scholarly journals From Microscopy to Nanoscopy: Defining an Arabidopsis thaliana Meiotic Atlas at the Nanometer Scale

2021 ◽  
Vol 12 ◽  
Author(s):  
Jason Sims ◽  
Peter Schlögelhofer ◽  
Marie-Therese Kurzbauer
Keyword(s):  
Arabidopsis Thaliana ◽  
New Technologies ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Great Promise ◽  
Nanometer Scale ◽  
Meiotic Chromosomes ◽  
Optimal Protocol ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

Visualization of meiotic chromosomes and the proteins involved in meiotic recombination have become essential to study meiosis in many systems including the model plant Arabidopsis thaliana. Recent advances in super-resolution technologies changed how microscopic images are acquired and analyzed. New technologies enable observation of cells and nuclei at a nanometer scale and hold great promise to the field since they allow observing complex meiotic molecular processes with unprecedented detail. Here, we provide an overview of classical and advanced sample preparation and microscopy techniques with an updated Arabidopsis meiotic atlas based on super-resolution microscopy. We review different techniques, focusing on stimulated emission depletion (STED) nanoscopy, to offer researchers guidance for selecting the optimal protocol and equipment to address their scientific question.

scholarly journals Laser-free super-resolution microscopy

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Kirti Prakash
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Correlative Imaging ◽  
Deep Blue ◽  
Meiotic Chromosomes ◽  
Set Up ◽  
Super Resolution Microscopy

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope set-up and a mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localization microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as ‘blinking’), detected and localized. 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 stimulated emission depletion and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

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 MINSTED fluorescence localization and nanoscopy

2021 ◽  
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 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.

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

scholarly journals How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology?

Nanoscale ◽  
2021 ◽  
Author(s):  
Adelaide Miranda ◽  
Ana I. Gómez-Varela ◽  
Andreas Stylianou ◽  
Liisa M. Hirvonen ◽  
Humberto Sánchez ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Super Resolution ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Detailed Picture

This review provides a detailed picture of the innovative efforts to combine atomic force microscopy and different super-resolution microscopy techniques to elucidate biological questions.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

scholarly journals pIgR and PECAM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion

2017 ◽  
Vol 214 (6) ◽  
pp. 1619-1630 ◽  
Author(s):  
Federico Iovino ◽  
Joo-Yeon Engelen-Lee ◽  
Matthijs Brouwer ◽  
Diederik van de Beek ◽  
Arie van der Ende ◽  
...  
Keyword(s):  
Pneumococcal Meningitis ◽  
Case Fatality ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Brain Invasion ◽  
Immunoglobulin Receptor ◽  
Platelet Endothelial Cell Adhesion ◽  
Lower Extent ◽  
Super Resolution Microscopy ◽  
The Brain

Streptococcus pneumoniae is the main cause of bacterial meningitis, a life-threating disease with a high case fatality rate despite treatment with antibiotics. Pneumococci cause meningitis by invading the blood and penetrating the blood–brain barrier (BBB). Using stimulated emission depletion (STED) super-resolution microscopy of brain biopsies from patients who died of pneumococcal meningitis, we observe that pneumococci colocalize with the two BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1). We show that the major adhesin of the pneumococcal pilus-1, RrgA, binds both receptors, whereas the choline binding protein PspC binds, but to a lower extent, only pIgR. Using a bacteremia-derived meningitis model and mutant mice, as well as antibodies against the two receptors, we prevent pneumococcal entry into the brain and meningitis development. By adding antibodies to antibiotic (ceftriaxone)-treated mice, we further reduce the bacterial burden in the brain. Our data suggest that inhibition of pIgR and PECAM-1 has the potential to prevent pneumococcal meningitis.

scholarly journals Expansion stimulated emission depletion microscopy (ExSTED)

2018 ◽  
Author(s):  
Mengfei Gao ◽  
Riccardo Maraspini ◽  
Oliver Beutel ◽  
Amin Zehtabian ◽  
Britta Eickholt ◽  
...  
Keyword(s):  
Excited State ◽  
Optical Resolution ◽  
Super Resolution ◽  
Stimulated Emission ◽  
High Fidelity ◽  
Sted Microscopy ◽  
Structural Details ◽  
Stimulated Emission Depletion ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

AbstractStimulated emission depletion (STED) microscopy is routinely used to resolve the ultra-structure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

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 Dynamics and nanoscale organization of the postsynaptic endocytic zone at excitatory synapses

2021 ◽  
Author(s):  
Lisa A.E. Catsburg ◽  
Manon Westra ◽  
Annemarie M. L. van Schaik ◽  
Harold D. MacGillavry
Keyword(s):  
Synaptic Plasticity ◽  
Actin Cytoskeleton ◽  
Glutamate Receptors ◽  
Super Resolution ◽  
Live Cell ◽  
Excitatory Synapses ◽  
Membrane Interactions ◽  
Synaptic Receptors ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

ABSTRACTAt postsynaptic sites of neurons, a prominent clathrin-coated structure, the endocytic zone (EZ), controls the trafficking of glutamate receptors and is essential for synaptic plasticity. Despite its importance, little is known about how this clathrin structure is organized to mediate endocytosis. We used live-cell and super-resolution microscopy techniques to reveal the dynamic organization of this poorly understood clathrin structure. We found that a subset of endocytic proteins only transiently appeared at postsynaptic sites. In contrast, other proteins, including Eps15, intersectin1L, and β2-adaptin, were persistently enriched and partitioned at the edge of the EZ. We found that uncoupling the EZ from the synapse led to the loss of most of these components, while disrupting the actin cytoskeleton or AP2-membrane interactions did not alter EZ positioning. We conclude that the EZ is a stable, highly organized molecular platform where components are differentially recruited and positioned to orchestrate the endocytosis of synaptic receptors.

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