Towards structural biology with super-resolution microscopy

Nanoscale ◽  
2018 ◽  
Vol 10 (35) ◽  
pp. 16416-16424 ◽  
Author(s):  
Julia Molle ◽  
Leonhard Jakob ◽  
Johann Bohlen ◽  
Mario Raab ◽  
Philip Tinnefeld ◽  
...  
Keyword(s):  
Structural Biology ◽  
Single Molecule ◽  
Structural Organization ◽  
Dna Nanotechnology ◽  
Super Resolution ◽  
Super Resolution Microscopy

The combination of DNA nanotechnology and single-molecule biochemistry allows the first step towards the investigation of the structural organization of a protein via SR microscopy.

Single Molecule Super-Resolution Microscopy Study on the Precision with Which DNA Nanostructures Can Orient Fluorescent Dyes

2020 ◽  
Author(s):  
Brett Michael Ward
Keyword(s):  
Single Molecule ◽  
Dna Nanotechnology ◽  
Super Resolution ◽  
Fluorescent Dye ◽  
Molecular Networks ◽  
Moderate Degree ◽  
Dna Nanostructures ◽  
Dye Molecules ◽  
Super Resolution Microscopy ◽  
Fluorescent Molecules

DNA nanotechnology enables the rapid, programmable self-assembly of novel structures and devices at the nanoscale. Utilizing the simplicity of Watson-Crick base pairing, DNA nanostructures are capable of assembling a variety of nanoparticles in arbitrary configurations with relative ease. Several emerging opto-electronic systems require a high degree of control of both the position and orientation of component fluorescent molecules, and while DNA nanostructures have demonstrated these capabilities, the precision with which DNA can orient fluorescent molecules is not well understood. Determining these bounds is critical in establishing the viability of DNA nanotechnology as a method of assembling fluorescent molecular networks. In this work, using a combination of single molecule emission dipole imaging and super-resolution microscopy techniques, we correlate the orientations of fluorescent dye molecules to the orientations of their DNA substrates along five degrees of freedom. Several species of dyes were embedded within a DNA sequence using either one or two covalent tethers. These strands were incorporated directly into DNA origami structures to investigate the dependence of the location and binding architecture of the dye on the orientational precision of DNA nanostructures. Dye functionalized strands were also folded into a simpler four-arm junction, which was then immobilized on an origami structure to study the influence of the DNA substrate on dye orientation. Correlated analysis of super-resolution images of origami structures and single molecule emission dipole images from the embedded fluorescent molecule within the same structure allowed us to directly measure the relative orientations of dye molecules within DNA nanostructures. The resulting measurements revealed a moderate degree of polar angle control but a large variation in azimuthal control for the majority of structures examined. These measurements establish a single-molecule method for measurement of correlated orientations and provide a powerful approach for future studies on increasing the precision in the orientational control of fluorescent dye molecule monomers by 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 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

Sodium sulfite as a switching agent for single molecule based super-resolution optical microscopy

2021 ◽  
Author(s):  
Anders K Engdahl ◽  
Oleg Grauberger ◽  
Mark Schüttpelz ◽  
Thomas Huser
Keyword(s):  
Single Molecule ◽  
Fluorescent Dyes ◽  
Sodium Sulfite ◽  
Super Resolution ◽  
Oxygen Scavenger ◽  
Dark State ◽  
Alexa Fluor ◽  
High Illumination ◽  
Human Osteosarcoma Cells ◽  
Super Resolution Microscopy

Photoinduced off-switching of organic fluorophores is routinely used in super-resolution microscopy to separate and localize single fluorescent molecules, but the method typically relies on the use of complex imaging buffers. The most common buffers use primary thiols to reversibly reduce excited fluorophores to a non-fluorescent dark state, but these thiols have a limited shelf life and additionally require high illumination intensities in order to efficiently switch the emission of fluorophores. Recently a high-index, thiol-containing imaging buffer emerged which used sodium sulfite as an oxygen scavenger, but the switching properties of sulfite was not reported on. Here, we show that sodium sulfite in common buffer solutions reacts with fluorescent dyes, such as Alexa Fluor 647 and Alexa Fluor 488 under low to medium intensity illumination to form a semi-stable dark state. The duration of this dark state can be tuned by adding glycerol to the buffer. This simplifies the realization of different super-resolution microscopy modalities such as direct Stochastic Reconstruction Microscopy (dSTORM) and Super-resolution Optical Fluctuation Microscopy (SOFI). We characterize sulfite as a switching agent and compare it to the two most common switching agents by imaging cytoskeleton structures such as microtubules and the actin cytoskeleton in human osteosarcoma cells.

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

2021 ◽  
Vol 33 (42) ◽  
pp. 2105719
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 Live cell single molecule-guided Bayesian localization super resolution microscopy

Cell Research ◽  
2016 ◽  
Vol 27 (5) ◽  
pp. 713-716 ◽  
Author(s):  
Fan Xu ◽  
Mingshu Zhang ◽  
Wenting He ◽  
Renmin Han ◽  
Fudong Xue ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Live Cell ◽  
Super Resolution Microscopy
Sign in / Sign up

Export Citation Format

Share Document