Temporal Dynamics ◽  
Growth Factor Receptor ◽  
Super Resolution ◽  
Actin Binding ◽  
Biological Knowledge ◽  
Data Set ◽  
Epidermal Growth ◽  
Molecular Brightness ◽  
Super Resolution Microscopy

AbstractSuper-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Learning the distribution of single-cell chromosome conformations in bacteria reveals emergent order across genomic scales

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joris J. B. Messelink ◽  
Muriel C. F. van Teeseling ◽  
Jacqueline Janssen ◽  
Martin Thanbichler ◽  
Chase P. Broedersz
Keyword(s):  
Single Cell ◽  
Caulobacter Crescentus ◽  
Model Organism ◽  
Super Resolution ◽  
Chromosome Organization ◽  
General Strategy ◽  
Cell Axis ◽  
Cellular Processes ◽  
Emergent Order ◽  
Genomic Clusters

AbstractThe order and variability of bacterial chromosome organization, contained within the distribution of chromosome conformations, are unclear. Here, we develop a fully data-driven maximum entropy approach to extract single-cell 3D chromosome conformations from Hi–C experiments on the model organism Caulobacter crescentus. The predictive power of our model is validated by independent experiments. We find that on large genomic scales, organizational features are predominantly present along the long cell axis: chromosomal loci exhibit striking long-ranged two-point axial correlations, indicating emergent order. This organization is associated with large genomic clusters we term Super Domains (SuDs), whose existence we support with super-resolution microscopy. On smaller genomic scales, our model reveals chromosome extensions that correlate with transcriptional and loop extrusion activity. Finally, we quantify the information contained in chromosome organization that may guide cellular processes. Our approach can be extended to other species, providing a general strategy to resolve variability in single-cell chromosomal organization.

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