Boosting the Localization Precision in Super-Resolution Microscopy: booSTORM

AbstractWe introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

Download Full-text

Heavy water: a simple solution to increasing the brightness of fluorescent proteins in super-resolution imaging

Chemical Communications ◽

10.1039/c5cc04575d ◽

2015 ◽

Vol 51 (70) ◽

pp. 13451-13453 ◽

Cited By ~ 15

Author(s):

Wei Qiang Ong ◽

Y. Rose Citron ◽

Joerg Schnitzbauer ◽

Daichi Kamiyama ◽

Bo Huang

Keyword(s):

Heavy Water ◽

Fluorescent Proteins ◽

Super Resolution ◽

Simple Solution ◽

Photon Yield ◽

Resolution Imaging ◽

Localization Precision ◽

Super Resolution Microscopy

D2O improves the photon yield of photoactivatable fluorescent proteins and thus the localization precision for super-resolution microscopy.

Download Full-text

Combining 3D single molecule localization strategies for reproducible bioimaging

10.1101/385799 ◽

2018 ◽

Cited By ~ 2

Author(s):

Clément Cabriel ◽

Nicolas Bourg ◽

Pierre Jouchet ◽

Guillaume Dupuis ◽

Christophe Leterrier ◽

...

Keyword(s):

Single Molecule ◽

Super Resolution ◽

3D Localization ◽

Point Spread ◽

Spread Function ◽

Multicolor Imaging ◽

Localization Precision ◽

Different Depths ◽

Super Resolution Microscopy ◽

Living Bacteria

We developed a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1 μm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently to provide a weakly anisotropic 3D resolution over the imaging range. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several-μm range by direct merging of multiple acquisitions at different depths.

Download Full-text

MINSTED fluorescence localization and nanoscopy

10.1101/2020.10.31.363424 ◽

2020 ◽

Author(s):

Michael Weber ◽

Marcel Leutenegger ◽

Stefan Stoldt ◽

Stefan Jakobs ◽

Tiberiu S. Mihaila ◽

...

Keyword(s):

Standard Deviation ◽

Super Resolution ◽

Diffraction Limit ◽

Stimulated Emission ◽

Far Field ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Molecular Scale ◽

Localization Precision ◽

Super Resolution Microscopy

AbstractWe introduce MINSTED, a stimulated-emission-depletion (STED) based fluorescence localization and super-resolution microscopy concept providing spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED donut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background, and the required number of fluorescence detections are low compared to most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200-1000 detections per fluorophore provide a localization precision of 1-3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement of far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

Download Full-text

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy

Molecules ◽

10.3390/molecules23123165 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3165 ◽

Cited By ~ 14

Author(s):

Philipp Blumhardt ◽

Johannes Stein ◽

Jonas Mücksch ◽

Florian Stehr ◽

Julian Bauer ◽

...

Keyword(s):

Binding Site ◽

Binding Sites ◽

Fluorescent Dyes ◽

Super Resolution ◽

Correlation Spectroscopy ◽

Reversible Binding ◽

Oxygen Scavenging ◽

Nanoscale Topography ◽

Localization Precision ◽

Super Resolution Microscopy

The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.

Download Full-text

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽

10.32607/2075851-2017-9-4-42-51 ◽

2017 ◽

Vol 9 (4) ◽

pp. 42-51

Author(s):

S. S. Ryabichko ◽

◽

A. N. Ibragimov ◽

L. A. Lebedeva ◽

E. N. Kozlov ◽

...

Keyword(s):

Cell Nucleus ◽

Super Resolution ◽

Structure And Function ◽

And Function ◽

Super Resolution Microscopy

Download Full-text

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

10.26434/chemrxiv.9209450.v1 ◽

2019 ◽

Author(s):

Jeffrey Chang ◽

Matthew Romei ◽

Steven Boxer

Keyword(s):

Rational Design ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Super Resolution ◽

Unit Cell Size ◽

Chlorine Substituent ◽

Gfp Chromophore ◽

The One ◽

Green Fluorescent ◽

Super Resolution Microscopy

Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of cis and trans rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the trans state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.

Download Full-text