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.

scholarly journals Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity

2021 ◽  
Author(s):  
Matthew D. Lycas ◽  
Aske L. Ejdrup ◽  
Andreas T. Sørensen ◽  
Nicolai O. Haahr ◽  
Søren H. Jørgensen ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Striatal Slices ◽  
D2 Autoreceptor ◽  
Molecular Components ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Excitatory Drive

SUMMARYThe nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.

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 Beginner’s guide to producing super-resolved images on a widefield fluorescence microscope

2020 ◽  
Vol 42 (4) ◽  
pp. 52-56
Author(s):  
Ilijana Vojnovic ◽  
Ulrike Endesfelder
Keyword(s):  
Fluorescence Microscopy ◽  
Sample Preparation ◽  
Single Molecule ◽  
Life Sciences ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Specialized Hardware ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

The development of super-resolution microscopy techniques, which are able to achieve resolutions in the nanometre range and as such allow the visualization of subcellular structures and dynamics, has considerably expanded the possibilities of fluorescence microscopy in the life sciences. While a majority of these techniques require highly specialized hardware, single-molecule localization microscopy (SMLM) can be implemented on conventional widefield fluorescence microscopes. Here, we describe what technical upgrades are necessary and discuss some of the difficulties that can be encountered during sample preparation and imaging.

scholarly journals Direct Observation of Anisotropy-Driven Formation of Amyloid Protein Core-Shell Structures in Real-time by Super-resolution Microscopy

2021 ◽  
Author(s):  
Min Zhang ◽  
Henrik Dahl Pinholt ◽  
Xin Zhou ◽  
Soeren S-R Bohr ◽  
Luca Banneta ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Growth Rates ◽  
Super Resolution ◽  
Average Growth ◽  
Single Molecule Level ◽  
Aggregation Processes ◽  
Morphological Transitions ◽  
Super Resolution Microscopy ◽  
Kinetics Of

Proteins misfolding and aggregation in the form of fibrils or amyloid containing spherulite-like structures, are involved in a spectrum of degenerative diseases. Current understanding of protein aggregation mechanism primarily relies on conventional spectrometric methods reporting the average growth rates and microscopy readouts of final structures, consequently masking the morphological and growth heterogeneity of the aggregates. Here we developed REal-time kinetics via binding and Photobleaching LOcalization Microscopy (REPLOM) super resolution method to observe directly and quantify the existence and abundance of diverse aggregation morphologies as well as the heterogeneous growth kinetics of each of them. Our results surprisingly revealed insulin aggregation is not exclusively isotropic, but it may also occur anisotropically. Combined with Machine learning we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape for each aggregation morphology. Our unifying framework of detection and analysis of spherulite growth can be extended to other protein systems and reveal their aggregation processes at single molecule level.

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 Graphene Energy Transfer for Single‐Molecule Biophysics, Biosensing, and Super‐Resolution Microscopy

2021 ◽  
pp. 2101099
Author(s):  
Izabela Kamińska ◽  
Johann Bohlen ◽  
Renukka Yaadav ◽  
Patrick Schüler ◽  
Mario Raab ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Super Resolution ◽  
Single Molecule Biophysics ◽  
Super Resolution Microscopy

scholarly journals Storm-Lite: An Inexpensive Tool to Demonstrate Stochastic Super Resolution Microscopy Techniques in the Classroom

2017 ◽  
Vol 112 (3) ◽  
pp. 464a
Author(s):  
Anthony Wu ◽  
Lark Moreno ◽  
Maxim Prigozhin ◽  
Sharlene Denos
Keyword(s):  
Super Resolution ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy
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