scholarly journals NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts

2017 ◽  
Author(s):  
Siân Culley ◽  
David Albrecht ◽  
Caron Jacobs ◽  
Pedro Matos Pereira ◽  
Christophe Leterrier ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Original Data ◽  
Focal Volume ◽  
Coated Pits ◽  
Superresolution Imaging ◽  
Cell Structures ◽  
Image Artefacts ◽  
Localisation Microscopy ◽  
Super Resolution Microscopy

Most super-resolution microscopy methods depend on steps that contribute to the formation of image artefacts. Here we present NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same focal volume, this approach generates a quantitative map of super-resolution defects, as well as methods for their correction. To illustrate its broad applicability to super-resolution approaches we apply our method to Localization Microscopy, STED and SIM images of a variety of in-cell structures including microtubules, poxviruses, neuronal actin rings and clathrin coated pits. We particularly focus on single-molecule localisation microscopy, where super-resolution reconstructions often feature imperfections not present in the original data. By showing the quantitative evolution of data quality over these varied sample preparation, acquisition and super-resolution methods we display the potential of NanoJ-SQUIRREL to guide optimization of superresolution imaging parameters.

scholarly journals Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Adrien Mau ◽  
Karoline Friedl ◽  
Christophe Leterrier ◽  
Nicolas Bourg ◽  
Sandrine Lévêque-Fort
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Uniform Illumination ◽  
Optical Sectioning ◽  
Coated Pits ◽  
Laser Illumination ◽  
Quantitative Analyses ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractNon-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and β2-spectrin in neurons. ASTER’s sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.

scholarly journals Left-handed DNA-PAINT for improved superresolution imaging in the nucleus

2020 ◽  
Author(s):  
H.J. Geertsema ◽  
G. Aimola ◽  
V. Fabricius ◽  
J.P. Fuerste ◽  
B.B. Kaufer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Hybridization ◽  
Super Resolution ◽  
Dna Oligomers ◽  
Superresolution Imaging ◽  
Left Handed ◽  
Nanoscale Topography ◽  
Target Molecules ◽  
Cellular Dna ◽  
Super Resolution Microscopy

AbstractDNA point accumulation in nanoscale topography (DNA-PAINT) advances super-resolution microscopy with superior resolution and multiplexing capabilities. However, cellular DNA may interfere with this single-molecule localization technique based on DNA-DNA hybridization. Here, we introduce left-handed DNA (L-DNA) oligomers that do not hybridize to naturally present R-DNA and demonstrate that L-DNA PAINT has the same specificity and multiplexing capability as R-DNA PAINT, but greatly improves specific visualization of nuclear target molecules.

scholarly journals Laser-free super-resolution microscopy

2017 ◽  
Author(s):  
Kirti Prakash
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Superresolution Microscopy ◽  
Correlative Imaging ◽  
Deep Blue ◽  
Meiotic Chromosomes ◽  
Enhanced Resolution ◽  
Blue Excitation ◽  
Localisation Microscopy ◽  
Super Resolution Microscopy

We report that single-molecule superresolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM), is an extension of single molecule localisation microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as “blinking”), detected and localised. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that STED and LFSM can be performed on the same biological sample using a simple imaging medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution.

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
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