Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

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Dynamic single-molecule counting for the quantification and optimization of nanoparticle functionalization protocols

Nanoscale ◽

10.1039/c9nr10218c ◽

2020 ◽

Vol 12 (6) ◽

pp. 4128-4136 ◽

Cited By ~ 3

Author(s):

Matěj Horáček ◽

Dion J. Engels ◽

Peter Zijlstra

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Medical Applications ◽

Single Molecule Level ◽

Microscopy Method ◽

Super Resolution Microscopy ◽

Direct Quantification

We provide a super-resolution microscopy method to characterize the chemical interface of nanoparticles at the single-molecule level. This provides a direct quantification and optimization of functionalization protocols for bio-medical applications.

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Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

10.1101/2020.06.26.174102 ◽

2020 ◽

Author(s):

Sven A. Szilagyi ◽

Moritz Burmeister ◽

Q. Tyrell Davis ◽

Gero L. Hermsdorf ◽

Suman De ◽

...

Keyword(s):

Single Molecule ◽

Signal To Noise Ratio ◽

Numerical Aperture ◽

Light Sheet ◽

Living Cells ◽

High Signal ◽

Active Optics ◽

Single Molecule Level ◽

Light Sheet Microscopy ◽

Reflected Light

AbstractObservation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

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Protein clustering and spatial organization in T-cells

Biochemical Society Transactions ◽

10.1042/bst20140316 ◽

2015 ◽

Vol 43 (3) ◽

pp. 315-321 ◽

Cited By ~ 5

Author(s):

Michael J. Shannon ◽

Garth Burn ◽

Andrew Cope ◽

Georgina Cornish ◽

Dylan M. Owen

Keyword(s):

T Cell ◽

Single Molecule ◽

Spatial Organization ◽

Super Resolution ◽

Cell Protein ◽

Single Molecule Level ◽

Composition And Structure ◽

Resolution Analysis ◽

And Function ◽

Super Resolution Microscopy

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.

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

10.1101/2021.08.20.457097 ◽

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.

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Cellular dynamics of the SecA ATPase at the single molecule level

Scientific Reports ◽

10.1038/s41598-021-81081-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anne-Bart Seinen ◽

Dian Spakman ◽

Antoine M. van Oijen ◽

Arnold J. M. Driessen

Keyword(s):

Protein Secretion ◽

Single Molecule ◽

Membrane Surface ◽

Super Resolution ◽

Single Molecule Level ◽

Cellular Environment ◽

A Minor ◽

Super Resolution Microscopy ◽

Dynamic Interplay ◽

Gain Access

AbstractIn bacteria, the SecA ATPase provides the driving force for protein secretion via the SecYEG translocon. While the dynamic interplay between SecA and SecYEG in translocation is widely appreciated, it is not clear how SecA associates with the translocon in the crowded cellular environment. We use super-resolution microscopy to directly visualize the dynamics of SecA in Escherichia coli at the single-molecule level. We find that SecA is predominantly associated with and evenly distributed along the cytoplasmic membrane as a homodimer, with only a minor cytosolic fraction. SecA moves along the cell membrane as three distinct but interconvertible diffusional populations: (1) A state loosely associated with the membrane, (2) an integral membrane form, and (3) a temporarily immobile form. Disruption of the proton-motive-force, which is essential for protein secretion, re-localizes a significant portion of SecA to the cytoplasm and results in the transient location of SecA at specific locations at the membrane. The data support a model in which SecA diffuses along the membrane surface to gain access to the SecYEG translocon.

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Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

Nature Communications ◽

10.1038/s41467-021-22002-9 ◽

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.

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Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

International Journal of Molecular Sciences ◽

10.3390/ijms22041903 ◽

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.

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